Citation
An investigation on how 8-OXO-7, 8-dihydroguanosine affects RNASE A, RNASE T1, and XRN1 rectivity with oligonucleotides of RNA and RNA structure using the theophylline binding aptamer as model

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Title:
An investigation on how 8-OXO-7, 8-dihydroguanosine affects RNASE A, RNASE T1, and XRN1 rectivity with oligonucleotides of RNA and RNA structure using the theophylline binding aptamer as model
Creator:
Kiggins, Courtney N.
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Master's ( Master of science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Chemistry, CU Denver
Degree Disciplines:
Chemistry
Committee Chair:
Resendiz, Marino J. E.
Committee Members:
Wang, Haobin
Ren, Xiaojun
Fisk, John D.

Notes

Abstract:
8-Oxo-7,8-dihydroguanosine (8-oxoG) is a result of oxidative damage on the nucleobase Guanosine. 8-OxoG has been well characterized in DNA, but little is known of its function in RNA. The purpose of this study was to understand how 8-oxoG influences enzyme reactivity, RNA structure, and RNA function. First, endoribonucleases Ribonuclease A (RNase A) and Ribonuclease T1 (RNase T1) were studied to understand 8-oxoG recognition. It was found that 8- oxoG becomes a substrate for RNase A, while it was not recognized by RNase T1. The functional group on C8 causes an anti to syn flip which exposes new hydrogen bonding patterns similar to that of Uridine, which allowed RNase A reactivity. The conformational change, though, causes 8-oxoG to lose its ability to be a substrate for RNase T1 due to sterics and adverse hydrogen bonding induced by the exocyclic functional group at C8. The information gathered from the endoribonucleases was then applied to study how 8-oxoG begets structural and functional changes to a well-studied aptamer, the theophylline binding aptamer. 8-OxoG insertion caused a 100-fold increase in dissociation constant (Kd) for the theophylline binding aptamers modified at positions G25 and G26, while a modification at G11 prevented the aptamer from binding to theophylline. RNase A and T1 degradation data also yielded different degradation sites in the modified aptamers compared to the canonical, indicating a change in structure. Following, the reactivity of exoribonuclease XRN1 was studied in the presence of oligomers containing one to three 8-oxoG modifications due to XRN1’s role in oxidized mRNA surveillance. XRN1 was found to stall at 8-oxoG sites as a function of number of 8-oxoG present when more than one modification was introduced. The data combined helps provide insights into how 8-oxoG may affect RNA decay and surveillance mechanisms

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University of Colorado Denver
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Auraria Library
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Copyright Courtney N. Kiggins. Permission granted to University of Colorado Denver to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Full Text
AN INVESTIGATION ON HOW 8-OXO-7,8-DMYDROGAUNOSINE AFFECTS RNASE
A, RNASE Ti, AND XRN1 REACTIVITY WITH OLIGONUCLEOTIDES OF RNA AND RNA STRUCTURE USING THE THEOPHYLLINE BINDING APTAMER AS MODEL
By
COURTNEY N. KIGGINS B.S., The Ohio State University, 2014
A Thesis submitted to the faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for a degree of Master of Science Chemistry Program
2019


This thesis for the Master of Science degree by Courtney N. Kiggins has been approved for the Chemistry Program by
Marino J.E. Resendiz, Chair Haobin Wang Xiaojun Ren John D. Fisk
Date: May 18, 2019


Kiggins, Courtney N. (M.S., Chemistry)
An Investigation on how 8-Oxo-7,8-dihydroguanosine Affects RNase A, Ti, and XRN1 Reactivity with Oligonucleotides of RNA and RNA Structure using the Theophylline Binding Aptamer as Model
Thesis directed by Assistant Professor Marino J.E. Resendiz
ABSTRACT
8-Oxo-7,8-dihydroguanosine (8-oxoG) is a result of oxidative damage on the nucleobase Guanosine. 8-OxoG has been well characterized in DNA, but little is known of its function in RNA. The purpose of this study was to understand how 8-oxoG influences enzyme reactivity, RNA structure, and RNA function. First, endoribonucleases Ribonuclease A (RNase A) and Ribonuclease Ti (RNase Ti) were studied to understand 8-oxoG recognition. It was found that 8-oxoG becomes a substrate for RNase A, while it was not recognized by RNase Ti. The functional group on C8 causes an anti to syn flip which exposes new hydrogen bonding patterns similar to that of Uridine, which allowed RNase A reactivity. The conformational change, though, causes 8-oxoG to lose its ability to be a substrate for RNase Ti due to sterics and adverse hydrogen bonding induced by the exocyclic functional group at C8. The information gathered from the endoribonucleases was then applied to study how 8-oxoG begets structural and functional changes to a well-studied aptamer, the theophylline binding aptamer. 8-OxoG insertion caused a 100-fold increase in dissociation constant (Kd) for the theophylline binding aptamers modified at positions G25 and G26, while a modification at G11 prevented the aptamer from binding to theophylline. RNase A and Ti degradation data also yielded different degradation sites in the modified aptamers compared to the canonical, indicating a change in structure. Following, the reactivity of exoribonuclease XRN1 was studied in the presence of oligomers containing one to three 8-oxoG modifications due to XRNl’s role in oxidized mRNA surveillance. XRN1 was
iii


found to stall at 8-oxoG sites as a function of number of 8-oxoG present when more than one modification was introduced. The data combined helps provide insights into how 8-oxoG may affect RNA decay and surveillance mechanisms.
The form and content of this abstract approved. I recommend its publication.
Approved: Marino J.E. Resendiz
IV


ACKNOWLEDGEMENTS
I am incredibly grateful for this journey and know I have a lot of people to thank for my success over the past year and a half. First, I would like to thank the Air Force Institute of Technology (AFIT) and the Chemistry Department at the United States Air Force Academy (USAFA) for sponsoring my Master of Science in Chemistry. I am very grateful for this opportunity and am excited for the assignment to follow. I wanted to thank my professors and mentors from my time as an Undergraduate at the Ohio State University. In particular, I would like to thank Dr. Chris Callam and Dr. Prabir Dutta for always believing in me.
I would like to thank the Biophysics Core at Anschutz Medical Campus for providing me access to the Microscale Thermophoresis. I would like to thank Shaun Bevers from the Biophysics Core for training me on the MST, all the insightful conversations, and all the help on trying to figure out my MST data. Thank you to Dr. Eric Chapman from the University of Denver for the gift of the XRN1 enzyme—looking forward to see where these results lead.
Thank you to my professors and teachers at University of Colorado Denver, in particular Dr. Jeff Knight and Dr. Xiaojun Ren, for the mentorship and guidance along the way. I owe an enormous amount of gratitude to Dr. Marino Resendiz for accepting me into his lab and teaching me all my newfound skills that I can carry with me to USAFA and the rest of my career. I am very lucky to have had an advisor who has the best interest of the student and who is committed and dedicated to the success of the student every step of the way. I know trying to finish a Thesis based Master’s in a year and a half was going to be tricky, crazy even, and I am so grateful for his guidance and his dedication to make sure I would finish on time. I also am grateful for my lab mates—thanks making research more enjoyable!
v


When I think back on my application to the USAFA Faculty Pipeline program, it’s funny to think of how wild of an idea that seemed at the time and how I never would’ve applied if it weren’t for one person. Thank you to Capt Ryan Patrick for putting the idea in my head. I can say with all certainty that if he had never even mentioned the pipeline to me, I never would’ve known about it, and I literally would not be writing this acknowledgement section. Thank you to the leadership at SMC from Kirtland AFB for supporting me in my application to the pipeline and doing everything they could so that I could pursue this endeavor.
Thank you to all the amazing friends in my life, especially my peers who have gone through this journey with me—Chelsea O’Hara, Sam Kent, Johanna Denne, Eunji Im, Kyle Brown, and Brian Shelton. They’ve provided me tremendous help, friendship, and laughs to curb our impending stress. Thank you to all my friendships outside of school and for providing me places to crash in Denver after I moved! Kate Treadwell and Kelsey Robb, your support has been incredible. Thank you to Capt Paige Pluemer and Capt Gabby Quirao for being listening ears and amazing friends. Thank you to Major Jonny Hoang for all your help and advice trying to figure out my PCS and preparing me for my next assignment.
Thank you to my mom, dad, and brother for always supporting me and believing in me. Fm very grateful for all you’ve done for me. Who would’ve thought the girl who could barely stay focused enough in Undergrad would go on to be teaching at USAFA shortly! Last, and certainly not least, I want to extend my never-ending gratitude to my love Dan Higgins. I could NOT have kept any ounce of sanity during this, especially after increasing my commute by an hour+, without your support and love. You have done so much for me during this, and I can’t express how grateful and lucky I am to have you.
vi


TABLE OF CONTENTS
I: INTRODUCTION.....................................................................1
8-Oxo-7,8-dihydrogaunosine (8-oxoG)...............................................1
Endoribonucleases................................................................2
Ribonuclease A.................................................................2
Ribonuclease Ti................................................................3
Theophylline Binding Aptamer......................................................5
XRN1 Exoribonuclease..............................................................7
Microscale Thermophoresis.........................................................8
II: EXPERIMENTAL...................................................................11
Materials.......................................................................11
Procedure.......................................................................12
RNase Free Water..............................................................12
RNA Oligomer Synthesis........................................................12
Deprotection of oligonucleotides..............................................13
Purification of RNA...........................................................13
Radiolabeling of RNA..........................................................14
5’ end Labeling using P-32....................................................14
3’ end Labeling using PCP.....................................................16
Enzymatic Degradation Experiments.............................................16
Microscale Thermophoresis (MST)...............................................18
Structure Characterization....................................................20
vii


Ill: RESULTS
22
Endoribonuclease Degradation of Oligonucleotides Containing 8-oxo-G..........22
RNase A...................................................................22
RNase Ti..................................................................26
Theophylline Binding Aptamer Studies.........................................27
Degradation studies.......................................................27
Small Molecule Binding Affinity Studies...................................31
XRN1 Exoribonuclease.........................................................34
IV: DISCUSSION.................................................................36
Enzymatic Degradation of Oligonucleotides Containing 8-oxo-G.................36
RNase A Reactivity........................................................36
RNase Ti reactivity.......................................................38
Theophylling Binding Aptamer Studies.........................................39
XRN1 Exoribonuclease.........................................................43
V: CONCLUSION..................................................................44
REFERENCES.....................................................................46
APPENDIX.......................................................................49
viii


LIST OF FIGURES
FIGURE
Figure 1. Hydrogen bonding patterns found in Guanine vs 8-oxoGua..........................2
Figure 2. RNase A active site.............................................................3
Figure 3. RNase Ti active site............................................................4
Figure 4. Xanthine derivatives............................................................5
Figure 5. Secondary and tertiary structures for the theophylline binding aptamer..........7
Figure 6. XRN1 active site................................................................8
Figure 7. Example of raw MST data........................................................10
Figure 8. Parameters for variable temperature measurements...............................21
Figure 9. RNase A Degradation of 8-oxoG sites in RNA1, RNA2, RNA3, and RNA4..............23
Figure 10. Comparing substrates at differing concentrations of RNase A...................24
Figure 11. RNase A degradation seen in RNA5 and RNA6.....................................24
Figure 12. RNase A degradation patterns for oligomers containing 8-oxoA..................25
Figure 13. RNase Ti degradation pattern for oligomers containing 8-oxoG..................26
Figure 14. RNase Ti degradation pattern for RNA1, RNA2, RNA5, and RNA6...................27
Figure 15. RNase A degradation patterns for the theophylline aptamers....................28
Figure 16. RNase Ti degradation patterns for the theophylline aptamers...................30
Figure 17. MST data of the theophylline binding aptamers in presence of theophylline.....32
Figure 18. Example of TP-RNA3 not binding to theophylline................................33
Figure 19. (A) MST data of TP-RNA1 and theobromine.......................................33
Figure 20. XRN1 degradation data of RNA1-4...............................................35
Figure 21. Proposed RNase A reactivity with 8-oxoG.......................................37
Figure 22. Proposed RNase Ti lack of reactivity with 8-oxoG..............................38
IX


Figure 23. Representation of TP-RNA1 structure......................................40
Figure 24. Representation of TP-RNA2 structure......................................41
Figure 25. Representation of TP-RNA3 structure......................................42
Figure SI. MALDI spectra of TP-RNAC.................................................49
Figure S2. MALDI spectra of TP-RNA1.................................................49
Figure S3. MALDI spectra of TP-RNA2.................................................49
Figure S4. MALDI spectra of TP-RNA3.................................................50
Figure S5. RNase A experiment diluting to concentrations............................50
Figure S6. Theophylline binding aptamers with TB....................................52
Figure S8. TP-RNAC CD melt and spectra Na2P2C>7buffer, pH 7.5.......................52
Figure S9. TP-RNA1 CD melt and spectra Na2P2C>7 buffer, pH 7.5......................53
Figure S10. TP-RNA2 CD melt and spectra Na2P2C>7 buffer, pH 7.5.....................53
Figure S11.TP-RNA3 CD melt and spectra Na2P2C>7 buffer, pH 7.5......................53
Figure S12. TP-RNAC melt TBS buffer, pH 7.6........................................54
Figure S13. TP-RNA1 melt TBS buffer, pH 7.6........................................54
Figure S14. TP-RNA2 melt TBS buffer, pH 7.6........................................55
Figure S15. TP-RNA3 melt TBS buffer, pH 7.6........................................55
Figure SI6. XRN1 degradation data (incubation for 2 hours)..........................56
Figure S17. Accurate RNase A Theophylline Binding Aptamer data......................58
x


LIST OF TABLES
TABLE
Table 1. Small molecules used in the binding affinity studies and the respective concentration
ranges...............................................................................19
Table 2. Small molecules used in the binding affinity studies and the respective concentration
ranges...............................................................................19
Table 3. Melting temperature of each aptamer in corresponding buffer.......................31
xi


CHAPTER I
INTRODUCTION
Oxidative damage has been widely studied in DNA, yet little is understood about its role in RNA [1], Oxidation, on both DNA and RNA, occurs readily on purines, where oxidation occurs on the 8th position of the nucleobase to yield 8-oxopurines. Purines have lower redox potentials than pyrimidines, making them more susceptible to oxidative damage. Studies of 8-oxo-purines have suggested that: 1). Genetic factors do not influence oxidation on DNA or RNA and 2). These lesions were not present in primordial RNA [1,2], This suggests that DNA and RNA oxidation occurs as a result of oxidative damage from endogenous and exogenous reactive oxygen species (ROS). Research is emerging which correlates oxidatively damaged RNA and disease, to include Alzheimer's, Type II Diabetes, and Parkinson’s. Interestingly, increased levels of a particular 8-oxo-purine, 8-oxo-Guanosine (8-oxoG), is found in the cerebrospinal fluid in Alzheimer’s patients and in the urine of Type II Diabetes sufferers [1], Other than being highly prevalent as a byproduct in inflammatory diseases, 8-oxoG is also correlated with protein misfolding and synthesis, suggesting that the lesion may alter RNA structure and function in tRNA and/or mRNA [4], These findings served as the motivation to understand 8-oxoG’s influence on RNA degradation, structure, and interactions with small molecules and biomolecules.
8-Oxo-7,8-dihydrogaunosine (8-oxoG)
It has been established that the oxidative lesion begets a different hydrogen bonding pattern than unaffected Guanosine [3], Substantial research has shown that functional groups at the C8 position of purines cause rotation along the glycosidic bond from an anti to syn
1


conformation [4], The conformational change exposes new hydrogen bonding patterns and may
2


lead to a base pair mismatch (figure 1). The 8-oxoG syn conformer adopts hydrogen bonding similar to that of uridine.
Watson-Crick
bonding
HO
H
HO
o OH
0=P-0^
6- 'f'
H
HN
Base pair mismatch
y
Guanosine
(anti
conformer)
8-oxo-
Gua nosine (syjx conformer)
Figure 1. Hydrogen bonding patterns found in Guanosine vs 8-oxoG. Guanosine naturally is in the anti conformation, which allows traditional Watson-Crick H-bonding with its nucleoside pair Cytidine. Functional group on C8 causes a confonnational flip around the glyocosidic bond and causes 8-oxoG to be in the syn confonner. The syn confonner introduces new H-bonds to allow H-bonding to Adenosine and resulting in a base pair mismatch.
The resulting mismatch has been well established and studied in DNA, along with mechanisms that control for oxidative damage. In order to better understand this relationship in RNA, ribonucleases known to degrade RNA were used to determine how 8-oxoG affects enzyme selectivity, RNA structure, and RNA function.
Endoribonucleases:
Ribonuclease A:
Ribonuclease A (RNase A), also known as bovine pancreatic ribonuclease, is widely used for protein studies due to its nature in degrading single stranded RNA at pyrimidine sites. The active site of RNase A has been studied extensively and contains three main residues that play a role in oligonucleotide hydrolysis: His 12, His 119, and Lys41 [6,7], The base His 12 deprotonates the TOH group on the ribose backbone, which allows the nucleophilic 02’ attack the 3’
3


phosphate resulting in transesterification. Next, the acidic Hisl 19 donates a proton to the 05’ to allow the hydrolysis of the 3’ phosphate. Lys41 stabilizes the 02’ to catalyze the acid-base reaction. Thr45 stabilizes the nucleoside within the active cleft of the enzyme and recognizes pyrimidines due to its ability to H-bond to N3 and the carbonyl at C2; this type of configuration is found in both cytidine and uridine (figure 2).
Recognition Site
H N |
0=p—
Hydrolysis Reaction 6 ^
Figure 2. Active site of RNase A with a cytidine as substrate. The hydrolysis reaction is included, with Hisl2 catalyzing the nucleophilic attack of 02. Thr45 recognizes pyrimidines within the active site due to its H-bonding specificity.
Ribomtclease I):
Ribonuclease Ti (RNase Ti) has a similar function as RNase A in that the enzyme catalyzes the hydrolysis at the 3’ end of the ribose backbone. The active site, though, expresses different residues that hydrogen bond specifically with guanosine over other nucleotides. The active site of RNase Ti includes residues His40, His92, Arg77, and Glu58 [8], Glu58 acts as the base, deprotonating the 2’0H group, which allows the nucleophilic attack of the phosphate. His40 stabilizes the T OH in order to catalyze the base reaction. His92 protonates the phosphate
4


group, which allows for the hydrolysis of the nucleotide. Arg77 stabilizes the phosphate, and thus helps to catalyze the protonation reaction (figure 3).
Figure 3. Active site of RNase Ti with a guanosine as substrate. The enzyme has specific Glu and Asn residues that selectively H-bond with guanosine and recognize it for hydrolysis. The enzyme induces a syn confonnational change for recognition.
The selection of guanosine over other nucleotides lies in the hydrogen bonding patterns of the active site. Glu46 and Asn residues stabilizes G to promote hydrolysis via H-bonding. The exocyclic functional groups also promote this selectivity as the C=0 is stabilized through H-bonding promoted through water and Asn residues, and the NH2 is in a similar manner.
Once a relationship was established with endo-ribonucleases and 8-oxoG, the enzymes were used to help determine the secondary structure of RNA containing the modification. The theophylline binding and the PreQlbinding aptamers were used as a model to determine how this modification influenced their structure.
Recognition Site
Hydrolysis Reaction
5


