Browsing by Author "Al-Hashimi, Hashim M"

Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. The Watson-Crick base pairs that ensure the fidelity of the genome generation after generation adopt a Watson-Crick geometry that is defined by their shape complementarity as a function of their hydrogen bonding patterns and their inter-nucleotide distance. The ability of a base pair to adopt the Watson-Crick geometry can determine its ability to be incorporated by the polymerase. Conversely, mismatches which can adopt a Watson-Crick-like geometry can also be incorporated by the polymerases. These Watson-Crick-like mismatches have been hypothesized to be lowly-populated and short-lived alternative conformations, which are difficult to characterize and visualize with most biophysical techniques. In this thesis, using NMR spectroscopy and kinetic simulations, we determined the ground state conformation of the A•G mismatch, characterized the formation of Watson-Crick-like Hoogsteen excited state conformations in DNA duplexes and investigated their role in DNA replication errors. By including a DNA-dynamics-driven step into a minimal kinetic mechanism for correct incorporation after the initial nucleotide binding step, we were able to accurately recapitulate the rate with which A•dGTP mismatches are misincorporated for polymerase β, across different pH conditions, and for the most damaged form of the mismatch involving 8-oxoguanine. We further find that the introduction of 8-oxoguanine to A•G does not stabilize the mismatch to only one conformation but rather redistributes the dynamic ensemble of the mismatch to favor the mutagenic conformation as the ground state. Through direct and indirect targeting of the 8-oxoguanine and adenine, respectively, the lesion not only affects the mismatch itself but also rescues long-range dynamics of the DNA duplex that was previously repressed by the unmodified mismatch. We also characterize the sequence-dependent dynamics of the A•G mismatch and find that the nearest-neighbor base changes can significantly alter the dynamic ensemble. These sequence-dependent modulations to the conformational landscape can explain the sequence-dependent behavior of MutY repair enzyme.

RNA recognition frequently results in conformational changes that optimize

intermolecular binding. As a consequence, the overall binding affinity of RNA

to its binding partners depends not only on the intermolecular interactions

formed in the bound state, but also on the energy cost associated with changing

the RNA conformational distribution. Measuring these conformational penalties

is however challenging because bound RNA conformations tend to have equilibrium

populations in the absence of the binding partner that fall outside detection by

conventional biophysical methods.

In this work we employ as a model system HIV-1 TAR RNA and its interaction with

the ligand argininamide (ARG), a mimic of TAR’s cognate protein binding partner,

the transactivator Tat. We use NMR chemical shift perturbations (CSP) and NMR

relaxation dispersion (RD) in combination with Bayesian inference to develop a

detailed thermodynamic model of coupled conformational change and ligand

binding. Starting from a comprehensive 12-state model of the equilibrium, we

estimate the energies of six distinct detectable thermodynamic states that are

not accessible by currently available methods.

Our approach identifies a minimum of four RNA intermediates that differ in terms

of the TAR conformation and ARG-occupancy. The dominant bound TAR conformation

features two bound ARG ligands and has an equilibrium population in the absence

of ARG that is below detection limit. Consequently, even though ARG binds to TAR

with an apparent overall weak affinity ($\Kdapp \approx \SI{0.2}{\milli

\Molar}$), it binds the prefolded conformation with a $K_{\ch{d}}$ in the nM

range. Our results show that conformational penalties can be major determinants

of RNA-ligand binding affinity as well as a source of binding cooperativity,

with important implications for a predictive understanding of how RNA is

recognized and for RNA-targeted drug discovery.

Additionally, we describe in detail the development of our approach for fitting

complex ligand binding data to mathematical models using Bayesian

inference. We provide crucial benchmarks and demonstrate the

robustness of our fitting approach with the goal of application

to other systems. This thesis aims to provide new insight into

the dynamics of RNA-ligand recognition as well as provide new

methods that can be applied to achieve this goal.

