1,721,051 research outputs found
Visualizing Rare Watson-Crick-Like Tautomeric and Anionic Mismatches in DNA and RNA
No full textThe central dogma of molecular biology relies on the correct Watson-Crick (WC) geometry of canonical deoxyribonucleic acid (DNA) dG•dC and dA•dT base pairs to replicate and transcribe genetic information with speed and an astonishing level of fidelity. In addition, the Watson-Crick geometry of canonical ribonucleic acid (RNA) rG•rC and rA•rU base pairs is highly conserved to ensure that proteins are translated with high fidelity. However, numerous other potential nucleobase tautomeric and ionic configurations are possible that can give rise to entirely new pairing modes between the nucleotide bases. Very early on, James Watson and Francis Crick recognized their importance and in 1953 postulated that if bases adopted one of their less energetically disfavored tautomeric forms (and later ionic forms) during replication it could lead to the formation of a mismatch with a Watson-Crick-like geometry and could give rise to “natural mutations.” Since this time numerous studies have provided evidence in support of this hypothesis and have expanded upon it; computational studies have addressed the energetic feasibilities of different nucleobases’ tautomeric and ionic forms in siico; crystallographic studies have trapped different mismatches with WC-like geometries in polymerase or ribosome active sites. However, no direct evidence has been given for (i) the direct existence of these WC-like mismatches in canonical DNA duplex, RNA duplexes, or non-coding RNAs; (ii) which, if any, tautomeric or ionic form stabilizes the WC-like geometry. This thesis utilizes nuclear magnetic resonance (NMR) spectroscopy and rotating frame relaxation dispersion (R1ρ RD) in combination with density functional theory (DFT), biochemical assays, and targeted chemical perturbations to show that (i) dG•dT mismatches in DNA duplexes, as well as rG•rU mismatches RNA duplexes and non-coding RNAs, transiently adopt a WC-like geometry that is stabilized by (ii) an interconnected network of rapidly interconverting rare tautomers and anionic bases. These results support Watson and Crick’s tautomer hypothesis, but additionally support subsequent hypotheses invoking anionic mismatches and ultimately tie them together. This dissertation shows that a common mismatch can adopt a Watson-Crick-like geometry globally, in both DNA and RNA, and whose geometry is stabilized by a kinetically linked network of rare tautomeric and anionic bases. The studies herein also provide compelling evidence for their involvement in spontaneous replication and translation errors.</p
Targeting Alternative Conformational States of the HIV-1 Rev Response Element Stem IIB using Small Molecules
No full textRNAs 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.</p
Development of NMR methodology for probing functional dynamics in RNA.
No full textOver last two decades, several new RNA functions have been discovered completely transforming how we view its role in gene expression and regulation. A key feature of many of these new RNA functions is that they almost always require or involve dramatic conformational changes in the RNA that typically occur in response to protein or ligand recognition. Though it is clear that RNA structures must change to perform their functions, the mechanism by which this occurs remains poorly understood. A central problem is that the dynamical properties of nucleic acids remain poorly understood especially in comparison to proteins. This dissertation describes the development of solution nuclear magnetic resonance (NMR) spectroscopic methods for characterizing dynamics in nucleic acids at timescales ranging between picoseconds and milliseconds. The methods are applied to uncover the molecular basis by which the transactivation response element (TAR) RNA from the human immunodeficiency virus type 1 (HIV-1) genome adaptively changes its structure and thereby binds to dramatically different molecular targets. A major problem in the application of NMR towards the study of RNA dynamics is that internal motions can lead to correlated changes in overall motions making analysis of NMR parameters intractable. This limitation is addressed by analytical methods for the case of magnetic field induced residual dipolar couplings (RDCs) and by chemical elongation of a target helix in the case of ordering media induced RDCs and spin relaxation measurements. The combined application of RDCs and spin relaxation allowed the characterization of the entire dynamical spectrum of TAR from picosecond to millisecond timescales. Results show that the two helices in TAR undergo super large amplitude motions at suprananosecond timescales. The helices twist and bend in a highly correlated manner and allow free TAR to dynamically visit seven distinct ligand bound conformation. The helix motions are activated by organized local motions in the bulge linker and neighboring residues where small molecules and ligands bind. Our results strongly suggest that small molecules capture existing TAR conformations that are appreciably populated in the free state rather than induce new ones via the conventional induced-fit mechanism.PhDBiochemistryPhysical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126984/2/3287667.pd
Measurements of Conformational Penalties in Nucleic Acids
No full textBiomolecules 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.
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Structure and Dynamics Based Methods Targeting RNA
No full textAs 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.</p
Structural and Dynamic Studies of RNA Bulge Motifs Utilizing Nuclear Magnetic Resonance
No full textBulges 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 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.</p
Resolving Atomic-resolution Nucleic Acid Ensembles Using Solution State Nuclear Magnetic Resonance
No full textNucleic 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.
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DNA Conformational Equilibria in Replication Fidelity
No full textAll 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.</p
Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids
No full textNucleic 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.</p
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