Theophylline Binding Aptamer:
Aptamers are oligomers or proteins that bind selectively to a particular small molecule or biomolecule of interest. One intensely studied RNA aptamer will be discussed, the Theophylline binding aptamer. The theophylline (TP) binding aptamer was developed in 1994 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), and the aptamer has since then been more thoroughly investigated [11], The aptamer binds selectively to theophylline, as the name suggests, and discriminates from other xanthine derivatives. Theophylline is unmethylated at N7, which differs from theobromine (TB) (methylated at N7) and caffeine (completely methylated at the amine functional groups) (figure 4).
Figure 4. Xanthine derivatives. In order from left to right: Theophylline, Theobromine, and Caffeine.
The aptamer is 33-nt long and has three levels of scaffolding which provides support for its binding pocket (figure 5). The binding pocket utilizes 14 nucleotides of the aptamer to form a platform/lower loop for the small molecule to set in, hydrogen bond to theophylline, and create a ceiling to stabilize the small molecule. The theophylline aptamer induces an S-turn and maintains a linear shape to bring the lower and upper loops of the conserved region into proximity to intercalate and form this binding pocket [12, 13], Ei23 H-bonds to A28, due to the S-turn in its tertiary structure, which allows for a base-triple interaction with U6 to form the floor of the binding pocket. C22 and U24 H-bond with theophylline directly, and another base triple interaction between A7, C8, and G26 form the ceiling of the binding pocket. Along with H-bond
6


interactions, 7i-stacking interactions act as the glue to maintain the integrity of the binding pocket. On one side of the pocket, C21, A7, C22, and U6 71-stack to form an interwoven network that intercalates the upper residues with the lower residues to facilitate H-bond interactions. On the other side of the core, G26 intercalates between G25 and U24, which is thought to stabilize the sharp bend found in the tertiary structure to allow C22 to intercalate between U6 and A7.
A10 was also found to 71-stack with G25 and G11 71-stacks with A10, to further stabilize the binding core (figure 5). An important piece to note is that A7 is displaced from its typical A-form helical position, which facilitates theophylline binding since it allows space for the molecule to fit in the pocket [12], Bound theophylline promotes this S-turn, which continues to add to the high specificity of this aptamer. Something in particular to note is C22’s highly discriminatory nature toward theophylline against other derivatives; this is driven via H-bonding, in which C22 is able to interact withNH7 and the C=0 group at position C6 of Theophylline.
(A)
A15 A G A C -G C -G G-C20 C
/
A10
\
C
U
u
C -G25 C -G
A C
U A
A5 G
G -C30
C -G G -U G -C
5' 3'
(B)
7


Figure 5. (A) Secondary structure of the theophylline binding. Nucleotides are colored depending on how they are grouped together in the tertiary structure. (B) Representation of the base triplets and stacking interaction in the binding pocket of the aptamer. Dashed lines represent Hydrogen bonding, while hexagons represent %-% stacking interactions.
Research has shown that substitution of nucleobases outside of this conserved region do not affect the aptamer’s ability to selectively bind to theophylline nearly as much as the ones in the conserved region [13], further implying a change to this region could dramatically alter the aptamer’s binding affinity toward xanthine derivatives.
XRN1 Exoribonuclease:
XRN1 (also known as PACMAN) is an exoribonuclease present in eukaryotes that non-selectively degrades/hydrolyzes single-stranded 5’ monophosphorylated RNA in a 5’ ->3’ direction [9], XRN1 is found to degrade Messenger RNA (mRNA), which is essential for protein transcription, and having this function is crucial in preventing defective mRNA from being transcribed. A lack of XRN1 expression is correlated with growth, developmental, and hormonal response defects [9,10]; thus, this enzyme plays a pivotal role in animal cell maturation. The active site of XRN1 has a small opening, which prevents double stranded and/or tri-phosphorylated RNA to enter. The small opening to the active site also requires the oligomer to be at least four nucleotides long so that the enzyme can effectively dock the RNA into its active site. Four conserved residues (Lys93, Gln97, ArglOO, and ArglOl) recognize the 5’ monophosphate to help position the nucleotide. The nucleotides all n-n stack with one another inside the active site, which is important to allow His41 and Trp540 to hold the nucleotides in place. The conserved residues of XRN1, His41 and Trp540, hold the nucleotides in place as the phosphate bond of the ribose backbone is exposed to two Mg2+ ions. The Mg2+ ions interact with acidic residues that ultimately facilitate the hydrolysis of the first nucleotide by stabilizing a
8


water molecule in position to nucleophilic attack the scissile phosphate (figure 6). The conserved residues 71-stack with the trinucleotide insertion and pull the next nucleotide into the active site using a ratchet like mechanism, which ultimately allows the hydrolysis of the oligonucleotide to continue [9,10,14],
0-"'H
Asp86
'Mg
+2
H O
''a
Asp205
Asp288 Asp207
1/ » O
Figure 6. The active site of XRN1 with an oligomer of three cytidines used for clarity. Blue dashed lines represent the 7i-stacking interactions between His41 and Trp540 and the nucleobases. A basic pocket of Arg residues dock the 5’-monophosphate into the active site. An acidic pocket of Asp and Glu residues coordinate with two Mg+2 ions, which facilitate the hydrolysis reaction of the terminal nucleotide.
Microscale Thermophoresis
Microscale Thermophoresis (MST) is a technique that allows the determination of the dissociation constant (Kd) between an aptamer and its cognate ligand in free solution and with
very minimal sample consumption [16], As the name suggests, the technique is based off thermophoresis, which is the study of how molecules traverse across a temperature gradient. MST uses an infrared (IR) laser to create this temperature gradient by exciting water molecules
9


in the buffer solution at single point, which heats the water molecules in the localized position; this effect creates a temperature gradient extending out from the focal point of the laser. As the aptamer binds to the ligand, the solvation shell and hydration radius changes around the complex compared to its unbound state, which then changes its thermodiffusion across the gradient [17], The aptamer is tracked using a fluorophore, and the fluorescence is measured as the aptamer migrates across the gradient. The MST measures fluorescence two ways in order to get the normalized fluorescence (Fn0rm). Before the IR laser is shot, the initial fluorescence (Fo) of the fluorophore is measured. When the laser is turned “ON” (t=lsec), the fluorescence is subdued and tracked over the course of the thermodiffusion until the laser is turned “OFF”; this is the fluorescence after thermodiffusion (Fi). The change in fluorescence is due to the unbound aptamer or the complex moving away from the focal point of the laser, where the initial fluorescence is measured. The fluorescence is then normalized by dividing Fi by Foto get Fn0rm [16] (figure 7). Fn0rmthen provides quantitative data points to describe how the complex is behaving in the temperature gradient. The Monolith 1.15 MST can measure up to 16 capillaries in a given experiment. A dilution series of the ligand is carried out in the presence of a constant concentration, and all 16 data sets are compared to provide the Kd of the bound complex.
10


ON Fo I
Time (sec)
Figure 7. Example of raw MST data indicating how the fluorescence behaves once the IR laser is turned “ON” until it is turned “OFF”. The F0 and Fi regions are labeled. Once the IR laser is turned “OFF”, the aptamer begins to diffuse back along the concentration gradient.
11


CHAPTER II
EXPERIMENTAL
Materials
The respective RNA oligomers were synthesized for enzyme degradation and structural probing studies:
*Note: G* denotes an 8-OxoG modification. Ghl denotes an 8-BromoG modification, and GMe<1 denotes an 8-
MethoxyG modification
RNA1: 5’-AAG AGG GAU GAC-3’
RNA2: 5’-AAG AGG* GAU GAC-3’
RNA3: 5’-AAG AG*G* GAU GAC-3’
RNA4:5’-AAG AG*G* G*AU GAC-3’
RNA5: 5’-AAG AGGBr GAU GAC-3 ’
RNA6: 5 ’ - AAG AGGMe° GAU GAC-3 ’
RNA7: 5’-AGA AGG GAG AAG-3’
RNA8: 5’-AGA AGC GAG AAG-3’
RNA9: 5’-AGA AGU GAG AAG-3’
RNA10: 5’-AGA AGG* GAG AAG-3’
RNA11: 5’-AGA AG*G GAG AAG-3’
RNA12: 5’-UUG GAA GAC A-3’
RNA13: 5’-UUGGAA* GAC A-3’
RNA14: 5’-UUG GA*A* GAC A-3’
RNA15: 5’-UUGGA*A* GA*C A-3’
TP-RNAC: 5’-GGC GAU ACC AGC CGA AAG GCC CUU GGC AGC GUC-3’
TP-RNA1: 5’-GGC GAU ACC AGC CGA AAG GCC CUU G*GC AGC GUC-3’
TP-RNA2: 5’-GGC GAU ACC AGC CGA AAG GCC CUU GG*C AGC GUC-3’
12


TP-RNA3: 5’-GGC GAU ACC AG*C CGA AAG GCC CUU GGC AGC GUC-3’
**TP denotes “Theophylline” for the “Theophylline Binding Aptamer”
All organic solvents used during the oligomer synthesis portion were HPLC grade unless otherwise noted. All water used was RNase Free unless otherwise noted. The endoribonucleases were obtained from ThermoScientific. The T4 PNK and T4 RNA Ligase I were obtained from New England Bio Labs. The XRN1 exoribonuclease was a gift from Dr. Eric Chapman from University of Denver. All experiments were carried out in triplicate.
Procedures
RNase Free Water Preparation
Before conducting any experiments with RNA, ribonucleases needed to be removed from the water, hence the name. RNase free water is generated from MilliQ water. A one liter glass jug was filled with MilliQ water. Next, 0.5% Diethyl Pyrocarbonate was added to the water and shook at 37°C overnight. The water was then autoclaved next day and cooled to RT before use. It’s important to note that the cap on the glass jug needs to be slightly unscrewed before being autoclaved; this prevents the glass from cracking.
RNA Oligomer Synthesis
Oligomers were synthesized using an Applied Biosystems 394 Synthesizer. Phosphoramidites were weighed out into respective vials, according to calculations of 0.1 M, and dissolved with respective volumes of acetonitrile under argon gas. The vials were placed into the synthesizer and synthesized through C or G 3’ columns. For the Theophylline binding aptamers used for Microscale Thermophoresis, the aptamers were synthesized through Cy-5 columns,
13


which fluorescently tagged the 3’ end. Following synthesis, each oligomer was deprotected and purified by PAGE and characterized by MALDI.
Deprotection of oligonucleotides:
Each column, containing the resin, was dried under argon before tapping the resin into 2mL eppendorfs.
Deprotection of Dimethyl Formamidine, Acetyl, and Benzoyl groups: A 1:1 mixture of methylamine and ammonia was added to the resin (700uL total volume). The eppendorfs were parafilmed and vortexed proceeding incubation at 60°C for 1.5 hrs. Following incubation, the resin was vortexed and cooled to room temperature. Once cooled, the solution was centrifuged and the supernatant was carefully removed. The RNA solution was frozen in liquid nitrogen and dried under speed vacuum for ~2.5 hrs until the RNA pellet formed.
Deprotection ofTBDMS groups'. 350uL of a mixture containing pyrrodiline (1.5mL), triethyl amine (750uL), and hydrofluoride (lmL) was added to the RNA pellet and mixed. The eppendorfs were parafilmed and vortexed proceeding incubation at 60°C for 1.5 hrs. Following incubation, the solution was vortexed and cooled to room temperature. Once cooled to room temperature, 40uL of 3M NaOAc and lmL of Ethanol were added to each solution. Next, each solution was cooled in a dry ice/Ethanol bath for 25 min. Once cooled, the Eppendorf tubes were centrifuged for ~7 min at RT. The supernatant was removed from each, without disturbing the pellet, and discarded. The pellet was then dried under speed vacuum on low heat for -5-10 minutes.
Purification of RNA:
Purification using PAGE: Once dried, 200uL of LB (90% formamide, ImM EDTA) was added and mixed until the pellet went into solution. The solution was then added to PAGE for
14


purification and ran for 15-18 hours. Once the gel finished running, the purified oligomer was located using UV light and cut and crushed in 50mL Falcon tubes. 15mL of EB (0.1M NaCl. ImM EDTA) was added to each and the tubes were shaken at 37°C between 24-42 hours. Separation of analytes using Sep-Pak columns'. The contents of the 50mL conical tube were transferred into 15mL conical tubes with as little of gel transferred as possible. The conical tubes were centrifuged for 10 minutes. Meanwhile, the Sep-Pak columns were prepared and rinsed, using a lOmL syringe, with: lOmL HPLC grade acetonitrile; lOmL RNase Free Water; and 3.5-4mL of Ammonia Acetate. Next, the solution containing the RNA (~8-12mL) was carefully transferred into the syringe, without any gel particles, and rinsed through the column. The column was then washed with 3xl0mL of RNase Free water and then the RNA was eluted and captured into eppendorfs using 3.5-4mL of 60% Methanol. The samples were then frozen and dried under speed vacuum for 3-4 hours. Once dried, 300uL of RNase Free water was added to the pellet. A 10-fold dilution of the oligomer stock solution was prepared to determine the concentration of the stock solution using UV-Vis via the NanoDrop.
Radiolabeling of RNA 5’ end Labeling using P-32:
Exchange reaction'. A cocktail containing: 1). 32.5uL of RNase Free water; 2). IOuL of lOuM RNA; 3). 5uL of T4 PNK Enzyme buffer; 4). 1.5uL of gamma P-32 labeled ATP; and 5). luL of T4 PNK Enzyme was prepared and incubated for 45 minutes at 37°C.
G-25 Sephadex solution'. The G-25 Sephadex solution was prepared by dissolving 30g of G-25 Sephadex in 250mL of TE buffer (pH 8, lOmM Tris-HCl, ImM EDTA). The solution was mixed overnight and stored at 4°C.
15


G-25 Sephadex Column: The plunger from a lmL syringe was removed and the syringe was placed into a standard test tube. The syringe was plugged with a small ball of glass wool. lmL of the G-25 Sephadex solution was pipetted into the syringe. The solution was centrifuged for ~7-8 minutes to flush out the liquid from the Sephadex syringe column. Following, the syringe was inserted into a 0.6mL eppendorf in order to capture the solution and 50uL of RNase Free MilliQ water was added to the column to rinse. The column was centrifuged for 5 minutes and the 50uL of water, that was captured in the eppendorf, was save for later use (to use for scintillation counts). The column was then inserted into a new 0.6mL eppendorf.
Purification of Radiolabeled RNA using PAGE: The cocktail was added directly into the Sephadex column. The column was centrifuged for 5 minutes to desalt the labeled RNA solution. 0.5uL of the radiolabeled RNA was added to the saved 50uL RNase Free MilliQ water for analysis via the Beckman LS 6500. 40uL of LB of was added to the remaining radiolabeled RNA solution and then succinctly added to PAGE for purification. The gel ran for approximately 3.5-4 hours (until the methylene blue ran halfway down the gel). The gel was wrapped in two layers of saran wrap (radioactive permanent marker was drawn on the first layer surrounding the area where the RNA oligomer was presumed to be, while the second layer protected the cassette from moisture) and placed into a Amersham Bioscience Exposure Casette for 15 minutes. The film was developed using a Storm 860 Phosphoimager. The development was printed and aligned underneath the gel to locate the RNA oligomers. The gel was cut and crushed in a 1.5mL Eppendorf.
For RNA shorter than 30nt long, the EB (0.1M NaCl, 400uL) was added immediately and shook at 37°C overnight. For RNA 30nt and longer, the crushed gel was stored in the -20°C
16


fridge overnight. The next day, the RNA was thawed and EB (0.1M NaCl, 400uL) was added. The mixture shook for 2-2.5hrs at room temperature.
Precipitation of Purified Radiolabeled RNA \ The Eppendorf was placed into a Chromatography Column and centrifuged for 7-8 minutes to separate the gel from the solution containing radiolabeled RNA. The solution was captured in a new 1.5mL Eppendorf and frozen briefly in liquid nitrogen. The RNA was then dried under speed vacuum on medium heat for 2-2.5hrs. A precipitation buffer (0.3M NaOAc, ph 5.5, lOOuL) was added to the RNA pellet and mixed to dissolve the pellet. 250uL of Ethanol was added to the solution and then chilled in a dry ice/ethanol bath for 25-30 minutes. The oligomers were centrifuged for 5 minutes, ideally at 4°C. The supernatant was carefully removed from the pellet and discarded. The oligomers were pulsed briefly in the centrifuge to allow extraction of any remaining supernatant. The pellets were dried under speed vacuum on low heat for 15 minutes and were dissolved back into solution (50uL of RNase Free MilliQ water). The purified, radiolabeled RNA was stored in the -20°C to prevent degradation.
3’ end Labeling using PCP:
An 1 luL cocktail containing the following was prepared: 1). luL DMSO (10%) 2). 2uL of 2uM RNA 3). luL of T4 RNA Ligase I Buffer 4). 4uL PCP 5). luL RNase Inhibitor 6). 2uL T4 RNA Ligase I. The reaction was incubated at 20°C for 14 hours. The reaction, typically, was prepared in the evening and left overnight until the morning. Following the ligation reaction, the RNA was purified and precipitated following the same steps under “5’ End Labeling using P-32”.
Enzymatic Degradation Experiments:
A dilution series of RNase A (5000U/mg) and Ti (lOOOU/uL) was accomplished by first making a 12.5-fold dilution (labeled 1). For XRN1 (19ng/nL), a 10-fold dilution was first accomplished.
17


Next, 10-fold dilutions of the enzyme were carried out to obtain solutions with lower concentrations (labeled 2, then 3, then 4, etc..Specifically, 3 implies a 100-fold dilution and 4 implies a 1,000-fold dilution for the RNase A and RNase Ti studies. For XRN1, 2 implies a 100-fold dilution and 3 implies a 1000 fold dilution. Depending on the RNA, a specific concentration was halved by adding 20uL of the dilution to 20uL RNase free water to obtain the desired concentration (for example, 20uL of concentration 4 plus 20uL of RNase free water obtained 4.5).
Table 1. Amount of RNase used per experiment. “Z” as a dilution factor is described above.
Concentration lOOOU/uL lOmg/mL 1.9ng/nL
Z RNase Tl (U) RNase A (ug) XRN1 (ng)
1 2,000 20 0.19
2 200 2 0.019
3 20 0.2 0.0019
4 2 0.02 -
5 0.2 0.002 -
6 0.02 0.0002 "
7 0.002 2xl05 -
8 0.0002 2xl06 -
RNase A: A cocktail solution of RNA (3000-5000 counts) in lOmM Phosphate buffer pH 5.5 was made. A 1:1 mixture of the RNA and the enzyme was accomplished and incubated at rt for 1 hour. Following incubation, 7uL of loading Buffer (LB: 90% formamide, ImM EDTA) was added and 9-10uL of each respective mixture was added to a denaturing PAGE. For shorter oligomers (<20), a short gel was used. For longer oligomers, a long gel was used. The gels ran until the methylene blue dye ran halfway to three-quarter of the way down the gel.
18