All organisms must accurately replicate their genomic DNA in order to transmit genetic information from generation to generation. The cognate Watson-Crick base pairs (dA•dT, dG•dC) adopt near identical ‘Watson-Crick geometry’ as defined by the hydrogen bonding pattern (height and depth) and the distance between the nucleoside sugar C1’ atoms (width). The shape complementarity of Watson-Crick pairs is a significant determinant in the selection of a correct nucleotide for a given template base during replication. Since the discovery of the DNA double helix, more than 60 years ago, the formation of Watson-Crick-like mismatch base pairs, stabilized by rare, energetically less favorable tautomeric or anionic bases, has been hypothesized as a cause of spontaneous mutation. However, these proposed lowly-populated and short-lived Watson-Crick-like conformational ‘excited states’ are characterized by subtle movements of protons and π-bonds that have proven difficult to visualize experimentally, even with modern biophysical techniques.

We have utilized nuclear magnetic resonance relaxation dispersion experiments in conjunction with kinetic modeling and in vitro assays to characterize the formation of tautomeric and anionic Watson-Crick-like dG•dT excited states in DNA duplexes and investigate their involvement in DNA replication errors. Insertion of the sequence- dependent tautomerization or ionization step into minimal kinetic mechanisms for correct incorporation during replication after the initial binding of the nucleotide, leads to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the system is under thermodynamic control and that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10-2 to 10-3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT- dominant at pH 8.4 and above, or for some mutagenic nucleotides. Kinetic modeling reveals additional plausible pathways for dG•dT misincorporation in which the tautomerization event takes place prior to binding or in which the polymerase alters the kinetics of tautomerization within the active site.

The conformational landscape of the dA•dG mismatch has been characterized with the use of NMR relaxation dispersion in DNA duplexes. The mismatch has been shown to adopt three predominant forms: Aanti•Ganti, Asyn•Ganti, and A+anti•Gsyn. We have characterized sequence-specific conformational exchange between all three of these base pair forms in multiple sequence contexts. In addition, we find that nearest-neighbor base changes can alter the ground state conformation of the dA•dG base pair between Aanti•Ganti and Asyn•Ganti. Such sequence-specific alterations to the conformational landscape have been proposed to alter reaction rates of an adenine glycosylase repair protein, MutY. Notably, this work shows for the first time that the Asyn•Ganti base pair is able to form in solution both as a ground state and excited state base pair; and may influence the activity of MutY. In addition, two tautomeric forms of the dA•dG base pair have been proposed to form WC-like base pairs but R1ρ experiments targeting the NH2 functional groups of dA and dG have been thus far unable to observe these proposed states.

Ribonucleic acid (RNA) is a versatile and dynamic biomolecule that serves as an indispensable component in the central dogma of molecular biology. The realization that RNA plays a wide variety of roles in gene expression and regulation has been accompanied by the discovery that virtually all types of RNA are chemically modified. These modifications have profound effects on RNA metabolism. N6-Methyladenosine (m6A) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA−RNA interactions by altering RNA structure mainly by destabilizing base pairing. Here we sought to provide a broad and deep description of how m6A reshapes the folding and recognition landscape of RNA, which provides detailed mechanisms via which m6A exerts its biological functions.First, we show that when neighbored by a 5ʹ bulge, m6A stabilizes m6A–U base pairs and global RNA structure by ~1 kcal/mol. The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3ʹ neighbor through a conformation that is stabilized by Mg2+. A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While m6A embedded in duplex RNA is poorly recognized by the YTH domain reader protein and m6A antibodies, both readily recognize m6A in this newly identified motif. The results uncover potentially abundant and functional m6A motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data. In addition to altering RNA stability, m6A has also been shown to slow the kinetics of biochemical processes involving RNA-RNA interactions. However, little is known about how m6A affects the kinetic rates of RNA folding and conformational transitions that are important for RNA functions. We developed an NMR relaxation dispersion (RD) method to non-invasively and site-specifically measure nucleic acid hybridization kinetics. Using this method, we discovered that m6A selectively slows annealing rate while has minimal impact on melting rate in different sequence contexts and buffer conditions. To understand the mechanism of the m6A-induced slowdown of hybridization, we used NMR RD to dissect the kinetic pathways of duplex hybridization. We show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions and provides an explanation for why the modification robustly slows diverse cellular processes. Taken together, these results uncover the important role of m6A on modulating RNA-RNA and RNA-protein interactions through altering RNA structure and dynamics, highlighting the structural-dynamics-function relationship.