RNase Tf. A cocktail solution of RNA (3000-5000 counts) in lOmM Phosphate buffer pH 5.5 was made. A 1:1 mixture of the RNA and the enzyme was accomplished and incubated at 50°C for 45 min. Following incubation, 7uL of LB was added and 9-10uL of each respective mixture was added to a denaturing PAGE. For shorter oligomers (<20), a short gel was used. For longer oligomers (>20), a long gel was used. The gels ran until the methylene blue dye ran halfway-UTs of the way down the gel.
XRN1 exoribomiclease: The XRN1 (3ug/mL) was obtained from Dr. Eric Chapman’s lab at University of Denver. Before beginning the experiment, the 5’end of the RNA was phosphorylated following the same steps in “5’ End Labeling using P-32” through desalting (the only difference was ImM of ATP was used as opposed to the gamma P-32 ATP). The T4 PNK enzyme was then inactivated by heating at 65°C for 10 minutes. The cocktail solution of RNA (800-1000 counts) in 50% lxEC3 (lOOmMNacl, 10mMMgC12, 50mM Tris-HCl, ImMDTT, pH7.6-7.8) was made. Next, luL of the XRN1 enzyme was added and the mixture was incubated at 37°C for 6 hours. Following, 1 luL of LB was added and 15-18uL of each respective mixture was added to a denaturing PAGE. The gels ran until the methylene blue dye ran halfway down the gel.
Microscale Thermophoresis (MST)
Before beginning studies, the aptamer needed to be tagged with a dye at the 3’ end in order to track its diffusion through the thermal gradient. The Theophylline binding aptamers were tagged with the Cy-5 fluorophore, which was obtain from Glen Research.
Small Molecule Binding Studies:
The small molecule binding studies were performed with 16 two-fold dilutions of each small molecule as seen in the table below. The goal was to measure binding at varying
19


concentrations of small molecule with a constant concentration of RNA to determine the Kd. The
Monolith NT. 115 MST was used for these studies.
Table 2. Small molecules used in the binding affinity studies and the respective concentration ____________________________________ ranges.___________________________
Small Molecule Concentration Range (16 2-fold dilutions)
Theophylline (TP) 11,25mM to 343nM
Theobromine (TB) lOOOuMto 30nM
Caffeine 11,25mM to 343nM
First, 16 0.6mL microtubes were filled with lOuL of the buffer. For theophylline, the buffer was lxTBS (pH 7.5, 50mM Tris-HCL, 150mM NaCl), and for theobromine the buffer was lxTBS with 10% DMSO. Next, a 16 2-fold dilution series was prepared for each small molecule, with lOuL total volume in each eppendorf. lOuL of 20nM RNA was then added to each dilution to accomplish lOnM of RNA (this brought the buffer down to 5% DMSO for solutions with Theobromine in it). Following, the mixtures were incubated in ice for 20 minutes for mixtures containing theophylline and caffeine and at RT for 20 minutes for mixtures containing theobromine. Next, ~10uL of the mixture was captured in a capillary tube and placed onto the plate and covered with the magnetic strip to prevent movement. Each mixture was withdrawn one at a time, with the mixture containing the highest concentration of small molecule being placed in the “1” position and the succeeding mixtures following in numerical order. The Eppendorf and the capillary were held horizontally to prevent bubble formation, and the capillary was held on the very end so that the middle would not get smudged. Once all dilutions were withdrawn, the program ran for approximately 20 minutes using the Monolith NT. 115 MST. Triplicate experiments of RNA with the small molecule were carried out.
20


Binding Check'.
Binding checks were accomplished for TP-RNAC with TP and TP-RNA1 with TB. The binding checks were carried out to validate binding of the small molecule and aptamer, so it was not necessary for the other aptamers and small molecules that did not show binding in the “Small Molecule Binding” studies.
Eight samples were made total—four that had only the RNA aptamer and four that contained the RNA aptamer and the small molecule. The RNA aptamer concentration was constant at lOnM and the small molecule remained constant as well with the highest concentration in order to saturate the aptamer (for example, the theophylline concentration in the presence of TP-RNAC was 11.25uM). The samples sat at room temperature for ~10 min. Once those eight samples were ready, ~10uL of the sample was captured in a capillary tube (as described above) and were placed on the plate, with the sample of only RNA in rows 1-4 and the RNA and the small molecule in rows 5-8.
Structure Characterization
Circular Dichroism Spectroscopy.
The secondary structures of the aptamers were first validated using Circular Dichroism. A solution of Phosphate Buffer (lOmM Sodium Phosphate, lOmM NaCl, and 5mM MgCb. pH 7.5) and 1.3-4uM RNA was accomplished for a total volume of 160uL. Each respective solution was pipetted into clean cuvettes and placed into the CD. A blank of the buffer accompanied. Two measurements of each cuvette were performed to verify secondary structure. If secondary structure was present, melting temperatures of each RNA were measured.
Melting Temperature '.
21


The same solution used previously for CD was used for measuring the melting temp. Before beginning the experiment, a small layer of mineral oil was added to the top to prevent boiling. Once the oil was added, a cap was taped onto the cuvette using Teflon tape. Next, the parameters for the experiment were set (figure 8 The experiment ran for 2-4 hours depending on parameter settings. The experiment was checked periodically to verify the High Tension (HT) voltage did not exceed 600mV.
Z] Parameters Advanced
Temperature Start/End j General | Control j Irrfocmation I Data Start temperature: 4 C 0 Reverse Hold-ime 0
IntervaKC) TareeKC) aradient(C/mm Wait(sec)
1 0.2 95 12 0
2
J Halt temperature ramping during measurement.
Monitor sensor
:•> Holder © Cel

H] Parameters Advanced
Temperature [ Start/End General Control | Inlormation | Data

Photometric mode
Wavelength:
CD
200mdeg/1.0dOD
CD scale:
FL scale: 200 mdeg/1.0 dOD
D.l.T. 1 sec â–¼
Bandwidth 1 00 nm
Sfct width. 100
Channels Num: 3 : Wavelength [nm]
Channel 1: |CD 1 270.0
Channel 2: I HI - 2
Channel 3: |Abs -I
Figure 8. Parameters for variable temperature measurements use to perfonn melting temperature experiements. Once complete, the data was graphed and smoothed using the graphing software Origin. The first derivative was accomplished in order to target the Tm.
22


CHAPTER III
RESULTS
Endoribonuclease Degradation of Oligonucleotides Containing 8-oxo-G
RNase A
In order to first characterize how 8-oxoG influences gene regulation and RNA structure, degradation of oligonucleotides containing 12 nucleobases was first investigated in the presence of RNase A. RNase A targets pyrimidines, which are cytidine and uridine. 8-oxoG obtains a novel binding pattern as the functional group causes rotation around the glycosidic bond, resulting in the syn-conformation. The syn-conformation allows for hydrogen bonding with Adenosine, with a similar hydrogen bonding pattern as pyrimidines. In order to understand if 8-oxoG could act as a substrate for RNase A, four dodecamers containing zero to three 8-oxoG sites were synthesized (RNA1-4). RNA1 acted as the control, RNA2 had one modification,
RNA3 had two modifications, and RNA4 had three modifications. The oligomers were treated with RNase A respectively at concentrations Z= 6 and 7 (see table 1). The results showed that RNase A did cleave the RNA strand at 8-oxoG sites (figure 9). The sequence had five G sites in order to help distinguish from 8-oxoG degradation patterns and one Uridine (at position 9) to validate the position of subsequent degradation spots. The data confirmed that 8-oxoG acts as a substrate for RNase A as RNA3 and RNA4 showed two and three degradation bands respectively compared to that of RNA2 that displayed one. At the more dilute concentration Z=7 (see table 1), though, RNase A almost exclusively cleaved at U9. This prompted further investigation to RNase A’s selectively toward pyrimidines and 8-oxoG.
23


1 -4 5’- AAG AXX XAU GAC
X = G or 8-oxoG
| RNase A| - Z â– 
RNA I
RNase A
6
2
3
\ l 4 I + +
7
JL
2
“1
4
2
♦
U9
X7
Xfc
X5
Figure 9. 20% denaturing PAGE of RNA1-4 following treatment with RNase A at concentrations 6 and 7. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4]
Dodecamers with multiple A and G sites and either one pyrimidine or 8-oxoG site were synthesized to better characterize RNase A’s selectivity. The experiments were carried out in an enzyme concentration dependent manner. The results indicate oligomers with a C present had greater degradation at the position at lower enzyme concentrations than oligomers with either a U or 8-oxoG present, while dodecamers containing either a U or 8-oxoG had similar degradation patterns at the same enzyme concentrations (figure 10). This indicates that RNase A selects C over U and 8-oxoG, while U and 8-oxoG are similar substrates.
24


7 5’-AGA AGO GAG AAG
8 5’-AGA AGC GAG AAG
9 S’- AGA AGU GAG AAG
10 5’-AGA AGG GAG AAG
11 5’- AGA AG°X RNA
Z = RNase A
10
-234-456- 3 45-345
11
I » .
- 3 4 5
- + + + - + + + - + ++ -+++ - + + +

X6
X5
Figure 10. 20% denaturing PAGE of RNA7-11 following treatment with an RNase A concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4]
Functional groups, such as methoxy and bromine, were also positioned on the C8’ position to continue defining 8-oxoG’s reactivity with RNase A. Interestingly, RNase A detected 8-methoxyG sites and cleaved, just with lower reactivity than that of 8-oxoG (figure 11). RNA6 had a G6 8-methoxyG modification and RNA2 had a G6 8-oxoG modification. As seen below, the enzyme recognized 8-oxoG at very high RNase A concentrations, which is consistent with previous data; whereas RNA6 had much lower rates of degradation, but were still present.
RNA 2 5 6
z = - 0 1 1.5 2 ‘<1 I 1.5 2' 0 1 1.5 1 0 1 1.5 2
Br RNase A - + + + •f + + + 4- + + + + + + 4- +
O OH 0=P~Oj 6- * 8-bromoG N **-< l < y ch3 O OH U9 X6 0^. . 0 H» * ^ *,. m * Band of interest using 8-OMcG functionalized RNA
O-P-Oj 6" * 8-methoxyG •m* m*
Figure 11. 20% denaturing PAGE of RNA1, 2, 5, and 6 following treatment with an RNase A concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no
25


enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. 8-bromoG and 8-methoxyG are pictured to the left. [4]
RNA5 had a G6 8-BrG modification (a bromine at the C8’ position) and was not recognized by the enzyme.
Next, oligomers that were ten nucleotides long were synthesized, varying in 8-oxo-7,8-hydroadenosine (8-oxoA) sites, and tested with RNase A. 8-oxoA has similar structural features as 8-oxoG and was thought to potentially act as a substrate also. The sequence for the oligomers can be seen in Figure x. The results show that RNase A did not recognize 8-oxoA as a substrate, with degradation only occurring at C9 and U2 (figure 12). This validates that RNase A is specific toward 8-oxoG and even though 8-oxoA may form similar hydrogen bonding patterns due to its ability to rotate around the glycosidic bond, the amine at C2 may introduce adverse H-bond interactions within the active site of RNase A.
5’- UUG GAAGAC A 5’- UUG GAX’x’GAC A 5’- UUG GAoxoAoxoGAC A 5’- UUG GAoxoAOM,GAoxtt A
3 4
-I________ _______A_
12 13 14 \ 15 13 12 13 14 » 15 14
+ + + + - + + + + -
«. • • * •
U2
Figure 12. 20% denaturing PAGE of RNA12-15 following treatment with RNase A at concentrations 3 and 4. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. 8-oxoA is pictured to the left of the degradation data. [4]
26


RNase Ti
Experiments with RNase Ti, an enzyme that selects for guanosine specifically, were also performed to accomplish characterization of 8-oxoG degradation patterns. The same dodecamers from figure 9 were tested in the presence of RNase T1 using concentration Z=5 (see table 1). Surprisingly, RNase Ti did not hydrolyze 8-oxoG sites, which is evident from the lack of degradation between G3 and G10 for RNA4 (figure 13).
1 5’- AAG AGG GAU GAC
2 5’- AAG AGG°'"GAU GAC
3 5’- AAG AG"'°G"”'GAH GAC
4 5’- AAG Aft"' ft',"ft,",AU GAC
RNA 11234
RNase Ti - + + + +

X = G or 8-oxoG
Figure 13. 20% denaturing PAGE of R.NA1-4 following treatment with RNase Ti at concentrations 3 and 4. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4]
In order to probe the lack of reactivity with the syn conformer of 8oxo-G further, two other functionalized guanosine bases were synthesized: G-bromo and G-methoxy. When these bases were incorporated into the dodecamer at position six (RNA 5 and 6), the results indicated that RNase Ti did not recognize the sites as well (figure 14). The combined results suggest the exocyclic functional group at C8 may introduce adverse H-bonding interactions within the binding pocket of RNase Ti.
27


|RNaseT|
D
E
G
JL.
R\A RNase T
2 12 5 6 - + + + +
F
i—*—i i i
31256 41256
-++ + + . + + + +
Figure 14. 20% denaturing PAGE of RNA1, 2, 5, and 6 following treatment with an RNase Ti concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4]
Theophylline Binding Aptamer Studies
Degradation studies:
The combined information from the RNase A and RNase Ti experiments yielded the opportunity to use this technique and study of RNA aptamer degradation to infer information about its secondary structure. The Theophylline binding aptamer, which is 33 nucleotides long, was modified with 8-oxoG in three different positions, respectively: G25, G26, and Gil. The modifications were chosen due to their role in forming the binding pocket on the aptamer [12], First, an RNase A experiment was undertaken to develop a degradation pattern for all aptamers to compare the canonical to the modified aptamers. A hydrolysis ladder (NaHC03, pH 9.1)
accompanied the experiment to identify what sites were targeted by the enzyme. The ladder works by hydrolyzing every nucleobase from the 3’ end, allowing a spot that contains from 1-33 nucleosides of the aptamer. Compared to the canonical, the three modified RNAs (TP-RNA1,
28


TP-RNA2, and TP-RNA3), did provide different patterns at RNase A concentrations 7 and 7.5 (figure 15).
RNA Z =
RNase A
TP-RNAC TP-RNA1 TP-RNA2
- 7 7.5 - 7 7.5 - 7
-+ + L - + + L - + L
TP-RNA3 - 7 7.5
A15 A C. A ylll - Wf *» * n if
O M C -G C -G ■-! ■■ ©Is
Cj — L/u / Cc A’0 u \ u C -G« C -G a r V y* • * v V v V 9 1 V * ** 5 *3
A L U A A5 G G -C30 C -G G -U G -C 5' 3' i * • w • w V « If 1 • « t I A
tov
<- %
% •
(Ohs
• • A1S
• •
• < I ll
• • •
V i l
Figure 15. 20% denaturing PAGE of RNAs TP-RNAC, 1, 2, and 3 following treatment with an RNase A concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. See Appendix B for more accurate data.
Knowing how 8-oxoG can form different hydrogen bonding patterns to bond with A, this was to be expected. TP-RNA1 and TP-RNA2 had very similar degradation patterns as the canonical, but TP-RNA1 had no degradation at site 13 and had a new degradation site at 30. TP-RNA1 also had increased degradation at G25, which suggests new hydrogen bonding patterns with the modified nucleobase. TP-RNA2 also had no degradation at site 13, but had greater
29


degradation at C6 and a new spot at A5, which may indicate the modified G26 could open the lower half of the binding pocket with its new hydrogen bonding patterns. TP-RNA2 has a modified position at G26, and interestingly, has greater degradation at that site compared to the other three aptamers. The finding is consistent with RNase A selectivity of 8-oxoG and may suggest this modification disrupts the second level of scaffolding in the aptamer’s secondary structure (figure 15); this could insinuate the binding pocket is also disrupted. TP-RNA3 also lacked degradation at U32, which may indicate the hydrogen bonding of U32 and G2 strengthens. The most evident difference in degradation occurred in TP-RNA3 at G18 and G19, which isn’t present in the other three aptamers. This suggests not only novel binding patterns within the aptamer, but may suggest a completely different secondary structure compared to the canonical. Data from the RNase Ti experiment showed degradation at G18 and G19 in TP-RNA3 compared to that of the other three aptamers (figure 16), which is consistent with the RNase A data. RNase Ti degradation in TP-RNA3 dramatically increases in the upper loop compared to the other three aptamers: TP-RNA1 and TP-RNA2 have slight degradation, while the canonical has very little to no degradation of G sites, suggesting the G sites are Hydrogenbonding similar to the known structure. The data for TP-RNA3 suggests a new secondary structure that may disrupt the upper scaffolding of the aptamer, which is important for maintaining the integrity of the binding pocket. Degradation at A15 was consistent with all four aptamers. See Appendix B for more accurate RNase A data.
30


RNA TP-RNAC TP-RNA1 TP-RNA2 TP-RNA3
- 4 4.5 5 - 4 4.5 5 - 4 4.5 5 - 4 4.5 5
RNase A - + + + -++ + L - + + + -+ + + L

Figure 16. 20% denaturing PAGE of RNAs TP-RNAC, 1, 2, and 3 following treatment with an RNase Ti concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations.
To further probe the impact of potential new hydrogen bonding patterns due to 8-oxoG modifications, melting temperatures of each aptamers were taken using CD to compare a change in stability.
Melting Temperatures of Aptamers
Before taking the melting temperatures (Tm) of the aptamers, secondary structure was
confirmed using CD spectroscopy (Figure S8-15). The Tms of the aptamers were taken in a
lOmM Sodium Phosphate buffer (pH 7.5) and a 50mM Tris-HCl saline (TBS) buffer (pH 7.6).
31


Table 3. Melting temperature of each aptamer in the corresponding buffer. Refer to Appendix A
for Figures S8-S15.
Aptamer Tm (Sodium Phosphate Buffer) Tm (lxTBS Buffer)
TP-RNAC 71±0.5°C >75°C
TP-RNA1 71±0.5°C >75°C
TP-RNA2 74.8±0.6°C >75°C
TP-RNA3 73.5±1.5 °C >75°C
The data suggest stable aptamers. A modification at G26 and G11 increases the aptamer’s stability since the melting temperature increases. To further probe the impact of potential new hydrogen bonding patterns due to 8-oxoG modifications, the aptamers were subjected to binding affinity analysis in the presence of theophylline, theobromine, and caffeine using MST.
Small Molecule Binding Affinity Studies
All four aptamers were first analyzed using MST in the presence of varying
concentrations of theophylline. The small molecule was prepared making 16 2-fold dilutions in a
TBS buffer. TP-RNAC bound to theophylline with a Kcio£29uM, which is consistent with
literature [11] (figure 17).
32


Figure 17. MST data of the theophylline binding aptamers with varying concentrations of theophylline (11.25mM to 343nM). (A) MST Traces (left) of TP-RNAC-ligand binding interaction (green) vs no TP-RN AC-ligand binding interaction (black) with the middle eight data sets removed for clarity and the A>fit curve (right) of TP-RNAC with triplicate experiments (48 two-fold dilution concentrations of theophylline total) (B) Kd-fit curve for TP-RNA1 (A'd=2mM) done in triplicate (C) A>fit curve for TP-RNA2 (k)/=1.5mM) do in triplicate TP-RNA1 and TP-RNA2 weakly bound to theophylline with a 100-fold increase in Kd(Kd = 2mM and 1.5mM respectively) (figure 17). The incomplete sigmoidal curve and large error bars also indicate that the aptamers are weak binders to theophylline. The data supports a change in secondary structure, ultimately influencing the binding pocket but still allowing a weak association of the small molecule. TP-RNA3 did not bind to theophylline, which suggests the binding pocket has been completely disrupted and is consistent with the degradation data (figure 18).
33


Figure 18. Example of TP-RNA3 not binding to theophylline. The points are scattered, which indicates the MST cannot conclude binding.
Next, the binding affinity of the aptamers was investigated in the presence of theobromine and
caffeine, which are two xanthine derivatives of theophylline. The theophylline binding aptamer
has been previously reported to have a very weak to no association with theobromine, which was
the result of the canonical aptamer [11] (figure S6). Interestingly, TP-RNA1 was found to
possibly bind to theobromine, yet TP-RNA2 and TP-RNA3 did not (figures 19 and S6).
M W
Figure 19. (A) Binding check of TP-RNA1 and theobromine which indicates binding. (B) Incomplete A>fit curve of TP-RNA1 and theobromine due to solubility issues.
34


The 8-oxoG insertion at G25 in TP-RNA1 seems to allow for a slight change in the binding pocket, which lessens its ability to discriminate between xanthine derivatives and may allow for hydrogen bonding at N7 of theobromine. Theobromine had to be dissolved in 100% DMSO prior to experiments. The solution becomes too saturated at 10% DMSO or greater for the MST, so dilutions had to be accomplished to keep DMSO concentrations at 5% or less. Theobromine stopped becoming soluble in DMSO at 5mM, making the most concentrated dilution in the series ImM. A sigmoidal curve to validate TP-RNA1 ’s /f /with theobromine wasn’t accomplished due to solubility issues with the DMSO and theobromine; thus, the KdO? the bound system cannot be confidently determined. None of the aptamers bound to caffeine, which is consistent with literature in regard to the canonical [11] (figure S7). Caffeine, unlike the other two derivatives, is completely methylated on the amine groups, which prevents hydrogen bonding.
XRN1 Exoribonuclease
Results from the XRN1 degradation study indicate a change between the canonical and the oligomers containing 8-oxoG. The enzyme appears to degrade the canonical completely, which makes sense given the oligomer is single stranded, monophosphorylated at the 5’ end, and does not contain unique or bulky modified nucleobases that may interfere with the active site interactions. What was surprising was to see how the degradation pattern of oligonucleotides containing varying amounts of 8-oxoG presented sites that were upstream of the canonical, suggesting the enzyme stalled at 8-oxoG sites. RNA2, with one modification at G6, showed one degradation site that appeared one base unit lower than RNA3’s degradation site, which seems to be a G5. RNA4, which has three modifications from G5-G7, had a similar pattern as RNA3 (figure 20).
35


RNAl RNA2 RNA3 RNA4
- 2 2.5 3 - 2 2.5 3 - 2 2.5 3 - 2 2.5
+ + + + + + - + + + - + +
* 9 ~ ^
11 *
Figure 20. 20% denaturing PAGE of RNA1-4 following treatment with an XRN1 concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations.
Next, the dodecamers were incubated with the XRN1 enzyme for two hours as opposed to six hours. Degradation increased at presumably 8-oxoG sites as the number of 8-oxoG sites increased (figure SI7). The data further support XRN1 reactivity is affected by 8-oxoG sites.
36


CHAPTER IV
DISCUSSION
Overall, 8-oxoG was found to be a substrate for RNase A but not for RNase Ti. This data allowed the study for RNA secondary structure and how the modification, 8-oxoG, influenced structural changes. The data does suggest a change in structure that varies from the canonical (TP-RNAC), with TP-RNA3 obtaining the greatest change in structure due to the drastic changes in degradation patterns as seen in figure 15. Small molecule binding studies using MST also showed that TP-RNA3 did not bind to theophylline, which further supports a large change in secondary structure, particularly within the binding pocket of the aptamer. Lastly, these findings set the motivation to understand how 8-oxoG affects exoribonucleases, such as XRN1, as they are pivotal for cell maturation and longevity. 8-oxoG was found to dramatically stall XRNl’s hydrolysis of single stranded RNA with increasing number of lesions present.
Enzymatic Degradation of Oligonucleotides Containing 8-oxo-G
RNase A Reactivity'.
8-oxoG reacted as a substrate for RNase A in a similar manner as Uridine. The carbonyl on the C8 position allows the nucleotide to switch from an anti to syn conformation, exposing the 08 and N7 to the active site of RNase A (figure 21).
37


Figure 21. Proposed hydrogen bonding patterns within the active site of RNase A and 8-oxoG, with adverse itneractions shown for 8-methoxyG, 8-bromoG, and 8-oxoA. [4]
Similar to U, Thr45 now has the ability to stabilize the 08 position in order to stabilize the nucleotide within the active cleft of RNase A. This then allows the acid base reaction to occur to hydrolyze the nucleotide. Guanosines that were modified at the C8’ position with bromine were not recognized by the enzyme due to the nucleotides inability to H-bond to Thr 45. Guanosines modified with a methoxy functional group at C8 were recognized by RNase A, just to a much weaker extent even though steric hindrance was present, suggesting the methoxy was still able to H-bond to stabilize the nucleotide. The data suggests RNase A’s selective cleavage of pyrimidines over purines is more in part due to H-bonding patterns within the active cleft than due to size or steric hindrance. Figure 21 shows how there is room for a purine to fit inside the active cleft meriting on size alone. 8-oxoA, although similar in sterics due to the C6 functional group and similar in its ability to adopt similar H-bonding patterns as 8-oxoG, was not recognized more in part due to adverse hydrogen-bonding with the exocyclic amine and the surrounding residues. Altogether, the data suggests that H-bonding ultimately provides the enzyme’s selectivity toward specific nucleotides.
38


RNase Ti reactivity:
8-oxoG was hypothesized to be a substrate for RNase Ti due to how it selects for Guanosine monophosphate (GMP): N1 is stabilized via Glu46 H-bonding interactions and C=0 at C6 is stabilized through interactions driven by Asn residues. RNase Ti induces a syn conformational change of GMP to fit inside the cleft and allow those hydrogen bonding patterns to accomplish hydrolysis of the nucleotide, which led to the assumption the C=0 at C8 would not interfere with the enzyme’s selectivity. Contrary to expected results, 8-oxoG was not recognized by RNase Ti, which suggests the carbonyl at C8 may present adverse H-bonding interactions or steric hindrance. RNA oligomers with the incorporation of 8-methoxyG and 8-BrG also were not substrates for the enzyme, which indicates sterics may play more of a role in hydrolysis inhibition than hydrogen-bonding does. Figure 22. depicts the proposed adverse
interactions that a functional group at C8 may impose on the residues within the active site of RNase Ti.
Figure 22. Proposed adverse hydrogen bonding patterns within the active site of RNase Ti and 8-oxoG and adverse interactions due to the exocyclic functional group at C8 for 8-oxoG, 8-bromoG, and 8-methoxyG. [4]
39


Theophylling Binding Aptamer Studies
The results from the RNase A and RNase Ti reactivity studies suggested the use of these enzymes to probe for secondary structural changes in the theophylline binding aptamers possessing the 8-oxoG modification. The results indicate that a modification at G11 has the most drastic effect on the aptamer’s structure, while modifications at G25 and G26 alter the structure slightly and still allow binding of theophylline. TP-RNA1 seems to have a relatively similar structure as the canonical according to the RNase A and Ti data and similar Tms, but a change with the Hydrogen-bonding pattern of presumably 8-oxoG25 may create a change of the binding pocket (figure 23). The anti to syn flip for 8-oxoG25 allows it to hydrogen bond with A10, which then breaks the bond with C9. This allows G26 to H-bond with C9, breaking the bond with C8, which ultimately disrupts the ceiling for the theophylline binding pocket (figure 23). C8 is necessary for the aptamer’s high affinity to interact with theophylline [12], The 8-oxoG25 and A10 base pairing also disrupts the 7i-stacking interaction between G25 and A10, which then could destabilize the binding core and may displace conserved residues, such as C21 and C22. The large increase in Kd for TP-RNA1 is also consistent with a disruption in the binding pocket. Slight degradation seen at G25 could indicate the H-bond to A10 is relatively weak. TP-RNA1 also seems to bind to theobromine with a similar or potentially stronger affinity than as to theophylline. This could be due to the displacement of C22, which is integral in discriminating for theophylline. The conservation of U24 lacks the ability to discriminate theophylline from xanthine derivatives since N9 of both theophylline and theobromine are the same, and thus could explain why this aptamer may bind to theobromine equally or with more affinity.
40


H----^
C -G 77=71
| C21 | | A10 [;;. ;;| gg»o-G25
I C22 | | C9 G26
I Ut I I U” II ™ I
Figure 23. Representation of TP-RNA1 and how 8-oxoG25 may induce a unique tertiary structure that disrupts the binding pocket by H-bonding to A10.
TP-RNA2 was similar to TP-RNA1 in regards to the over 100-fold increase in K,j, but the structural differences seem to be more pronounced. According to the RNase A data, U6, which is pivotal in forming the floor of the binding pocket, does not seem to have nearly as strong of H-bonds with U23 and A28; this indicates U6 may not be interacting in the aptamers tertiary structure. The possible H-bond between 8-oxoG26 and A7 may displace or prevent U6’s interaction with its corresponding upper loop residues. The Tm (74.5 °C) of TP-RNA2 also indicates the aptamer becomes more stable than the canonical (71°C) due to this modification. This can be rationalized due to 8-oxoG26’s H-bond with A7, which seems to provide a large amount of stabilization to the aptamer’s structure. The aptamer seems to require a more flexible tertiary structure to allow binding of theophylline. As mentioned above, A7 needs the ability to move out of its A-form helix to create space for the small molecule, which is unachievable if it is H-bonding with 8-oxoG26. This novel H-bond also creates a new ceiling for the small molecule, as G25 can now H-bond with C8. Like TP-RNA1, the modification in TP-RNA2 disrupts the necessary component of the theophylline ceiling (figure 24). 8-oxoG’s H-bond with A7 may also disrupt the 7i-stacking interactions of C21, A7, C22, and U6 by losing its ability to intercalate
41


between C21 and C22; this could decrease the interaction between the upper loop and lower loop residues.
Figure 24. Representation of TP-RNA2 and how 8-oxoG26 may induce a unique tertiary structure that disrupts the binding pocket by H-bonding to A7.
Both TP-RNA1 and TP-RNA2 may retain the ability to bind to theophylline, although weakly, since U24 is still conserved in the core and seems to not be burdened by adverse Id-bonding or 7i-stacking interactions. Considering TP-RNA3’s modification at G11 and the role it plays in stacking with A10, it was reasonable to assume TP-RNA3 would behave similarly to TP-RNA1 and TP-RNA2 in the fact that it would weakly bind to theophylline. Surprisingly, TP-RNA3 wasn’t a receptor for theophylline in any regard and did not even weakly bind to the small molecule. This information suggests a change in its secondary/tertiary structure that either disrupts the binding core or a structure that is changed completely from the known structure. RNase A and RNase Ti data indicate apparent degradation patterns that are unique compared to the other theophylline aptamers. Degradation at G18 and G19 was not observed for other atapmers and is also consistent with the RNase Ti data that show spots at G18 and G19 (figure 25). Degradation of G25 and G26 has also dramatically decreased compared to TP-RNA1 and TP-RNA2 indicating the two are H-bonding and thus are protected from RNase degradation. The
42


data seem to suggest that the upper loop and scaffolding of the aptamer are affected due to this modification. 8-oxoGl 1 seems to break the upper scaffolding of the aptamer as it no longer bonds with C20. C12 also appears to not H-bond with G19, which further implies the scaffolding isn’t tightly bound. It is possible that 8-oxoGl 1 could H-bond with A16 or A17, which would explain why both G18 and G19 become exposed for degradation (figure 25).
c A15
c ..A
G*~ -A_
/ G G
A10
\ U U
C20
C
C
C -G25 C -G A C U A
G -C30 C -G G -U G -C 5' 3'
A10
G18
C22 U24

C21 □D— G25
C8
G26
U6 U23~[::| A28
A5 = G29
Figure 25. Representation of TP-RNA3 and how 8-oxoGl 1 may induce a unique tertiary structure by H-bonding to A16 or 17 and disrupts the binding pocket. See Appendix B for more accurate data to support this claim.
The data supports a disruption in the upper loop/scaffolding of the aptamer, which could prevent C21 and C22 to intercalate with A7 and U6. In turn, this disruption could also prevent U24 from the binding core and intercalating between G26 and A28, which would ultimately destabilize the S-turn that is needed to maintain the integrity of the binding pocket.
Degradation at site A15 for all four aptamers was unexpected. It has been well established that RNase A selectively targets pyrimidines due to how the nucleobase H-bonds within the active site. Research has shown, though, that a purine is able to fit inside the active cleft of the enzyme, in particular 8-oxoG, due to the syn conformer adopting H-bonding patterns
43


similar to Uridine [4]; so, it is not out of the question the enzyme could select for A15, especially due to the fact it is very exposed in its secondary and tertiary structure. In order to validate A15 degradation, future work will be accomplished by inserting a methylated Adenosine at position 15 into the aptamer. Appendix B addresses this finding and provides more accurate data that disputes degradation occurring at A15; thus, the future experiment to see whether RNase A targets a methylated Adenosine or not is no longer required.
XRN1 Exoribonuclease
Thus far, the exoribonuclease seems to stall once it hits an 8-oxoG site. It is well known that the conformational change around the glycosidic bond disrupts traditional Watson-Crick H-bonding and can influence enzyme reactivity [4], Degradation at 8-oxoG sites indicate the lesion does stall the enzyme, which could be due to this conformational change that can disrupt H-bonding within the active site or the conformational change may cause the specificity to dramatically decrease as the enzyme docks the nucleobases for hydrolysis [14], Future work to include the 8-bromoG modification will help determine if the nucleobase does disrupt H-bonding within the active site due to a conformational change.
44


CHAPTER V
CONCLUSION
This work has helped establish how 8-oxoG may affect RNA decay and recognition by various ribonucleases. 8-OxoG becomes a substrate for RNase A due to the anti to syn flip which exposes interactions similar to that of Uridine. On the contrary, these interactions prevent RNase Tirecognition as it exposes adverse Hydrogen bonding interactions with the backbone of Asp41. This ultimately gives insight into how ribonucleases are affected by the conformational change presented by 8-oxoG.
Next, the reactivity with endoribonucleases helped demonstrate how 8-oxoG induces structural changes within an aptamer’s secondary and tertiary structure. The theophylline binding aptamers modified at G25 and G26 both presented minimal structural changes in comparison to the canonical, but it was evident these minimal changes had a big impact on the binding pocket of the aptamer as the Kd for the aptamers increased 100-fold. 8-OxoG at position G11 had the biggest impact, which was evident according to the degradation data and due to the aptamer’s loss in ability to bind to theophylline, which indicates the binding pocket was completely disrupted. MALDI experiments to characterize degraded sites from an RNase A experiment will be performed.
XRN1 data, preliminary as it is, showed some promising findings that demonstrate 8-oxoG causes the exoribonuclease to stall and reduce in efficiency. Future studies to determine how exactly the lesion causes this will be to study RNA with an 8-bromoG modification in the presence of XRN1. 8-bromoG induces a conformational change around the glycosidic bond from anti to syn, and information regarding how XRN1 is affected in the presence of this nucleobase will help determine how the conformational change could be affecting the enzyme. An experiment with RNase Ti will help determine at which position the enzyme stalls at, which will
45


ultimately validate that the enzyme is stalling at 8-OxoG sites. Lastly, a G step experiment will help determine with certainty that XRN1 does stall at 8-OxoG sites regardless of position in the oligomer. This is an important finding due to the proposed role of XRN1 to maintain mRNA integrity [18],
46


REFERENCES
[1] Poulsen, H. E., Specht, E.; Broedbaek, K., Henriksen, T.; Ellervik, C., Mandrup-Poulsen, T., Tonnesen, M., Nielsen, P. E., Andersen, H. El., and Weimann, A. (2012) RNA modifications by oxidation: A novel disease mechanism? Free Rad. Biol. Med. 52, 1353-1361.
[2] Kim, S. C., O’Flaherty, D. K., Zhou, L., Lelyveld, V. S., and Szostak, J. W. (2018) Inosine, but none of the 8-oxo-purines, is a plausible component of a primordial version of RNA. PNAS. 115(52)13318-13323
[3] Cheng, X., Kelso, C., Hornak, V., de los Santos, C., Grollman, A. P., and Simmerling, C. (2005). Dynamic behavior ofDNA base pairs containing 8-oxogaunine. J Am Chem Soc. 127(40):13906-18.
[4] Herbert, C., Dzowo, Y. K., Elrban, A., Kiggins, C. N., and Resendiz, M. J. E. (2018) Reactivity and Specificity ofRNase Ti, RNase A, and RNase H Towards Oligonucleotides of RNA Containing 8-Oxo-7,8-dihydrogaunosine. Biochemistry. 57, 2971-2983
[5] Raines, R. T. (1998) Ribonuclease A. Chem. Rev. 98 (3): 1045-1066.
[6] Borkakoti, N. (1983) The active site of ribonuclease A from the crystallographic studies of ribonuclease-A-inhibitor complexes. Eur. J. Biochem. 132, 89-94.
[7] Zegers, F; Maes, D.; Dao-Thi, M. H.; Poortmans, F.; Palmer, R.; Wyns, L. (1994) The structures ofRNase A complexed with 3 ’-CMP and d(CpA): active site conformation and conserved water molecules. Protein Sci. 3, 2322-2339
[8] Heydenreich, A., Koellner, G., Choe, H-W., Cordes, F., Kisker, C., Schindelin, H., Adamiak, R., Hahn, El., and Saenger, W. (1993) The complex between ribonuclease Tiand 3 ’GMP suggests geometry of enzymic reaction path. Eur. J. Biochem. 218, 1005-1012
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[9] Nagarajan, V. K., Jones, C. I., Newbury, S. F., and Green, P. J. (2013) XRN5 ’—>3 ’ exoribonucleases: Structure, mechanisms andjunctions. Biochim Biophys Acta. 1829(0): 590-603.
[10] Jones, C. I., Zabolotskaya, M. V., and Newbury, S. F. (2012) The 5 ’—>3 ’ exoribonuclease XRNl/Pacman and its function in cellular processes and development. WIREs RNA. 3:455-468
[11] Jenison, R. D., Gill, S. C., Pardi, A., and Polisky, B. (1994) High-Resolution Molecular Discrimination by RNA. Science. 263: 1425-1428
[12] Zimmermann, G. R., Jenison, R. D., Wick, C. L., Simorre, JP., and Pardi, A. (1997) Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA. Nature Structural Biology. 4(8): 644-649.
[13] Lee, S. W., Zhao, L., Pardi, A., and Xia, T. (2011). Ultrafast Dynamics Show That the Theophylline and 3-Methylxanthine Aptamers Employ a Conformational Capture Mechanism for Binding Their Ligands. Biochemistry. 49(13): 2942-2951.
[14] Jinek, M., Coyle, S. M., and Doudna, J. A. (2011). Coupled 5’ Nucleotide Recognition and Processivity in XRN 1-MediatedmRNA Decay. Molecular Cell. 41(5): 600-608.
[15] Avir, R., Heinemann, U. Tokuoka, R., and Saenger, W. (1988). Three-dimensional Structure of the Ribonuclease Ti *2 ’-GMP Complex at 1.9A Resolution. The Journal of Biological Chemistry. 263(30):15358-15368
[16] Seidel, S. A.i>, Mijkman, M. P., Lea, W. A., Bogaart, G.V.D., Jerabek-Willemsen, M., Lazic, A., Joseph, J. S., Srinivasan, P., Baaske, P., Simeonov, A., Katrich, I., Melo, F. A., Ladbury, J. E., Schreiber, G., Watts, A., Braun, D., and Duhr, S. (2013). Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods. 59: 301-315.
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[17] Moon, M. H., Hilimire, T. A., Sanders, A. M. and Schneekloth, J. S. Jr. (2018).Measuring RNA-LigandInteractions with Microscale Thermophoresis. Biochemistry. 57(31): 4638-4643.
[18] Simms, L. C., Hudson, B. H., Mosior, J. W., Rangwala, A. S., and Zaher, H. S. (2014). An active role for the ribosome in determining the fate of oxidized mRNA. Cell Rep. 9(4): 1256-1264.
49