Biomolecules are dynamic entities that adopt a variety of conformations in solution. Conformational changes in biomolecules routinely take place when they take part in biochemical processes such as binding and catalysis. The energetic cost or conformational penalty to adopt an alternative conformation, which typically is paid for by thermal fluctuations or inter-molecular contacts with a partner molecule, can be an important determinant of these efficacy and selectivity of these biochemical processes. These conformational penalties can also be modulated by changes in physiological conditions and chemical modifications, enabling fine control over these biochemical processes, and aberrant changes to these conformational penalties can also be associated with disease. Thus, measurement of conformational penalties in biomolecules and how they can be tuned by external cues are essential to understand the role of conformational dynamics in biology.In this thesis, a combination of experimental and computational techniques such as NMR spectroscopy, UV melting and MD simulations are used to measure conformational penalties in nucleic acids and how they are modulated by post-transcriptional and epigenetic modifications, with particular applications to the formation of Watson-Crick like mismatches in DNA, and Hoogsteen base pairs in RNA and DNA. Improved methods that enable measurements of these conformational penalties with increased throughput and sensitivity, involving the use of NMR spectroscopy and UV melting, are also presented.

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.

The structure and biological functions of the DNA double helix have been understood in the light of Watson-Crick (WC) base pairs (bps). However, bps can adopt an alternative base pairing conformation termed as the Hoogsteen (HG) bps in double-stranded DNA. The WC bps have been shown to exist in dynamic equilibrium with short-lived low-populated HG bps in vitro. This thesis studies the modulation of the WC-HG equilibrium on DNA recognition, the potential role of HG bps in DNA damage, and also develops new sequencing approaches to map the HG bps in vivo.To study how the WC / HG dynamics was modulated upon recognition of duplex DNA by small molecule drugs, we applied NMR relaxation dispersion experiments and molecular dynamics simulations to a bisintercalator echinomycin and a monointercalator actinomycin D. In both cases, DNA recognition resulted in the quenching of HG dynamics at bps involved in intermolecular base-specific hydrogen bonds. In the case of echinomycin, the HG population increased 10-fold for bps flanking the chromophore most likely due to intermolecular stacking interactions, whereas actinomycin D minimally affected HG dynamics at other sites. The results revealed that modulation of HG dynamics at binding interfaces could be a general phenomenon with important implications for DNA-ligand and DNA-protein recognition. To explore the thermodynamic propensities of DNA to adopt minor conformations such as HG bps, a simple, fast, and cost-effective approach called “delta-melt” was developed that combining optical melting experiments with chemical modifications and mutations. With this approach, we obtained unique insights into the thermodynamic cooperativity in HG formation at adjacent bps. This finding was verified by NMR experiments and provides an explanation for the frequent observation of tandem HG bps in crystal structures of DNA duplexes containing HG bps. Because HG bps expose the WC faces of the purine nucleobases to the solvent, we reasoned that they could potentially lead to damage to the WC faces of purines. To test this hypothesis, we developed a biochemical assay that combines chemical probing, dot-blot, and primer extension to assess HG-mediated damage to the WC faces of nucleobases. The results indicated that the reactivity to dimethyl sulfate (DMS) of adenine-N1 in HG A-T bps is ~2-4 fold higher than that in WC A-T bps, suggesting potential roles of HG bps in cytotoxic damage induction in double-stranded DNA. The unique DMS reactivity signature of HG bps provided a means to discriminate HG from WC bps. By extending this DMS chemical probing method and combining it with the next-generation sequencing (NGS) technology, we developed a new DNA sequencing method, “2dDMS-seq” to comprehensively map the non-canonical structures of genomic DNA in living cells strand-specifically and at single nucleotide resolution. The method utilizes both N1-methylated adenine (m1A) and N3-methylated adenine (m3A) produced by DMS treatment as probes for non-canonical DNA structures. Applied to the yeast Saccharomyces cerevisiae, this approach probed single-stranded DNA (ssDNA) regions, nucleosome (NCP) positioning, transcription factors (TF)-binding sites, autonomously replicating sequences (ARS), and potential HG A-T bps, and thus provided in-depth insights into the structure, accessibility, damage, repair, and protein-binding landscapes of genomic DNA in vivo.