APPENDIX A
Please refer to reference [4] for supplemental figures for RNase A, RNase T i, and XRN1 studies for RNA’s 1-15
Theophylline Binding Aptamer Studies:
Final - Shots 500 - Plate D: Label C4
Figure SI. MALDI spectra of TP-RNAC
Final - Shots 500 - Plate D: Label C5
O-i
o-
o-
0-
0-
0-
0-
o-
0-
5563.8315(R1048 ,S31)


Mass fm/z)
1130.4570(R923 ,S68)

11657.4
Figure S2. MALDI spectra of TP-RNA1
Final - Shots 500 - Plate D: Label C6
100-, 90 80-70 60-50 40-30 20-10


5563.8447 (R669.S113)
5595.2554(R639, S23)


7826.2 9741.8
__________________Mass (m/z)__________________
11130.4834 (R797, S137)

11192.8184 (R711 ,S27)
V
Figure S3. MALDI spectra of TP-RNA2
50


Final - Shots 500 - Plate D: Label C7
Figure S4. MALDI spectra of TP-RNA3
RNA Z = RNase A
TP-RNAC TP-RNA1
7 7.5 8 -7 7.S 8
+ ++ L - + + +L
TP-RNA2 TP-RNA3
7 7.5 8 - 7 7.5 8
+ ++ L -+ + + L
Figure S5. RNase A experiment diluting to concentration Z=8. which shows minimal to no degradation (see table 1)
51


776
777 776 775 774
r
i772
|
771
770
769
766
767 766
Target C Complex C' *
1.0E-08 1.01-07 106-06 1.06-05 1OE-04 IOE-03
Ligand Concentration
Figure S6. (Top left) CF with TB not binding (bottom left) Shows binding with IF and TB; (top right) 2F with TB
not binding (bottom right) 3F with TB not binding
52


Figure S7. All theophylline aptamers with caffeine: TP-RNAC (top left), TP-RNAf (top right), TP-RNA2 (bottom left), and TP-RNA3 (bottom right). The plots indicate the small molecule did not bind (signal to noise ratio was 3 or under).
Figure S8. TP-RNAC CD melt and spectra Na;P;0- buffer. pH 7.5.
53


25-,
Temperature (°C)
Figure S9. TP-RNA1 CD melt and spectra Na;P;0- buffer. pH 7.5
Figure S10. TP-RNA2 CD melt and spectra Na;P;0- buffer. pH 7.5.
Figure S11.TP-RNA3 CD melt and spectra Na;P;0- buffer. pH 7.5.
54


CD[mdeg] w CDjmdegl
10 -
X 35 40 45 50 55 60 65 70 75 80 85 90 95 100105
Temperature (°C)
S12. TP-RNAC melt TBS buffer, pH 7.6.
X 35 40 45 50 55 60 65 70 75 80 85 90 95 100105
Temperature (°C)
Figure S13. TP-RNA1 melt TBS buffer, pH 7.6.
55


10 -
8 -
a)
1 6-
o
u
2 -
X 35 40 45 50 55 60 65 70 75 80 85 90 95 100105 Temperature (°C)
Figure S14. TP-RNA2 melt TBS buffer, pH 7.6.
Temperature (°C)
Figure S15. TP-RNA3 melt TBS buffer, pH 7.6.
56


RNAl RNA2 RNA3 RNA4
- 2 2.5 3 - 2 2.5 3 - 2 2.5 3 - 2 2.5 3
- + + + + + + - + + + - + + +
% ^ t •» ♦ - «►* ***
Figure S16. 20% denaturing PAGE of RNA1-4 following treatment with an XRN1 concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. The incubation of the RNAs with XRN1 lasted for 2 hours.
57


APPENDIX B
Upon further examination and replications of the theophylline binding aptamer experiments in the presence of RNase A, a more accurate data set was accomplished that defines what sites were targeted in the aptamers (figure SI6). Each aptamer showed degradation at site U6, which appears to not have an effect on the aptamers’ affinity toward theophylline and the xanthine derivatives. For TP-RNA1 and TP-RNA2, G25 or G26 do not seemed to be a target for RNase A degradation, and instead appear to be C21 and C22, which is consistent with what is known with RNase A selectivity (targets pyrimidines). For TP-RNA3, the sites actually targeted by RNase A appear to be C12 and Cl3, which is still consistent with the RNase T1 data and the proposed structure. Also, the new structure seems to prevent C21 and C22 degradation for the aptamer with the G11 modification. The data for TP-RNA3 still indicate a structural change that disrupts the binding pocket of the aptamer. RNase A also seems to target C9 in all four aptamers, which was presumed to be A15 from the incorrect RNase A data. The finding disputes RNase A reactivity with A15 and instead suggests reactivity with C9, which is consistent with what is known about RNase A. The data is still consistent for what was described in Chapter IV for the proposed structural changes to each aptamer.
58


RNA Z = RNase A
A'5 A G A C -G C -G G-C20
A C
U A
A5 G
G -C30 C -G G -U G -C
TP-RNAC TP-RNA1 TP-RNA2
- 7 7.25 7.5 - 7 7.25 7.5 - 7 7.25 7.5
- + ++ L - + + +L - + ++ L
/ cc : i : 5 V
A10 u V
\ u V y0
C -G25 C -G . . . *
•»»* !•••
Figure SI7. 20% denaturing PAGE of RNAs TP-RNAC-3 following treatment with an RNase A concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. The theophylline binding aptamer is picture with the 8-oxoG modification highlighted for each.
59


Full Text

PAGE 1

AN INVESTI G ATION ON HOW 8 OXO 7,8 DIHYDROGAUNOSINE AFFECTS RNASE A, RNASE T 1 , AND XRN1 REACTIVITY WITH OLIGONUCLEOTIDES OF RNA AND RNA STRUCTURE USING THE THEOPHYLLINE BINDING APTAMER AS MODEL By COURTNEY N. KIGGINS B.S., The Ohio State University, 2014 A Thesis submitted to the faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for a degree of Master of Science Chemistry Program 2019

PAGE 2

ii This thesis for the Master of Science degree by Courtney N. Kiggins h as been approved for the Chemistry Program by Marino J.E. Resendiz, Chair Haobin Wang Xiaojun Ren John D. Fisk Date: May 18, 2019

PAGE 3

iii Kiggins, Courtney N . (M.S., Chemistry) An Investigation on how 8 Oxo 7,8 dihydroguanosine Affects RNase A, T 1 , and XRN1 Reactivity with Oligonucleotides of RNA and RNA Structure using the Theophylline Binding Aptamer as Model Thesis directed by Assistant P rofessor Marino J.E. Resendiz ABSTRACT 8 Oxo 7,8 dihydroguanosine (8 oxoG) is a result of oxidative damage on the nucleobase Guanosine. 8 OxoG has been well characterized in DNA, but little is known of its function in RNA. The purpose of this stud y was to understand how 8 oxoG influences enzyme reactivity, RNA structure, and RNA function. First, endoribonucleases Ribonuclease A (RNase A) and Ribonuclease T 1 (RNase T 1 ) were studied to understand 8 oxoG recognition. It was found that 8 oxoG becomes a substrate for RNase A, while it was not recognized by RNase T 1 . The functional group on C8 causes an anti to syn flip which exposes new hydrogen bonding patterns similar to that of Uridine, which allowed RNase A reactivity. The conformational change, tho ugh, causes 8 oxoG to lose its ability to be a substrate for RNase T 1 due to sterics and adverse hydrogen bonding induced by the exocyclic functional group at C8. The information gathered from the endoribonucleases was then applied to study how 8 oxoG bege ts structural and functional changes to a well studied aptamer, the theophylline binding aptamer. 8 OxoG insertion caused a 100 fold increase in dissociation constant ( K d ) for the theophylline binding aptamers modified at positions G25 and G26, while a mod ification at G11 prevented the aptamer from binding to theophylline. RNase A and T 1 degradation data also yielded different degradation sites in the modified aptamers compared to the canonical, indicating a change in structure. Following, the reactivity of exoribonuclease XRN1 was studied in the presence of oligomers containing one to three 8

PAGE 4

iv found to stall at 8 oxoG sites as a function of number of 8 oxoG present wh en more than one modification was introduced. The data combined helps provide insights into how 8 oxoG may affect RNA decay and surveillance mechanisms. The form and content of this abstract approved. I recommend its publication. Approved: Marino J.E. Resendiz

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v ACKNOWLEDGEMENTS I am incredibly grateful for this journey and know I have a lot of people to thank for my success over the past year and a half. First, I would like to thank the Air Force Institute of Technol ogy (AFIT) and the Chemistry Department at the United States Air Force Academy (USAFA) for sponsoring my Master of Science in Chemistry. I am very grateful for this opportunity and am excited for the assignment to follow. I wanted to thank my professors an d mentors from my time as an Undergraduate at the Ohio State University. In particular, I would like to thank Dr. Chris Callam and Dr. Prabir Dutta for always believing in me. I would like to thank the Biophysics Core at Anschutz Medical Campus for providi ng me access to the Microscale Thermophoresis. I would like to thank Shaun Bevers from the Biophysics Core for training me on the MST, all the insightful conversations, and all the help on trying to figure out my MST data. Thank you to Dr. Eric Chapman fro m the University of Denver for the gift of the XRN1 enzyme looking forward to see where these results lead. Thank you to my professors and teachers at University of Colorado Denver, in particular Dr. Jeff Knight and Dr. Xiaojun Ren, for the mentorship and guidance along the way. I owe an enormous amount of gratitude to Dr. Marino Resendiz for accepting me into his lab and teaching me all my newfound skills that I can carry with me to USAFA and the rest of my career. I am very lucky to have had an advisor wh o has the best interest of the student and who is committed and dedicated to the success of the student every step of the way. I know trying to finish a Thesis based his guidance and his dedication to make sure I would finish on time. I also am grateful for my lab mates thanks making research more enjoyable!

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vi to think of say with all certainty that if he had never even mentioned the pipeline to me, known about it, and I literally would not be writing this acknowledgement section. Thank you to the leadership at SMC from Kirtland AFB for supporting me in my application to the pipeline and doing everything they could so that I could pu rsue this endeavor. Thank you to all the amazing friends in my life, especially my peers who have gone through this journey with me dship, and laughs to curb our impending stress. Thank you to all my friendships outside of school and for providing me places to crash in Denver after I moved! Kate Treadwell and Kelsey Robb, your support has been incredible. Thank you to Capt Paige Plueme r and Capt Gabby Quirao for being listening ears and amazing friends. Thank you to Major Jonny Hoang for all your help and advice trying to figure out my PCS and preparing me for my next assignment. Thank you to my mom, dad, and brother for always supporting me and believing in me. stay focused enough in Undergrad would go on to be teaching at USAFA shortly! Last, and certainly not least, I want to extend my never ending gratitude to my love Dan Higgins. I could NOT have kept any ounce of sanity during this, especially after increasing my commute by an express how grat eful and lucky I am to have you.

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vii TABLE OF CONTENTS I: INTRODUCTION ................................ ................................ ................................ ...................... 1 8 Oxo 7,8 dihydrogaunosine (8 oxoG) ................................ ................................ ...................... 1 Endoribonucleases ................................ ................................ ................................ ...................... 2 Ribonuclease A ................................ ................................ ................................ ....................... 2 Ribonuclease T 1 ................................ ................................ ................................ ...................... 3 Theophylline Binding Aptamer ................................ ................................ ................................ .. 5 XRN1 Exoribonuclease ................................ ................................ ................................ .............. 7 Microscale Thermophoresis ................................ ................................ ................................ ........ 8 II: EXPERIMENTAL ................................ ................................ ................................ ................... 11 Materials ................................ ................................ ................................ ................................ ... 11 Procedure ................................ ................................ ................................ ................................ .. 12 RNase Free Water ................................ ................................ ................................ ................. 12 RNA Oligomer Synthesis ................................ ................................ ................................ ..... 12 Deprotection of oligonucleotides ................................ ................................ .......................... 13 Purification of RNA ................................ ................................ ................................ .............. 13 Radiolabeling of RNA ................................ ................................ ................................ .......... 14 using P 32 ................................ ................................ ................................ ... 14 using PCP ................................ ................................ ................................ .... 16 Enzymatic Degradation Experiments ................................ ................................ ................... 16 Mic roscale Thermophoresis (MST) ................................ ................................ ...................... 18 Structure Characterization ................................ ................................ ................................ .... 20

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viii III: RESULTS ................................ ................................ ................................ ............................... 22 Endoribonuclease Degradation of Oligonucleotides Containing 8 oxo G ............................... 22 RNase A ................................ ................................ ................................ ................................ 22 RNase T 1 ................................ ................................ ................................ ............................... 26 Theophylline Binding Aptamer Studies ................................ ................................ .................... 27 Degradation studies ................................ ................................ ................................ ............... 27 Small Molecule B inding Affinity Studies ................................ ................................ ............. 31 XRN1 Exoribonuclease ................................ ................................ ................................ ............ 34 IV: DISCUSSION ................................ ................................ ................................ ......................... 36 Enzymatic Degradation of Oligonucleotides Containing 8 oxo G ................................ ........... 36 RNase A Reactivity ................................ ................................ ................................ ............... 36 RNase T 1 reactivity ................................ ................................ ................................ ............... 38 Theophylling Binding Aptamer Studies ................................ ................................ ................... 39 XRN1 Exoribonuclease ................................ ................................ ................................ ............ 43 V: CONCLUSION ................................ ................................ ................................ ........................ 44 REFER E NCES ................................ ................................ ................................ ............................. 46 APPENDIX ................................ ................................ ................................ ................................ ... 4 9

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ix LIST OF FIGURES FIGURE Figure 1. Hydrogen bonding patterns found in Guanine vs 8 oxoGua ................................ ........... 2 Figure 2. RNase A active site ................................ ................................ ................................ .......... 3 Figure 3. RNase T 1 active site ................................ ................................ ................................ ......... 4 Figure 4. Xanthine derivatives ................................ ................................ ................................ ........ 5 Figure 5. Secondary and tertiary structures for the theophylline binding aptamer ......................... 7 Figure 6. XRN1 active site ................................ ................................ ................................ .............. 8 Figure 7. Example of raw MST data ................................ ................................ ............................. 10 Figure 8. Parameters for variable temperature measurements ................................ ...................... 21 Figure 9. RNase A Degradation of 8 oxoG sites in RNA1, RNA2, RNA3, and RNA4 ............... 23 Figure 10. Comparing substrates at differing concentrations of RNase A ................................ .... 24 Figure 11. RNase A degradation seen in RNA5 and RNA6 ................................ ......................... 24 Figure 12. RNase A degradation patterns for oligomers containing 8 oxoA ................................ 25 Figure 13. RNase T 1 degradation pattern for oligomers containing 8 oxoG ................................ 26 Figure 14. RNase T 1 degradation pattern for RNA1, RNA2, RNA5, and RNA6 ......................... 27 Figure 15. RNase A degradation patterns for the theophylline aptamers ................................ ..... 28 Figure 16. RNase T 1 degradation patterns for the theophylline aptamers ................................ ..... 30 Figure 17. MST data of the theophylline binding aptamers in presence of theophylline ............. 32 Figur e 18. Example of TP RNA3 not binding to theophylline ................................ ..................... 33 Figure 19. (A) MST data of TP RNA1 and theobromine ................................ ............................. 33 Figure 20. XRN1 degradation data of RNA1 4. ................................ ................................ ........... 35 Figure 21. Proposed RNase A reactivity with 8 oxoG ................................ ................................ . 37 Figure 22. Proposed RNase T 1 lack of reactivity with 8 oxoG ................................ ..................... 38

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x Figure 23. Representation of TP RNA1 structure ................................ ................................ ........ 40 Figure 24. Representation of TP RNA2 structure ................................ ................................ ........ 41 Figure 25. Representation of TP RNA3 structure ................................ ................................ ........ 42 Figure S1. MALDI spectra of TP RNAC ................................ ................................ ..................... 49 Figure S2. MALDI spectra of TP RNA1 ................................ ................................ ...................... 49 Figure S3. MALDI spectra of TP RNA2 ................................ ................................ ...................... 49 Figure S4. MALDI spectra of TP RNA3 ................................ ................................ ...................... 50 Figure S5. RNase A experiment diluting to concentration8 ................................ ......................... 50 Figure S6. Theophylline binding aptamers with TB ................................ ................................ ..... 52 Figure S8. TP RN AC CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5. ................................ ........... 52 Figure S9. TP RNA1 CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5 ................................ ............. 53 Figure S10. TP RNA2 CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5. ................................ .......... 53 Figure S11.TP RNA3 CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5. ................................ ........... 53 Figure S12. TP RNAC melt TBS buffer, pH 7.6. ................................ ................................ ......... 54 Figure S13. TP RNA1 melt TBS buffer, pH 7.6. ................................ ................................ ......... 54 Figure S14. TP RNA2 melt TBS buffer, pH 7.6. ................................ ................................ ......... 55 Figure S15. TP RNA3 melt TBS buffer, pH 7.6. ................................ ................................ ......... 55 Figure S16. XRN1 degradation data (incubation for 2 hours) ................................ ...................... 56 Fi gure S17. Accurate RNase A Theophylline Binding Aptamer data ................................ .......... 58

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xi LIST OF TABLES TABLE Table 1. Small molecules used in the binding affinity studies and the respective concentration ranges ................................ ................................ ................................ ................................ .... 19 Table 2. Small molec ules used in the binding affinity studies and the respective concentration ranges ................................ ................................ ................................ ................................ .... 19 Table 3. Melting temperature of each aptamer in corresponding buffer ................................ ....... 31

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1 CHAPTER I INTRODUCTION Oxidative damage has been widely studied in DNA, yet little is understood about its role in RNA [1]. Oxidation, on both DNA and RNA, occurs readily on purines, where oxidation occurs on the 8th position of the nucleobase to yield 8 oxopurines. Purines have lower redox potentials than pyrimidines, making them more susceptible to oxidative damage. Studies of 8 oxo purines have suggested that: 1). Genetic factors do not influence oxidation on DNA or RNA and 2). These lesions were not present in primordial RNA [1,2]. This suggests that DNA and RNA oxidation occu rs as a result of oxidative damage from endogenous and exogenous reactive oxygen species (ROS). Research is emerging which correlates oxidatively damaged RNA and evels of a particular 8 oxo purine, 8 oxo Guanosine (8 oxoG), is found in the cerebrospinal fluid in prevalent as a byproduct in inflammatory diseases, 8 oxoG is also correlated with protein misfolding and synthesis, suggesting that the lesion may alter RNA structure and function in tRNA and/or mRNA [4]. These findings served as the motivation to understand 8 influence on RNA degradation, structure, and i nteractions with small molecules and biomolecules. 8 Oxo 7,8 dihydrogaunosine (8 oxoG) It has been established that the oxidative lesion begets a different hydrogen bonding pattern than unaffected Guanosine [3]. Substantial research has shown that functi onal groups at the C8 position of purines cause rotation along the glycosidic bond from an anti to syn

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2 conformation [4]. The conformational change exposes new hydrogen bonding patterns and may

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3 lead to a base pair misma tch (figure 1). The 8 oxoG syn conformer adopts hydrogen bonding similar to that of uridine. Figure 1. Hydrogen bonding patterns found in Guanosine vs 8 oxoG. Guanosine naturally is in the anti conformation, which allows traditional Watson Crick H bonding with its nucleoside pair Cytidine. Functional group on C8 causes a conformational flip around the glyocosidic bond and causes 8 oxoG to be in the syn conformer. The syn conformer introduces new H bonds to allow H bonding to Adenosine and resulting in a base pair mismatch. The resulting mismatch has been well established and studied in DNA, along w ith mechanisms that control for oxidative damage. In order to better understand this relationship in RNA, ribonucleases known to degrade RNA were used to determine how 8 oxoG affects enzyme selectivity, RNA structure, and RNA function. Endoribonucleases : Ribonuclease A : Ribonuclease A (RNase A), also known as bovine pancreatic ribonuclease, is widely used for protein studies due to its nature in degrading single stranded RNA at pyrimidine sites. The active site of RNase A has been studied extensively and contains three main residues that play a role in oligonucleotide hydrolysis: His12, His119, and Lys41 [6,7]. The base His12 deprotonates

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4 phosphate resultin base reaction. Thr45 stabilizes the nucleoside within the active cleft of the enzyme an d recognizes pyrimidines due to its ability to H bond to N3 and the carbonyl at C2; this type of configuration is found in both cytidine and uridine (figure 2). Figure 2. Active site of RNase A with a cytidine as substrate. The hydrolysis reaction is included, with His12 catalyzing the nucleophilic attack of O2. Thr45 recognizes pyrimidines within the active site due to its H bonding specificity. Ribonuclease T 1 : Ribonuclease T 1 (RNase T 1 ) has a similar function as RNase A in that the enzyme different residues that hydrogen bond specifically with guanosine over other nucleotides. The active si te of RNase T 1 includes residues His40, His92, Arg77, and Glu58 [8]. Glu58 acts as the ates the phosphate

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5 group, which allows for the hydrolysis of the nucleotide. Arg77 stabilizes the phosphate, and thus helps to catalyze the protonation reaction (figure 3). Figure 3. Active site of RNase T 1 with a guanosine as substrate. T he enzyme has specific Glu and Asn residues that selectively H bond with guanosine and recognize it for hydrolysis. The enzyme induces a syn conformational change for recognition. The selection of guanosine over other nucleotides lies in the hydrogen bondi ng patterns of the active site. Glu46 and Asn residues stabilizes G to promote hydrolysis via H bonding. The exocyclic functional groups also promote this selectivity as the C=O is stabilized through H bonding promoted through water and Asn residues, and the NH2 is in a similar manner. Once a relationship was established with endo ribonucleases and 8 oxoG, the enzymes were used to help determine the secondary structure of RNA containing the modification. The theophylline binding and the PreQ1binding aptame rs were used as a model to determine how this modification influenced their structure.

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6 Theophylline Binding Aptamer : Aptamers are oligomers or proteins that bind selectively to a particular small molecule or biomolecule of interest. One inten sely studied RNA aptamer will be discussed, the Theophylline binding aptamer. The theophylline (TP) binding aptamer was developed in 1994 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), and the aptamer has since then been more thor oughly investigated [11]. The aptamer binds selectively to theophylline, as the name suggests, and discriminates from other xanthine derivatives. Theophylline is unmethylated at N7, which differs from theobromine (TB) (methylated at N7) and caffeine (completely methylated at the amine functional groups) (figure 4). Figure 4. Xanthine derivatives. In order from left to right: Theophylline, Theobromine, and Caffeine. The aptamer is 33 nt long and has three levels of scaffolding which provides support for its binding pocket (figure 5). The binding pocket utilizes 14 nucleotides of the aptamer to form a platform/lower loop for the small molecule to set in, hydrogen bond to theophylline, and create a ceiling to stabilize the small molecule. The theophylline aptamer induces an S turn an d maintains a linear shape to bring the lower and upper loops of the conserved region into proximity to intercalate and form this binding pocket [12, 13]. U23 H bonds to A28, due to the S turn in its tertiary structure, which allows for a base triple inter action with U6 to form the floor of the binding pocket. C22 and U24 H bond with theophylline directly, and another base triple interaction between A7, C8, and G26 form the ceiling of the binding pocket. Along with H bond

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7 stack ing interactions act as the glue to maintain the integrity of the binding stack to form an interwoven network that intercalates the upper residues with the lower residues to facilitate H bond intera ctions. On the other side of the core, G26 intercalates between G25 and U24, which is thought to stabilize the sharp bend found in the tertiary structure to allow C22 to intercalate between U6 and A7. stacks with A10, to further stabilize the binding core (figure 5). An important piece to note is that A7 is displaced from its typical A form helical position, which facilitates theophylline binding since it allows space for the molecule to fit in the pocket [12 ]. Bound theophylline promotes this S turn, which continues to add to the nature toward theophylline against other derivatives; this is driven via H bonding, i n which C22 is able to interact with NH7 and the C=O group at position C6 of Theophylline.

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8 Figure 5. ( A ) Secondary structure of the theophylline binding. Nucleotides are colored depending on how they are grouped together in the tertiary str ucture. ( B ) Representation of the base triplets and stacking interaction in the interactions. Research has shown that substitution of nucleobases outside of this conserved region do not conserved region [13], further implying a change to this region could dramatically alter the ty toward xanthine derivatives. XRN1 Exoribonuclease : XRN1 (also known as PACMAN) is an exoribonuclease present in eukaryotes that non selectively degrades/hydrolyzes single direction [9]. XRN1 is found to degrade Messenger RNA (mRNA), which is essential for protein transcription, and having this function is crucial in preventing defective mRNA from being transcribed. A lack of XRN1 expression is correlated with growth, developmental, and hormonal response d efects [9,10]; thus, this enzyme plays a pivotal role in animal cell maturation. The active site of XRN1 has a small opening, which prevents double stranded and/or tri phosphorylated RNA to enter. The small opening to the active site also requires the olig omer to be at least four nucleotides long so that the enzyme can effectively dock the RNA into its inside the active site, which is important to allow His41 and Trp540 to hold the nucleotides in place. The conserved residues of XRN1, His41 and Trp540, hold the nucleotides in place as the phosphate bond of the ribose backbone is exposed to two Mg2+ ions. The Mg2+ ions interact with acidic residues that ultimately facilitate the hydrolysis of the first nucleotide by stabilizing a

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9 water molecule in position to nucleophilic attack th e scissile phosphate (figure 6). The conserved stack with the trinucleotide insertion and pull the next nucleotide into the active site using a ratchet like mechanism, which ultimately allows the hydrolysis of the oligonucleotide to continue [9, 10,14]. Figure 6. The active site of XRN1 with an oligomer of three cytidines used for clarity. Blue dashed lines represent stacking interactions between His41 and Trp540 and the nucleobases. A basic pocket of Arg residues dock the monophosphate into the active site. An acidic pocket of Asp and Glu residues coordinate with two Mg +2 ions, which facilitate the hydrolysis reaction of the terminal nucleotide. Microscale Thermophoresis Microscale Thermophoresis (MST) is a technique that allows the determination of the dissociation constant ( K d ) between an aptamer and its cognate ligand in free solution and with very minimal sample consumption [16]. As the name suggests, the technique is based off thermophoresis, which is the study of how molecules traverse across a temperature gradient. MST uses an infrared (IR) laser to create this temperature gradient by exciting water molecules

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10 in the buffer solution at single point, whic h heats the water molecules in the localized position; this effect creates a temperature gradient extending out from the focal point of the laser. As the aptamer binds to the ligand, the solvation shell and hydration radius changes around the complex compa red to its unbound state, which then changes its thermodiffusion across the gradient [17]. The aptamer is tracked using a fluorophore, and the fluorescence is measured as the aptamer migrates across the gradient. The MST measures fluorescence two ways in o rder to get the normalized fluorescence (F norm ). Before the IR laser is shot, the initial fluorescence (F 0 ) of the and tracked over the course of the thermodiffusi fluorescence after thermodiffusion (F 1 ). The change in fluorescence is due to the unbound aptamer or the complex moving away from the focal point of the laser, where the initial fluorescence is measured. The fluorescence is then normalized by dividing F 1 by F 0 to get F norm [16] (figure 7). F norm then provides quantitative data points to describe how the complex is behaving in the temperature gradient. The Monolith 1.15 MST can measure up to 16 capillaries in a given experiment. A dilution series of the ligand is carried out in the presence of a constant concentration, and all 16 data sets are compared to provide the K d of the bound complex.

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11 0 and F 1 diffuse back along the concentr ation gradient.

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12 CHAPTER II EXPERIMENTAL Materials The respective RNA oligomers were synthesized for enzyme degradation and structural probing studies: *Note: G* denotes an 8 OxoG modification, G Br denotes an 8 BromoG modification, and G MeO denotes an 8 MethoxyG modification AAG AGG GAU GAC AAG AGG* GAU GAC AAG AG*G* GAU GAC AAG AG*G* G*AU GAC AAG AGG Br GAU GAC AAG AGG MeO GAU GAC AGA AGG GA G AAG AGA AGC GAG AAG AGA AGU GAG AAG AGA AGG* GAG AAG AGA AG*G GAG AAG UUG GAA GAC A UUG GAA* GAC A UUG GA*A* GAC A UUG GA*A* GA*C A TP GGC G AU ACC AGC CGA AAG GCC CUU GGC AGC GUC TP GGC GAU ACC AGC CGA AAG GCC CUU G*GC AGC GUC TP GGC GAU ACC AGC CGA AAG GCC CUU GG*C AGC GUC

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13 TP GGC GAU ACC AG*C CGA AAG GCC CUU GGC AGC GUC **TP denotes All organic solvents used during the oligomer synthesis portion were HPLC grade unless otherwise noted. All water used was RNase Free unless otherwise noted. The endoribonucleases were obtained from Th ermoScientific. The T4 PNK and T4 RNA Ligase I were obtained from New England Bio Labs. The XRN1 exoribonuclease was a gift from Dr. Eric Chapman from University of Denver. All experiments were carried out in triplicate. Procedures RNase Free Water Prepa ration Before conducting any experiments with RNA, ribonucleases needed to be removed from the water, hence the name. RNase free water is generated from MilliQ water. A one liter glass jug was filled with MilliQ water. Next, 0.5% Diethyl Pyrocarbonate was added to the water and shook at 37ºC overnight. The water was then autoclaved next day and cooled to RT before use. autoclaved; this prevents the glass from c racking. RNA Oligomer Synthesis Oligomers were synthesized using an Applied Biosystems 394 Synthesizer. Phosphoramidites were weighed out into respective vials, according to calculations of 0.1 M, and dissolved with respective volumes of acetonitrile und er argon gas. The vials were placed into the used for Microscale Thermophoresis, the aptamers were synthesized through Cy 5 columns,

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14 which purified by PAGE and characterized by MALDI. Deprotection of oligonucleotides: Each column, containing the resin, was dried under argon before tapping the resin into 2mL eppendorfs. Deprotection of Dimethyl Formamidine, Acetyl, and Benzoyl groups: A 1:1 mixture of methylamine and ammonia was added to the resin (700uL total volume). T he eppendorfs were parafilmed and vortexed proceeding incubation at 60 o C for 1.5 hrs. Following incubation, the resin was vortexed and cooled to room temperature. Once cooled, the solution was centrifuged and the supernatant was carefully removed. The RNA solution was frozen in liquid nitrogen and dried under speed vacuum for ~2.5 hrs until the RNA pellet formed. Deprotection of TBDMS groups : 350uL of a mixture containing pyrrodiline (1.5mL), triethyl amine (750uL), and hydrofluoride (1mL) was added to the RNA pellet and mixed. The eppendorfs were parafilmed and vortexed proceeding incubation at 60 o C for 1.5 hrs. Following incubation, the solution was vortexed and cooled to room temperature. Once cooled to room temperature, 40uL of 3M NaOAc and 1mL of Ethano l were added to each solution. Next, each solution was cooled in a dry ice/Ethanol bath for 25 min. Once cooled, the Eppendorf tubes were centrifuged for ~7 min at RT. The supernatant was removed from each, without disturbing the pellet, and discarded. The pellet was then dried under speed vacuum on low heat for ~5 10 minutes. Purification of RNA: Purification using PAGE : Once dried, 200uL of LB (90% formamide, 1mM EDTA) was added and mixed until the pellet went into solution. The solution was then added t o PAGE for

PAGE 26

15 purification and ran for 15 18 hours. Once the gel finished running, the purified oligomer was located using UV light and cut and crushed in 50mL Falcon tubes. 15mL of EB (0.1M NaCl. 1mM EDTA) was added to each and the tubes were s haken at 37 o C between 24 42 hours. Separation of analytes using Sep Pak columns : The contents of the 50mL conical tube were transferred into 15mL conical tubes with as little of gel transferred as possible. The conical tubes were centrifuged for 10 minutes . Meanwhile, the Sep Pak columns were prepared and rinsed, using a 10mL syringe, with: 10mL HPLC grade acetonitrile; 10mL RNase Free Water; and 3.5 4mL of Ammonia Acetate. Next, the solution containing the RNA (~8 12mL) was carefully transferred into the syringe, without any gel particles, and rinsed through the column. The column was then washed with 3x10mL of RNase Free water and then the RNA was eluted and captured into eppendorfs using 3.5 4mL of 60% Methanol. The samples were then frozen and dried und er speed vacuum for 3 4 hours. Once dried, 300uL of RNase Free water was added to the pellet. A 10 fold dilution of the oligomer stock solution was prepared to determine the concentration of the stock solution using UV Vis via the NanoDrop. Radiolabeling of RNA 32: Exchange reaction : A cocktail containing: 1). 32.5uL of RNase Free water; 2). 10uL of 10uM RNA; 3). 5uL of T4 PNK Enzyme buffer; 4). 1.5uL of gamma P 32 labeled ATP; and 5). 1uL of T4 PNK Enzyme was prepared and incubated for 45 minutes at 37 o C. G 25 Sephadex solution : The G 25 Sephadex solution was prepared by dissolving 30g of G 25 Sephadex in 250mL of TE buffer (pH 8, 10mM Tris HCl, 1mM EDTA). The solution was mixed overnight and stored at 4 o C.

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16 G 25 Sephadex Column : The plunger from a 1mL syringe was removed and the syringe was placed into a standard test tube. The syringe was plugged with a small ball of glass wool. 1mL of the G 25 Sephadex solution was pipetted into the syringe. The solution was centrifuged for ~7 8 minutes to flush out the liquid from the Sephadex syringe column. Following, the syringe was inserted into a 0.6mL eppendorf in order to capture the solution and 50uL of RNase Free MilliQ water was added to the column to rinse. The col umn was centrifuged for 5 minutes and the 50uL of water, that was captured in the eppendorf, was save for later use (to use for scintillation counts). The column was then inserted into a new 0.6mL eppendorf. Purification of Radiolabeled RNA using PAGE : The cocktail was added directly into the Sephadex column. The column was centrifuged for 5 minutes to desalt the labeled RNA solution. 0.5uL of the radiolabeled RNA was added to the saved 50uL RNase Free MilliQ water for analysis via the Beckman LS 6500. 40uL of LB of was added to the remaining radiolabeled RNA solution and then succinctly added to PAGE for purification. The gel ran for approximately 3.5 4 hours (until the methylene blue ran halfway down the gel). The gel was wrapped in two layers of saran wra p (radioactive permanent marker was drawn on the first layer surrounding the area where the RNA oligomer was presumed to be, while the second layer protected the cassette from moisture) and placed into a Amersham Bioscience Exposure Casette for 15 minutes. The film was developed using a Storm 860 Phosphoimager. The development was printed and aligned underneath the gel to locate the RNA oligomers. The gel was cut and crushed in a 1.5mL Eppendorf. For RNA shorter than 30nt long, the EB (0.1M NaCl, 400uL) was added immediately and shook at 37ºC overnight. For RNA 30nt and longer, the crushed gel was stored in the 20ºC

PAGE 28

17 fridge overnight. The next day, the RNA was thawed and EB (0.1M NaCl, 400uL) was added. The mixture shook for 2 2.5hrs at room te mperature. Precipitation of Purified Radiolabeled RNA : The Eppendorf was placed into a Chromatography Column and centrifuged for 7 8 minutes to separate the gel from the solution containing radiolabeled RNA. The solution was captured in a new 1.5mL Eppendo rf and frozen briefly in liquid nitrogen. The RNA was then dried under speed vacuum on medium heat for 2 2.5hrs. A precipitation buffer (0.3M NaOAc, ph 5.5, 100uL) was added to the RNA pellet and mixed to dissolve the pellet. 250uL of Ethanol was added to the solution and then chilled in a dry ice/ethanol bath for 25 30 minutes. The oligomers were centrifuged for 5 minutes, ideally at 4ºC. The supernatant was carefully removed from the pellet and discarded. The oligomers were pulsed briefly in the centrifu ge to allow extraction of any remaining supernatant. The pellets were dried under speed vacuum on low heat for 15 minutes and were dissolved back into solution (50uL of RNase Free MilliQ water). The purified, radiolabeled RNA was stored in the 20ºC to pr event degradation. An 11uL cocktail containing the following was prepared: 1). 1uL DMSO (10%) 2). 2uL of 2uM RNA 3). 1uL of T4 RNA Ligase I Buffer 4). 4uL PCP 5). 1uL RNase Inhibitor 6). 2uL T4 RNA Ligase I. The reaction was incubated at 20 o C for 14 hours. The reaction, typically, was prepared in the evening and left overnight until the morning. Following the ligation reaction, the RNA was Enzymatic Degradation Experiments: A dilution series of RNase A (5000U/mg) and T 1 (1000U/uL) was accomplished by first making a 12.5 fold dilution (labeled 1). For XRN1 (19ng/nL), a 10 fold dilution was first accomplished.