Nucleic acid molecules do not fold into static 3D structures but rather adopt various 3D conformations that interconvert over a wide range of timescales from pico-seconds to seconds under solution conditions. These conformational transitions are oftentimes involved in many fundamental biological processes, such as nucleic acid recognition and catalysis. A collection of these inter-converting 3D conformations with their Boltzmann-weights is referred to as a ‘dynamic ensemble’. Determining dynamic ensembles is important for elucidating the biological roles of nucleic acids, but this remains very difficult due to the enormous gap between the data required to describe an ensemble versus the experimental data that we can bring to bear. This dissertation develops new methods to determine nucleic acid dynamic ensembles at atomic resolution using solution state nuclear magnetic resonance (NMR) spectroscopy and applies it to three model systems.We developed a new approach to determine the ground state ensembles of RNAs with specific application to the helix-junction-helix motif the HIV-1 transactivation response element (TAR). The approach directly generates starting ensembles from RNA secondary structures using a structure prediction method, Rosetta’s Fragment Assembly of RNA with Full-Atom Refinement (FARFAR). The ensemble is then refined by using NMR residual dipolar couplings (RDCs). By testing the ensemble accuracy using quantum calculations of chemical shifts, comparison to existing crystal structures and atomic mutagenesis, we demonstrated that by starting from a FARFAR ensemble, a more accurate ground state ensemble for TAR is obtained relative to a previously determined ensemble generated using molecular dynamics (MD) simulations. We applied a similar approach to determine dynamic ensembles for lowly populated short-lived states of nucleic acids with specific application to A-T Hoogsteen base pairs (bps) in duplex DNA. We describe a strategy to resolve the dynamic ensembles of such low-abundant short-lived conformational states by combining chemical mutagenesis, NMR relaxation dispersion (RD) and RDCs, MD simulations and quantum calculations of chemical shifts. The dynamic ensembles reveal key structural features of Hoogsteen bps: the DNA helix is more constricted and kinked towards the major groove direction and this is accompanied by local sugar and backbone deformations. These unique structural fingerprints could subsequently be used to identify 13 A(syn)-T and 4 G(syn)-C+ Hoogsteen bps in protein-DNA complexes in the Protein Data Bank (PDB) which were mismodeled as Watson-Crick, revealing a greater tendency to form Hoogsteen bps near chemically or structurally stressed DNA regions. NMR methods have also been developed to study the hybridization kinetics of DNA and RNA duplexes. This non-invasive approach relies on NMR RD to measure the kinetics of nucleic acid hybridization and structurally assign the melted species of DNA and RNA duplexes at high temperature. With this approach, we show that the epitranscriptomic modification m6A slows the annealing rate of RNA duplexes, without substantially affecting the melting rate, potentially explaining how m6A slows down a variety of biologically important transitions such as the tRNA selection during mRNA translation, and the NTP incorporation during DNA replication and reverse transcription.