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18 Next, 10 fold dilut ions of the enzyme were carried out to obtain solutions with lower fold dilution and 4 implies a 1,000 fold dilution for the RNase A and RNase T 1 studies. For XRN1, 2 implies a 100 fold dilution and 3 implies a 1000 fold dilution. Depending on the RNA, a specific concentration was halved by adding 20uL of the dilution to 20uL RNase free water to obtain the desired concentration (for example, 20uL of concentration 4 plus 20uL of RNase free water obtained 4.5). Concentration 1000U/uL 10mg/mL 1.9ng/nL Z RNase T1 (U) RNase A (ug) XRN1 (ng) 1 2,000 20 0.19 2 200 2 0.019 3 20 0.2 0.0019 4 2 0.02 5 0.2 0.002 6 0.02 0.0002 7 0.002 2x10 5 8 0.0002 2x10 6 RNase A : A cocktail solution of RNA (3000 5000 counts) in 10mM Phosphate buffer pH 5.5 was made. A 1:1 mixture of the RNA and the enzyme was accomplished and incubated at rt for 1 hour. Following incubation, 7uL of loading Buffer (LB: 90% formamide, 1mM EDTA) was added and 9 10uL of each respective mixture was added to a denaturing PAGE. For shorter oligomers (<20), a short gel was used. For longer oligomers, a long gel was used. The gels ran until the methylene blue dye ran halfway to three quarter of the way dow n the gel.

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19 RNase T 1 : A cocktail solution of RNA (3000 5000 counts) in 10mM Phosphate buffer pH 5.5 was made. A 1:1 mixture of the RNA and the enzyme was accomplished and incubated at 50ºC for 45 min. Following incubation, 7uL of LB was added and 9 10uL of each respective mixture was added to a denaturing PAGE. For shorter oligomers (<20), a short gel was used. For longer oligomers (>20), a long gel was used. The gels ran until the methylene blue dye ran halfway of the way down the gel. XRN 1 exoribonuclease only difference was 1mM of ATP was used as opposed to the gamma P 32 ATP). The T4 PNK enzyme was then inactivated by heating at 65ºC for 10 minutes. The cocktail solution of RNA (800 1000 counts) in 50% 1xEC3 (100mM Nacl, 10mM MgCl2, 50mM Tris HCl, 1mM DTT , pH7.6 7.8) was made. Next, 1uL of the XRN1 enzyme was added and the mixture was incubated at 37ºC for 6 hours. Following, 11uL of LB was added and 15 18uL of each respective mixture was added to a denaturing PAGE. The gels ran until the methylene blue dy e ran halfway down the gel. Microscale Thermophoresis (MST) track its diffusion through the thermal gradient. The Theophylline binding aptamers were tagged with the Cy 5 fluorophore, which was obtain from Glen Research. Small Molecule Binding Studies : The small molecule binding studies were performed with 16 two fold dilutions of each small molecule as seen in the table below. The goal was to measure binding at varying

PAGE 31

20 concentrations of small molecule with a constant concentration of RNA to determine the K d . The Monolith NT.115 MST was used for these studies. Table 2. Small molecules used in the binding affinity studies and the respective concentration ranges. Small Molecule Concentration Range (16 2 fold dilutions) Theophylline (TP) 11.25mM to 343nM Theobromine (TB) 1000uM to 30nM Caffeine 11.25mM to 343nM First, 16 0.6mL microtubes were filled with 10uL of the buffer. For theophylline, the buffer was 1xTBS (pH 7.5, 50mM Tris HCL, 150mM NaCl), and for theobromine the buffer was 1xTBS with 10% DMSO. Next, a 16 2 fold dilution series was prepared for each smal l molecule, with 10uL total volume in each eppendorf. 10uL of 20nM RNA was then added to each dilution to accomplish 10nM of RNA (this brought the buffer down to 5% DMSO for solutions with Theobromine in it). Following, the mixtures were incubated in ice f or 20 minutes for mixtures containing theophylline and caffeine and at RT for 20 minutes for mixtures containing theobromine. Next, ~10uL of the mixture was captured in a capillary tube and placed onto the plate and covered with the magnetic strip to preve nt movement. Each mixture was withdrawn one at a time, with the mixture containing the highest concentration of small molecule being Eppendorf and the capillary were h eld horizontally to prevent bubble formation, and the capillary was held on the very end so that the middle would not get smudged. Once all dilutions were withdrawn, the program ran for approximately 20 minutes using the Monolith NT.115 MST. Triplicate exp eriments of RNA with the small molecule were carried out.

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21 Binding Check : Binding checks were accomplished for TP RNAC with TP and TP RNA1 with TB. The binding checks were carried out to validate binding of the small molecule and aptamer, so it was Eight samples were made total four that had only the RNA aptamer and four that contained the RNA aptamer and the small molecule. The RNA aptamer concentration was constant at 10nM and the small molecule remained constant as well with the highest concentration in order to saturate the aptamer (for example, the theophylline concentration in the presence of TP RNAC was 11.25uM). The samples sat at room temperature for ~10 min. Once those eight samples were ready, ~10uL of the sample was captured in a capillary tube (as described above) and were placed on the plate, with the sample of only RNA in rows 1 4 and the RNA and the small molecule in rows 5 8. Structure Characterization Circular Dichroism Spectroscop y: The secondary structures of the aptamers were first validated using Circular Dichroism. A solution of Phosphate Buffer (10mM Sodium Phosphate, 10mM NaCl, and 5mM MgCl 2, pH 7.5) and 1.3 4uM RNA was accomplished for a total volume of 160uL. Each respective solution was pipetted into clean cuvettes and placed into the CD. A blank of the buffer accompanied. Two measurements of each cuvette were performed to verify seco ndary structure. If secondary structure was present, melting temperatures of each RNA were measured. Melting Temperature :

PAGE 33

22 The same solution used previously for CD was used for measuring the melting temp. Before beginning the experiment, a sma ll layer of mineral oil was added to the top to prevent boiling. Once the oil was added, a cap was taped onto the cuvette using Teflon tape. Next, the parameters for the experiment were set (figure 8 The experiment ran for 2 4 hours depending on parameter settings. The experiment was checked periodically to verify the High Tension (HT) voltage did not exceed 600mV. Figure 8. Parameters for variable temperature measurements use to perform melting temperature experiements. Once complete, the data was graphe d and smoothed using the graphing software Origin. The first derivative was accomplished in order to target the Tm.

PAGE 34

23 CHAPTER III RESULTS Endoribonuclease Degradation of Oligonucleotides Containing 8 oxo G RNase A In order to first characterize how 8 oxoG influences gene regulation and RNA structure, degradation of oligonucleotides containing 12 nucleobases was first investigated in the presence of RNase A. RNase A targets pyrimidines, which are cytidine and uridine. 8 oxoG obtains a novel binding pattern as the functional group causes rotation around the glycosidic bond, resulting in the syn conformation. The syn conformation allows for hydrogen bonding with Adenosine, with a similar hydrogen bonding pattern as pyrimidines. In order to understand if 8 oxoG could act as a substrate for RNase A, four dodecamers containing zero to three 8 oxoG sites were synthesized (RNA1 4). RNA1 acted as the control, RNA2 had one modification, RNA3 had two modifications, and RNA4 had three modificatio ns. The oligomers were treated with RNase A respectively at concentrations Z= 6 and 7 (see table 1). The results showed that RNase A did cleave the RNA strand at 8 oxoG sites (figure 9). The sequence had five G sites in order to help distinguish from 8 oxo G degradation patterns and one Uridine (at position 9) to validate the position of subsequent degradation spots. The data confirmed that 8 oxoG acts as a substrate for RNase A as RNA3 and RNA4 showed two and three degradation bands respectively compared to that of RNA2 that displayed one. At the more dilute concentration Z=7 (see table 1), though, RNase A almost exclusively cleaved at U9. This prompted further investigation to 8 oxoG.

PAGE 35

24 Figure 9. 20% denaturing PAGE of RNA1 4 following treatment with RNase A at concentrations 6 and 7. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4] Dodecamers with multiple A and G sites and either one pyrimidine or 8 oxoG site were enzyme concentration dependent manner. The results indicate oligomers with a C present had greater degradation at the position at lower enzyme concentrations than oligomers with either a U or 8 oxoG present, while dodecamers containing either a U or 8 oxoG had similar degradation patterns at t he same enzyme concentrations (figure 10). This indicates that RNase A selects C over U and 8 oxoG, while U and 8 oxoG are similar substrates.

PAGE 36

25 Figure 10. 20% denaturing PAGE of RNA7 11 following treatment with an RNase A concentration gradie nt. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4] Functional groups, such as methoxy and bromine, we position to continue defining 8 8 methoxyG sites and cleaved, just with lower reactivity than that of 8 oxoG (figure 11). RNA6 had a G6 8 methoxyG modification an d RNA2 had a G6 8 oxoG modification. As seen below, the enzyme recognized 8 oxoG at very high RNase A concentrations, which is consistent with previous data; whereas RNA6 had much lower rates of degradation, but were still present. Figure 11. 20% denaturing PAGE of RNA1, 2, 5, and 6 following treatment with an RNase A concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no

PAGE 37

26 enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. 8 bromoG and 8 methoxyG are pictured to the left. [4] RNA5 had a G6 8 ognized by the enzyme. Next, oligomers that were ten nucleotides long were synthesized, varying in 8 oxo 7,8 hydroadenosine (8 oxoA) sites, and tested with RNase A. 8 oxoA has similar structural features as 8 oxoG and was thought to potentially act as a s ubstrate also. The sequence for the oligomers can be seen in Figure x. The results show that RNase A did not recognize 8 oxoA as a substrate, with degradation only occurring at C9 and U2 (figure 12). This validates that RNase A is specific toward 8 oxoG an d even though 8 oxoA may form similar hydrogen bonding patterns due to its ability to rotate around the glycosidic bond, the amine at C2 may introduce adverse H bond interactions within the active site of RNase A. Figure 12. 20% denaturing PAGE of RNA12 15 following treatment with RNase A at concentrations 3 and 4. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrati ons. 8 oxoA is pictured to the left of the degradation data. [4]

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27 RNase T 1 Experiments with RNase T 1 , an enzyme that selects for guanosine specifically, were also performed to accomplish characterization of 8 oxoG degradation patterns. The sam e dodecamers from figure 9 were tested in the presence of RNase T 1 using concentration Z=5 (see table 1). Surprisingly, RNase T 1 did not hydrolyze 8 oxoG sites, which is evident from the lack of degradation between G3 and G10 for RNA4 (figure 13). Figure 13. 20% denaturing PAGE of RNA1 4 following treatment with RNase T 1 at concentrations 3 and 4. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4] In order to probe the lack of reactivity with the syn conformer of 8oxo G further, t wo other functionalized guanosine bases were synthesized: G bromo and G methoxy. When these bases were incorporated into the dodecamer at position six (RNA 5 and 6), the results indicated that RNase T 1 did not recognize the sites as well (figure 14). The c ombined results suggest the exocyclic functional group at C8 may introduce adverse H bonding interactions within the binding pocket of RNase T 1 .

PAGE 39

28 Figure 14. 20% denaturing PAGE of RNA1, 2, 5, and 6 following treatment with an RNase T 1 conce ntration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. [4] Theophylline Binding Aptamer Studi es Degradation studies : The combined information from the RNase A and RNase T 1 experiments yielded the opportunity to use this technique and study of RNA aptamer degradation to infer information about its secondary structure. The Theophylline binding ap tamer, which is 33 nucleotides long, was modified with 8 oxoG in three different positions, respectively: G25, G26, and G11. The modifications were chosen due to their role in forming the binding pocket on the aptamer [12]. First, an RNase A experiment was undertaken to develop a degradation pattern for all aptamers to compare the canonical to the modified aptamers. A hydrolysis ladder (NaHCO3, pH 9.1) accompanied the experiment to identify what sites were targeted by the enzyme. The ladder works by hydroly 33 nucleosides of the aptamer. Compared to the canonical, the three modified RNAs (TP RNA1,

PAGE 40

29 TP RNA2, and TP RNA3), did provide different patterns at RNase A concentrati ons 7 and 7.5 (figure 15). Figure 15. 20% denaturing PAGE of RNAs TP RNAC, 1, 2, and 3 following treatment with an RNase A concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indic ates the addition of enzyme at the respective concentrations. See Appendix B for more accurate data. Knowing how 8 oxoG can form different hydrogen bonding patterns to bond with A, this was to be expected. TP RNA1 and TP RNA2 had very similar degradation p atterns as the canonical, but TP RNA1 had no degradation at site 13 and had a new degradation site at 30. TP RNA1 also had increased degradation at G25, which suggests new hydrogen bonding patterns with the modified nucleobase. TP RNA2 also had no degrada tion at site 13, but had greater

PAGE 41

30 degradation at C6 and a new spot at A5, which may indicate the modified G26 could open the lower half of the binding pocket with its new hydrogen bonding patterns. TP RNA2 has a modified position at G26, and i nterestingly, has greater degradation at that site compared to the other three aptamers. The finding is consistent with RNase A selectivity of 8 oxoG and may stru cture (figure 15); this could insinuate the binding pocket is also disrupted. TP RNA3 also lacked degradation at U32, which may indicate the hydrogen bonding of U32 and G2 strengthens. The most evident difference in degradation occurred in TP RNA3 at G18 a nd G19, within the aptamer, but may suggest a completely different secondary structure compared to the canonical. Data from the RNase T 1 experiment showed degrad ation at G18 and G19 in TP RNA3 compared to that of the other three aptamers (figure 16), which is consistent with the RNase A data. RNase T 1 degradation in TP RNA3 dramatically increases in the upper loop compared to the other three aptamers: TP RNA1 and TP RNA2 have slight degradation, while the canonical has very little to no degradation of G sites, suggesting the G sites are Hydrogen bonding similar to the known structure. The data for TP RNA3 suggests a new secondary structure that may disrupt the up per scaffolding of the aptamer, which is important for maintaining the integrity of the binding pocket. Degradation at A15 was consistent with all four aptamers. See Appendix B for more accurate RNase A data.

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31 Figure 16. 20% denaturing PAGE of RNAs TP RNAC, 1, 2, and 3 following treatment with an RNase T 1 concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme a t the respective concentrations. To further probe the impact of potential new hydrogen bonding patterns due to 8 oxoG modifications, melting temperatures of each aptamers were taken using CD to compare a change in stability. Melting Temperatures of Aptam ers Before taking the melting temperatures (Tm) of the aptamers, secondary structure was confirmed using CD spectroscopy (Figure S8 15). The Tms of the aptamers were taken in a 10mM Sodium Phosphate buffer (pH 7.5) and a 50mM Tris HCl saline (TBS) buffer (pH 7 .6).

PAGE 43

32 Table 3. Melting temperature of each aptamer in the corresponding buffer. Refer to Appendix A for Figures S8 S15. stability since the melting temperature increases. To further probe the impact of potential new hydrogen bonding patter ns due to 8 oxoG modifications, the aptamers were subjected to binding affinity analysis in the presence of theophylline, theobromine, and caffeine using MST. Small Molecule Binding Affinity Studies All four aptamers were first analyzed using MST in the presence of varying concentrations of theophylline. The small molecule was prepared making 16 2 fold dilutions in a TBS buffer. TP RNAC bound to theophylline with a K d of 29uM, which is consistent with literature [11] (figure 17). Aptamer Tm (Sodium Phosphate Buffer) Tm (1xTBS Buffer) TP RNAC 71±0.5ºC >75ºC TP RNA1 71±0.5ºC >75ºC TP RNA2 74.8±0.6ºC >75ºC TP RNA3 73.5±1.5 ºC >75ºC

PAGE 44

33 Figure 17. MST data of the theophylline binding aptamers with varying concentrations of theophylline (11.25mM to 343nM). (A) MST Traces (left) of TP RNAC ligand binding interaction (green) vs no TP RNAC ligand b inding interaction (black) with the middle eight data sets removed for clarity and the K d fit curve (right) of TP RNAC with triplicate experiments (48 two fold dilution concentrations of theophylline total) (B) K d fit curve for TP RNA1 ( K d =2mM) done in tri plicate (C) K d fit curve for TP RNA2 ( K d =1.5mM) do in triplicate TP RNA1 and TP RNA2 weakly bound to theophylline with a 100 fold increase in K d ( K d = 2mM and 1.5mM respectively) (figure 17). The incomplete sigmoidal curve and large error bars also indicate that the aptamers are weak binders to theophylline. The data supports a change in secondary structure, ultimately influencing the binding pocket but still allowing a weak association of the small molecule. TP RNA3 did not bind to theophylline, which suggests the binding pocket has been completely disrupted and is consistent with the degradation data (figure 18).

PAGE 45

34 Figure 18. Example of TP RNA3 not binding to theophylline. The points are scattered, which indicates the MST cannot conclude binding. Next, the binding affinity of the aptamers was investigated in the presence of theobromine and caffeine, which are two xanthine derivatives of theo phylline. The theophylline binding aptamer has been previously reported to have a very weak to no association with theobromine, which was the result of the canonical aptamer [11] (figure S6). Interestingly, TP RNA1 was found to possibly bind to theobromine , yet TP RNA2 and TP RNA3 did not (figures 19 and S6). Figure 19. (A) Binding check of TP RNA1 and theobromine which indicates binding. (B) Incomplete K d fit curve of TP RNA1 and theobromine due to solubility issues.

PAGE 46

35 The 8 oxoG insertion at G25 in TP RNA1 seems to allow for a slight change in the binding pocket, which lessens its ability to discriminate between xanthine derivatives and may allow for hydrogen bonding at N7 of theobromine. Theobromine had to be dissolved in 100% DM SO prior to experiments. The solution becomes too saturated at 10% DMSO or greater for the MST, so dilutions had to be accomplished to keep DMSO concentrations at 5% or less. Theobromine stopped becoming soluble in DMSO at 5mM, making the most concentrated dilution in the series 1mM. A sigmoidal curve to validate TP K d to solubility issues with the DMSO and theobromine; thus, the K d of the bound system cannot be confidently determined. None of the aptamers bou nd to caffeine, which is consistent with literature in regard to the canonical [11] (figure S7). Caffeine, unlike the other two derivatives, is completely methylated on the amine groups, which prevents hydrogen bonding. XRN1 Exoribonuclease Results from t he XRN1 degradation study indicate a change between the canonical and the oligomers containing 8 oxoG. The enzyme appears to degrade the canonical completely, do es not contain unique or bulky modified nucleobases that may interfere with the active site interactions. What was surprising was to see how the degradation pattern of oligonucleotides containing varying amounts of 8 oxoG presented sites that were upstream of the canonical, suggesting the enzyme stalled at 8 oxoG sites. RNA2, with one modification at G6, showed one be a G5. RNA4, which has three modifications fro m G5 G7, had a similar pattern as RNA3 (figure 20).

PAGE 47

36 Figure 20. 20% denaturing PAGE of RNA1 4 following treatment with an XRN1 concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. Next, the dodecamers were incubated with the XRN1 enzyme for two hours as opposed to six hours. Degradation increased at presumably 8 oxoG sites as the number of 8 oxoG sites increased (figure S17). The data further support XRN1 reactivity is affected by 8 oxoG sites.

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37 CHAPTER IV DISCUSSION Overall, 8 oxoG was found to be a substrate for RNase A but not for RNase T 1 . This data allowed the study for RNA secondary structure and how the modification, 8 oxoG, influenced structural changes. The data does suggest a change in structure that varies from the canonical (TP RNAC), with TP RNA3 obtaining the greatest change in structure due to the drastic changes in degradation patterns as seen in figure 15. Small molecule binding studies using MST also showed that TP RNA3 did not bind to theophylline, which further supports a large change in secondary structure, particularly within the bind ing pocket of the aptamer. Lastly, these findings set the motivation to understand how 8 oxoG affects exoribonucleases, such as XRN1, as they are pivotal for cell maturation and longevity. 8 hydrolysis of single stranded RNA with increasing number of lesions present. Enzymatic Degradation of Oligonucleotides Containing 8 oxo G RNase A Reactivity : 8 oxoG reacted as a substrate for RNase A in a similar manner as Uridine. The carbonyl on the C8 position allows the nucleotide to switch from an anti to syn conformation, exposing the O8 and N7 to the active site of RNase A (figure 21).

PAGE 49

38 Figure 21. Proposed hydrogen bonding patterns within the active site of RNase A and 8 oxoG, with adverse itneractions shown for 8 methoxyG, 8 bromoG, and 8 oxoA. [4] Similar to U, Thr45 now has the ability to stabilize the O8 position in order to stabilize the nucleotide within the active cleft of RNase A. This then allows the acid base reaction to occur to hydrolyze the not recognized by the enzyme due to the nucleotides inability to H bond to Thr 45. Guanosines modified with a methoxy functional group at C8 were recognized by RNase A, just to a much weaker extent even though steric hindrance was present, suggesting the methoxy was still able to H pyrimidines over purines is more in part due to H bonding pattern s within the active cleft than due to size or steric hindrance. Figure 21 shows how there is room for a purine to fit inside the active cleft meriting on size alone. 8 oxoA, although similar in sterics due to the C6 functional group and similar in its abil ity to adopt similar H bonding patterns as 8 oxoG, was not recognized more in part due to adverse hydrogen bonding with the exocyclic amine and the surrounding residues. Altogether, the data suggests that H bonding ultimately provides the ity toward specific nucleotides.

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39 RNase T 1 reactivity : 8 oxoG was hypothesized to be a substrate for RNase T 1 due to how it selects for Guanosine monophosphate (GMP): N1 is stabilized via Glu46 H bonding interactions and C=O at C6 is stabilized through interactions driven by Asn residues. RNase T 1 induces a syn conformational change of GMP to fit inside the cleft and allow those hydrogen bonding patterns to accomplish hydrolysis of the nucleotide, which led to the assumption the C=O at C8 would n oxoG was not recognized by RNase T 1 , which suggests the carbonyl at C8 may present adverse H bonding interactions or steric hindrance. RNA oligomers with the incorporation of 8 met hoxyG and 8 BrG also were not substrates for the enzyme, which indicates sterics may play more of a role in hydrolysis inhibition than hydrogen bonding does. Figure 22. depicts the proposed adverse interactions that a functional group at C8 may impose on the residues within the active site of RNase T 1 . Figure 22. Proposed adverse hydrogen bonding patterns within the active site of RNase T 1 and 8 oxoG and adverse interactions due to the exocyclic functional group at C8 for 8 oxoG, 8 bromoG, and 8 methoxyG. [4]

PAGE 51

40 Theophylling Binding Aptamer Studies The results from the RNase A and RNase T 1 reactivity studies suggested the use of these enzymes to probe for secondary structural changes in the theophylline binding aptamers possessing the 8 oxoG modification. The results indicate that a modification at G11 has the most slightly and still allow binding of theophylline. TP RNA1 seems to have a r elatively similar structure as the canonical according to the RNase A and T 1 data and similar Tms, but a change with the Hydrogen bonding pattern of presumably 8 oxoG25 may create a change of the binding pocket (figure 23). The anti to syn flip for 8 oxoG2 5 allows it to hydrogen bond with A10, which then breaks the bond with C9. This allows G26 to H bond with C9, breaking the bond with C8, which ultimately disrupts the ceiling for the theophylline binding pocket (figure 23). C8 is s high affinity to interact with theophylline [12]. The 8 oxoG25 and stacking interaction between G25 and A10, which then could destabilize the binding core and may displace conserved residues, such as C21 and C22. The large increase in K d for TP RNA1 is also consistent with a disruption in the binding pocket. Slight degradation seen at G25 could indicate the H bond to A10 is relatively weak. TP RNA1 also seems to bind to theobromine with a similar or potentially stronge r affinity than as to theophylline. This could be due to the displacement of C22, which is integral in discriminating for theophylline. The conservation of U24 lacks the ability to discriminate theophylline from xanthine derivatives since N9 of both theoph ylline and theobromine are the same, and thus could explain why this aptamer may bind to theobromine equally or with more affinity.

PAGE 52

41 Figure 23. Representation of TP RNA1 and how 8 oxoG25 may induce a unique tertiary structure that disrupts the binding pocket by H bonding to A10. TP RNA2 was similar to TP RNA1 in regards to the over 100 fold increase in K d , but the structural differences seem to be more pronounced. According to the RNase A data, U6, which is pivotal in forming the floor of t he binding pocket, does not seem to have nearly as strong of H bonds with U23 and A28; this indicates U6 may not be interacting in the aptamers tertiary structure. The possible H bond between 8 interaction with i ts corresponding upper loop residues. The Tm (74.5 ºC) of TP RNA2 also indicates the aptamer becomes more stable than the canonical (71ºC) due to this modification. This can be rationalized due to 8 bond with A7, which seems to provide a large a tertiary structure to allow binding of theophylline. As mentioned above, A7 needs the ability to move out of its A form helix to create space for the small mole cule, which is unachievable if it is H bonding with 8 oxoG26. This novel H bond also creates a new ceiling for the small molecule, as G25 can now H bond with C8. Like TP RNA1, the modification in TP RNA2 disrupts the necessary component of the theophylline ceiling (figure 24). 8 bond with A7 may also stacking interactions of C21, A7, C22, and U6 by losing its ability to intercalate

PAGE 53

42 between C21 and C22; this could decrease the interaction between the upper loop and lower loop residues. Figure 24. Representation of TP RNA2 and how 8 oxoG26 may induce a unique tertiary structure that disrupts the binding pocket by H bonding to A7. Both TP RNA1 and TP RNA2 may retain the ability to bind to theophylline, although weak ly, since U24 is still conserved in the core and seems to not be burdened by adverse H stacking interactions. Considering TP plays in stacking with A10, it was reasonable to assume TP RNA3 would beha ve similarly to TP RNA1 and TP RNA2 in the fact that it would weakly bind to theophylline. Surprisingly, TP molecule. This information suggests a change in its secondary/tertiary structure that either disrupts the binding core or a structure that is changed completely from the known structure. RNase A and RNase T 1 data indicate apparent degradation patterns that are unique compared to the other theophylline apta mers. Degradation at G18 and G19 was not observed for other atapmers and is also consistent with the RNase T 1 data that show spots at G18 and G19 (figure 25). Degradation of G25 and G26 has also dramatically decreased compared to TP RNA1 and TP RNA2 indicating the two are H bonding and thus are protected from RNase degradation. The

PAGE 54

43 data seem to suggest that the upper loop and scaffolding of the aptamer are affected due to this modification. 8 oxoG11 seems to break the upper scaffolding o f the aptamer as it no longer bonds with C20. C12 also appears to not H bond with G19, which further implies the scaffolding oxoG11 could H bond with A16 or A17, which would explain why both G18 and G19 become exp osed for degradation (figure 25). Figure 25. Representation of TP RNA3 and how 8 oxoG11 may induce a unique tertiary structure by H bonding to A16 or 17 and disrupts the binding pocket. See Appendix B for more accurate data to support this claim. The data supports a disruption in the upper loop/scaffolding of the aptamer, which could prevent C21 and C22 to intercalate with A7 and U6. In turn, this disruption could also prevent U24 from the binding core and intercalating between G26 and A28, which would ultimately destabilize the S turn that is needed to maintain the integrity of the binding pocket. Degradation at site A15 for all four aptamers was unexpected. It has been well established that RNase A selectively targets pyrimidines due to how the nucleo base H bonds within the active site. Research has shown, though, that a purine is able to fit inside the active cleft of the enzyme, in particular 8 oxoG, due to the syn conformer adopting H bonding patterns

PAGE 55

44 similar to Uridine [4]; so, it is not out of the question the enzyme could select for A15, especially due to the fact it is very exposed in its secondary and tertiary structure. In order to validate A15 degradation, future work will be accomplished by inserting a methylated Adenosine at po sition 15 into the aptamer. Appendix B addresses this finding and provides more accurate data that disputes degradation occurring at A15; thus, the future experiment to see whether RNase A targets a methylated Adenosine or not is no longer required. XRN1 Exoribonuclease Thus far, the exoribonuclease seems to stall once it hits an 8 oxoG site. It is well known that the conformational change around the glycosidic bond disrupts traditional Watson Crick H bonding and can influence enzyme reactivity [4]. Degrad ation at 8 oxoG sites indicate the lesion does stall the enzyme, which could be due to this conformational change that can disrupt H bonding within the active site or the conformational change may cause the specificity to dramatically decrease as the enzym e docks the nucleobases for hydrolysis [14]. Future work to include the 8 bromoG modification will help determine if the nucleobase does disrupt H bonding within the active site due to a conformational change.

PAGE 56

45 CHAPTER V CONCLUSION This wor k has helped establish how 8 oxoG may affect RNA decay and recognition by various ribonucleases. 8 OxoG becomes a substrate for RNase A due to the anti to syn flip which exposes interactions similar to that of Uridine. On the contrary, these interactions p revent RNase T 1 recognition as it exposes adverse Hydrogen bonding interactions with the backbone of Asp41. This ultimately gives insight into how ribonucleases are affected by the conformational change presented by 8 oxoG. Next, the reactivity with endorib onucleases helped demonstrate how 8 oxoG induces aptamers modified at G25 and G26 both presented minimal structural changes in comparison to the canonical, bu t it was evident these minimal changes had a big impact on the binding pocket of the aptamer as the K d for the aptamers increased 100 fold. 8 OxoG at position G11 had the biggest impact, which was evident according to the degradation data and due to the ap loss in ability to bind to theophylline, which indicates the binding pocket was completely disrupted. MALDI experiments to characterize degraded sites from an RNase A experiment will be performed. XRN1 data, preliminary as it is, showed some promising findings that demonstrate 8 oxoG causes the exoribonuclease to stall and reduce in efficiency. Future studies to determine how exactly the lesion causes this will be to study RNA with an 8 bromoG modi fication in the presence of XRN1. 8 bromoG induces a conformational change around the glycosidic bond from anti to syn, and information regarding how XRN1 is affected in the presence of this nucleobase will help determine how the conformational change coul d be affecting the enzyme. An experiment with RNase T 1 will help determine at which position the enzyme stalls at, which will

PAGE 57

46 ultimately validate that the enzyme is stalling at 8 OxoG sites. Lastly, a G step experiment will help determine with certainty that XRN1 does stall at 8 OxoG sites regardless of position in the oligomer. This is an important finding due to the proposed role of XRN1 to maintain mRNA integrity [18].

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47 REFER E NCES [1] Poulsen, H. E., Specht, E.; Broedbaek, K., Hen riksen, T.; Ellervik, C., Mandrup Poulsen, T., Tonnesen, M., Nielsen, P. E., Andersen, H. U., and Weimann, A. (2012) RNA modifications by oxidation: A novel disease mechanism? Free Rad. Biol. Med. 52, 1353 1361. [2] hou, L., Lelyveld, V. S., and Szostak, J. W. (2018) Inosine, but none of the 8 oxo purines, is a plausible component of a primordial version of RNA . PNAS. 115 (52) 13318 13323 [3] Cheng, X., Kelso, C., Hornak, V., de los Santos, C., Grollman, A. P., and Simmer ling, C. (2005). Dynamic behavior of DNA base pairs containing 8 oxogaunine . J Am Chem Soc. 127(40):13906 18. [4] Herbert, C., Dzowo, Y. K., Urban, A., Kiggins, C. N., and Resendiz, M. J. E. (2018) Reactivity and Specificity of RNase T 1 , RNase A, and RNase H T owards Oligonucleotides of RNA Containing 8 Oxo 7,8 dihydrogaunosine . Biochemistry. 57, 2971 2983 [5] Raines, R. T. (1998) Ribonuclease A . Chem. Rev. 98 (3): 1045 1066. [6] Borkakoti, N. (1983) The active site of ribonuclease A from the crystallographic studies of ribonuclease A inhibitor complexes . Eur. J. Biochem. 132, 89 94. [7] Zegers, I.; Maes, D.; Dao Thi, M. H.; Poortmans, F.; Palmer, R.; Wyns, L. (1994) The CMP and d(CpA): active site conformati on and conserved water molecules . Protein Sci. 3, 2322 2339 [8] Heydenreich, A., Koellner, G., Choe, H W., Cordes, F., Kisker, C., Schindelin, H., Adamiak, R., Hahn, U., and Saenger, W. (1993) The complex between ribonuclease T 1 suggests geometry of enzymic reaction path . Eur. J. Biochem. 218, 1005 1012

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48 [9] Nagarajan, V. K., Jones, C. I., Newbury, S. F., and Green, P. J. (2013) -exoribonucleases: Structure, mechanisms and functions . Biochim Biophys Acta. 1829(0): 590 603. [10] Jones, C. I., Zabolotskaya, M. V., and Newbury, S. F. (2012) -XRN1/Pacman and its function in cellular processes and development . WIREs RNA. 3:455 468 [11] Jenison, R. D., Gill, S. C., Pardi, A., and Polisky, B. (1994) High Resol ution Molecular Discrimination by RNA . Science. 263: 1425 1428 [12] Zimmermann, G. R., Jenison, R. D., Wick, C. L., Simorre, JP., and Pardi, A. (1997) Interlocking structural motifs mediate molecular discrimination by a theophylline binding RNA . Nature Structural Biology. 4(8): 644 649. [13] Lee, S. W., Zhao, L., Pardi, A., and Xia, T. (2011). Ultrafast Dynamics Show That the Theophylline and 3 Methylxanthine Aptamers Employ a Conformational Capture Mechanism for Binding Their Ligands. Biochemistry . 49(13): 2 942 2951. [14] Jinek, M., Coyle, S. M., and Doudna, J. A. (2011). and Processivity in XRN1 Mediated mRNA Decay. Molecular Cell . 41(5): 600 608. [15] Avir, R., Heinemann, U. Tokuoka, R., and Saenger, W. (1988). Three dimensional Structure of the Ribonuclease T 1 GMP Complex at 1.9A Resolution. The Journal of Biological Chemistry . 263(30):15358 15368 [16] Seidel, S. A.i>, Mijkman, M. P., Lea, W. A., Bogaart, G.V.D., Jerabek Willemsen, M., Lazic, A., Joseph, J. S., Srinivasan, P., Baas ke, P., Simeonov, A., Katrich, I., Melo, F. A., Ladbury, J. E., Schreiber, G., Watts, A., Braun, D., and Duhr, S. (2013). Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions . Methods. 59: 301 315.

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49 [17] Moon, M. H., Hilimire, T. A., Sanders, A. M. and Schneekloth, J. S. Jr. (2018). Measuring RNA Ligand Interactions with Microscale Thermophoresis. Biochemistry . 57(31): 4638 4643. [18] Simms, L. C., Hudson, B. H., Mosior, J. W., Rangwala, A. S., a nd Zaher, H. S. (2014). An active role for the ribosome in determining the fate of oxidized mRNA . Cell Rep. 9(4): 1256 1264.

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50 APPENDIX A Please refer to reference [4] for supplemental figures for RNase A, RNase T 1 , and XRN1 studies 15 Theophylline Binding Aptamer Studies: Figure S1. MALDI spectra of TP RNAC Figure S2. MALDI spectra of TP RNA1 Figure S3. MALDI spectra of TP RNA2

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51 Figure S4. MALDI spectra of TP RNA3 Figure S5. RNase A experiment diluting to concentration Z=8, which shows minimal to no degradation (see table 1).

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52 Figure S6. (Top left) CF with TB not binding (bottom left) Shows binding with 1F and TB; (top right) 2F with TB not binding (bottom right) 3F with TB not binding

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53 Figure S7. All theophylline aptamers with caffeine: TP RNAC (top left), TP RNA1 (top right), TP RNA2 (bottom left), and TP RNA3 (bottom right). The plots indicate the small molecule did not bind (signal to noise ratio was 3 or under). 30 25 20 15 10 5 0 5 0 10 15 20 25 30 35 Figure S8. TP RNAC CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5. CD (mdeg) 200 250 Wavelength (nm) 300 35

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54 25 20 15 10 5 0 5 10 15 20 25 Figure S9. TP RNA1 CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5 20 15 10 5 0 5 10 15 20 0 Wavelength (nm) Figure S10. TP RNA2 CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5. 20 15 10 5 0 5 10 15 20 0 Wavelength (nm) Figure S11.TP RNA3 CD melt and spectra Na 2 P 2 O 7 buffer, pH 7.5. 200 250 300 350 Wavelength (nm) CD (mdeg) CD (mdeg) CD (mdeg) 200 250 300 35 200 250 300 35

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55 Figure S12. TP RNAC melt TBS buffer, pH 7.6. Figure S13. TP RNA1 melt TBS buffer, pH 7.6.

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56 Figure S14. TP RNA2 melt TBS buffer, pH 7.6. Figure S15. TP RNA3 melt TBS buffer, pH 7.6.

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57 Figure S16. 20% denaturing PAGE of RNA1 4 following treatment with an XRN1 concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective concentrations. The incubation of the RNAs with XRN1 lasted for 2 hours.

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58 APPENDIX B Upon further examination and replications of the theophylline binding aptamer experiments in the presence of RNase A, a more accurate data set was accomplished that defines what sites were targeted in the aptamers (figure S16). Each aptamer showed degradation at site xanthine derivatives. For TP RNA1 and TP RNA2, G25 or G26 do not seemed to be a target for RNase A degradation, and instead appear to be C21 and C22, which is consistent with what is known with RNase A selectivity (targets pyrimidines). For TP RNA3, the sites actually targeted by RNa se A appear to be C12 and C13, which is still consistent with the RNase T1 data and the proposed structure. Also, the new structure seems to prevent C21 and C22 degradation for the aptamer with the G11 modification. The data for TP RNA3 still indicate a st ructural change that disrupts the binding pocket of the aptamer. RNase A also seems to target C9 in all four aptamers, which was presumed to be A15 from the incorrect RNase A data. The finding disputes RNase A reactivity with A15 and instead suggests react ivity with C9, which is consistent with what is known about RNase A. The data is still consistent for what was described in Chapter IV for the proposed structural changes to each aptamer.

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59 Figure S17. 20% denaturing PAGE of RNAs TP RNAC 3 following treatment with an RNase A concentration gradient. Z corresponds to the order of magnitude in dilution factor (see Table 1). The minus sign signifies no enzyme and the addition sign indicates the addition of enzyme at the respective conc entrations. The theophylline binding aptamer is picture with the 8 oxoG modification highlighted for each.