Modern biomedical science enjoys an unprecedented ability to identify and describe viral pathogenic mechanisms, as well as characterize the biomolecular components that constitute them. However, we have not yet achieved a fully quantitative biophysical understanding in which modeling of component molecules is accurately predictive of viral functions in cells. Reconstitution, in which a cellular process is reduced to the in vitro behavior of its component parts, is a promising strategy for establishing such predictive relationships, but there is debate as to whether the structures and activities of RNAs observed in vitro transfer to the cellular context. My thesis work seeks to probe these predictive relationships between in vitro and in vivo activity of viral RNAs and apply them to viral RNA-targeted drug development. I first quantitatively examined the relationships among levels of reconstitution of the human immunodeficiency virus 1 (HIV-1) cellular process of transactivation. This process involves complex RNA-protein interactions between an HIV-1 RNA, the transactivation response element (TAR), and a complex of the viral Tat protein and host super-elongation complex proteins (SEC). I designed library of seventeen TAR mutants and quantitatively measured their activity using four different assays, including a gene-reporter cell-based assay, electrophoretic mobility shift assay, fluorescence-based binding assay, and interhelical stacking measurements using nuclear magnetic resonance imaging (NMR) to probe behavior at varying levels of reconstitution. I found strong agreement (r=0.96-0.86) when comparing each level such that cellular activity was predicted by the reconstituted protein complex binding in vitro, which was predicted by peptide binding, which was then predicted by the stacking dynamics of the unbound RNA. This work demonstrates that reconstitution of complex cellular processes at multiple levels of reduction is a viable strategy for establishing predictive relationships with high fidelity. After establishing this predictive relationship of HIV-1 TAR ensemble behavior in vitro and in cells, I looked to take advantage of this well studied system for drug-screening applications. In reviewing the history of small molecule (SM) TAR-binders and other viral helix-junction-helix (HJH) motifs, it struck me that very few of the many compounds that bound TAR with high affinity in vitro had significant if any activity in cells. This contrasts with what would be expected based on my previous study, which demonstrates a highly predictive relationship between in vitro and cellular activities of TAR. This must mean that the discrepancy in activities must not be due to TAR, but instead due to the small molecules themselves. I investigated this idea further by executing a study of small molecules that have been reported to bind to various helix-junction-helix motif RNAs, other than TAR, and measuring their activity in vitro and in cells against both TAR and another distinct HIV-1 HJH RNA, RRE. I found that all of the molecules bound both TAR and RRE, some with equivalent affinities to their intended target. To further investigate the structural basis of this nonspecific RNA-binding behavior, I performed a structural survey of all RNA-SM complexes in the Protein Data Bank (PDB). This revealed that RNA-SM interactions with known high specificity differed in hydrogen bonding (H-bond) patterns from known low specific interactions. High specificity interactions all had unique H-bond patterns, which low specific interactions were marked by a similar H-bond pattern to atoms that are exposed in canonical A-form RNA helices. The result of this study suggests that HIV-1 TAR in its ground state form is not necessarily a good candidate for drug targeting, as the SM that are able to bind to it will likely bind to many other cellular RNAs, making in vitro screening a poor predictor of cellular activity. With this knowledge, I instead focused my drug-targeting efforts on a structurally complex RNA with unique targetable motifs – the Zika virus (ZIKV) Xrn1-exonuclease resistant RNA element (xrRNA1). Using a pipeline consisting of structure-based virtual screening and experimental screening of viral attenuation and cellular function, I screened ~80,000 small molecules and found eight that significantly modified viral replication in cells, with modest effects on cellular activity of xrRNA1. I have also included a separate project I worked on during my PhD, development of a curriculum around cultural determinants of health and health disparities in the School of Medicine.

Bulges are ubiquitous building blocks of the three-dimensional structure of RNA. They help define the global structure of helices and points of flexibility allowing for functionally important dynamics, such as binding of proteins, ligands and small molecules to occur. This thesis utilizes a battery of nuclear magnetic resonance (NMR) methods and a model system of RNA bulge motifs, the transactivation response element (TAR) RNA from the human immunodeficiency virus type 1 (HIV-1), to characterize the dynamic energy landscape of bulges. Specifically investigating how it varies with bulge length, divalent cations, and in the presence of epi-transcriptomic modifications.

Deleting a single bulge residue (C24) from trinucleotide HIV-1 TAR bulge shifts a pre-existing equilibrium from the unstacked to a stacked conformation in which the bulge residues flip out of the helix and are highly flexible at the picosecond-to-nanosecond timescale. However, the mutation minimally impacts microsecond-to-millisecond conformational exchange directed towards two low-populated and short-lived excited conformational states that form through a reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond timescale. Therefore, minor changes in bulge topology preserve motional modes occurring over the picosecond-to-millisecond timescales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

The impact of more broadly varying the length of the TAR poly-pyrimidine bulge (n = 1, 2, 3, 4 and 7) on inter-helical dynamics has been studied across a range of Mg2+ concentrations. In the absence of Mg2+ (25 mM monovalent salt), n 3 bulges adopt predominantly unstacked conformations (stacked population <15%) whereas 1-bulge and 2-bulge motifs adopt predominantly stacked conformations (stacked population >85%). The 2-bulge motif is biased toward linear conformations and increasing the bulge length leads to broader inter-helical distributions and structures that are on average more kinked. In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >75%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition does not vary substantially (within 3-fold) with bulge length. In the absence of Mg2+, the difference between the free energy of inter-helical coaxial stacking across the bulge variants is estimated to be ~2.9 kcal/mol, based on an NMR chemical shift mapping approach, with stacking being more energetically disfavored for the longer bulges. This difference decreases to ~0.4 kcal/mol in the presence of 3 mM Mg2+. It is proposed that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thus increasing the number of co-axial conformations that can be sampled.

N6-Methyladenosine (m6A) and N1-Methylpurine (m1A and m1G) xx or just refer to m1G?xx are post-transcriptional RNA modifications that are proposed to influence RNA function through mechanisms that can involve modulation of RNA structure. m6A is thought to modulate RNA structure by destabilizing base pairing. Here, it is shown that m6A can stabilize A-U base pairing and overall RNA structure when placed within the context of a bulge motif. m1A has also been shown to potently destabilize RNA duplexes due to their inability to favorably accommodate Hoogsteen base pairing. It is shown that such Hoogsteen base pairs can form in RNA when placed in the context of a bulge motif.

Taken together, the studies show that the dynamic energy landscape of polypyridine bulges is highly robust with respect to changes in bulge length allowing for gradual variations in the population and energetics of common conformations. Mg2+ plays an important role in smoothening these variations most likely by diminishing electrostatic contributions that could vary significantly across bulges of different length. The results also show that the structural impact of epi-transcriptomic modifications can be greatly altered relative to duplex RNA when targeting bulge motifs.

As non-coding RNAs are increasingly implicated in cellular regulatory functions and disease states, there is a need to deepen our understanding of RNA structure-function relationships as well as to develop methods targeting RNA with small molecules. The transactivation response element (TAR) RNA from human immunodeficiency virus type 1 (HIV-1) is an established drug target for the development of anti-HIV therapeutics and has served as a model system for understanding RNA dynamics and RNA:ligand interactions. Like many RNAs, HIV-1 TAR is a highly flexible molecule that experiences dynamics ranging from local fluctuations in base orientation and interhelical angles to higher-order dynamics that transiently alter base pairing away from the ground state (GS) secondary structure. The work presented in this thesis is aimed at developing approaches targeting TAR with small molecules that integrate its broad range of structural dynamics.

First, nuclear magnetic resonance (NMR) chemical shift mapping is applied in concert with fluorescence binding assays and computational docking to efficiently characterize the TAR-binding modes of a focused library of amiloride derivatives. Through this work, amiloride is established as a novel RNA binding scaffold with interesting structure-activity relationships. Ultimately, this approach yielded ten novel TAR binders with demonstrated selectivity for TAR over tRNA and with up to a 100-fold increase in activity over the parent dimethyl amiloride compound.

Next, we demonstrate that ensemble-based virtual screening (EBVS) is a powerful approach to predict ligand binding for flexible RNA targets. Here, we generate a library to evaluate EBVS enrichment by subjecting HIV-1 TAR to experimental high-throughput screening against ~100,000 drug-like small molecules. EBVS against a dynamic ensemble of the TAR GS determined previously by combining NMR spectroscopy data and molecular dynamics (MD) simulations scores hits and non-hits with an area under the receiver operator characteristic curve of ~0.85-0.94 and with ~40-75% of all hits falling within the top 2% of scored molecules. Importantly, the enrichment was shown to depend on the accuracy of the ensemble.

Finally, we explore the novel strategy of specifically targeting non-native RNA excited state conformations inspired by the fact that their altered secondary structures are likely functionally inactive and highly unique. We use a mutational stabilize-and-rescue approach to demonstrate that TAR ES2 dramatically inhibits TAR activity in cells, suggesting that stabilizing the ES conformation with small molecules would similarly inhibit activity. To pursue TAR ES2 as a potential target, we have determined the first-ever dynamic ensemble of an RNA ES using a combination of MD and NMR residual dipolar couplings (RDCs) measured on a highly accurate ES2-stabilizing mutant. This dynamic ensemble was subjected to our validated EBVS approach to identify small molecules that bind and stabilize TAR ES2. Using NMR chemical shift fingerprinting, we have identified molecules that bind the TAR ES2 structure, including two that induce significant broadening in wtTAR consistent with chemical exchange and two that show a preference for TAR ES2 over the GS.

Together, this work explores multiple novel strategies for structure-specific RNA targeting.

RNAs are growing in their importance as regulators in multiple biological processes. A deep understanding of how RNAs function within cells requires an understanding of their dynamic behavior to enable the targeting of RNA in drug discovery as therapeutics. The HIV-1 Rev response element (RRE) RNA which is a known drug target mediates the export of incompletely-spliced viral RNA to express viral proteins required for HIV-1 replication and spread. Rev protein first recognizes the purine-rich region on RRE stem IIB (RREIIB), and then other Rev monomers cooperatively assemble on RRE to form ribonucleoprotein complex. Conformational flexibility at this stem IIB region has been shown to be important for Rev binding. In addition, m6A modifications on HIV-1 RNA has shown their critical roles to HIV-1 replication, and two probably essential m6A sites on purine-rich region on RREIIB was discovered. However, the nature of the flexibility and m6A modification to RREIIB have remained elusive. The work in this thesis is aiming to characterize the conformational dynamics of RRE stem IIB as potential drug targets, and to discover small molecules binding to RRE using computational and experimental drug screening.

First, nuclear magnetic resonance (NMR) techniques are applied to identify the native and non-native conformations of RREIIB. Including relaxation dispersion NMR and a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger stem II three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of around 20%. The ESs disrupt the Rev binding site by changing local secondary structure and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15 to 80 fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play important roles in the cooperative Rev binding and the development of anti-HIV therapeutics.

Secondly, m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the RREIIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. We show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev-ARM using optical melting experiments, NMR spectroscopy, and in vitro binding assays. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA.

Next, to confirm the RRE ESs ensemble visualized in vitro can recapitulate in cells, we show that stabilizing ESs using point substitution mutations leads to potent conformation-dependent inhibition of RNA cellular activity. The point substitution mutations with invert the equilibrium so that the ES becomes the dominant (>50% population) conformation and secondary rescue mutations to restore the GS conformation and control for sequence effects. We then demonstrate that the degree to which increasing the population of the ES at the expense of the GS leads to a corresponding decrease in cellular activity. The results also support that stabilizing non-native ESs potentially provides an alternative therapeutic strategy for targeting RNA.

Finally, we construct atomic resolution ensembles for the RRE ground state using RDC-SAS; perform computational docking against the ensemble of RRE GS ,and validate selected hits using in vitro and cell-based assays. We will also generate the dynamic ensembles of RRE ESs as alternative targets for ensemble-based virtual screening. The final goal is to identify small molecules that stabilize RRE GS or inactive ESs and thereby inhibit Rev-RRE interaction.