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1.
Nucleic Acids Res ; 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34792150

RESUMO

Watson-Crick base pairs (bps) are the fundamental unit of genetic information and the building blocks of the DNA double helix. However, A-T and G-C can also form alternative 'Hoogsteen' bps, expanding the functional complexity of DNA. We developed 'Hoog-finder', which uses structural fingerprints to rapidly screen Hoogsteen bps, which may have been mismodeled as Watson-Crick in crystal structures of protein-DNA complexes. We uncovered 17 Hoogsteen bps, 7 of which were in complex with 6 proteins never before shown to bind Hoogsteen bps. The Hoogsteen bps occur near mismatches, nicks and lesions and some appear to participate in recognition and damage repair. Our results suggest a potentially broad role for Hoogsteen bps in stressed regions of the genome and call for a community-wide effort to identify these bps in current and future crystal structures of DNA and its complexes.

2.
Nat Commun ; 12(1): 5201, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465779

RESUMO

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, 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 at elevated temperatures 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.


Assuntos
Adenosina/análogos & derivados , DNA/química , DNA/metabolismo , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Pareamento de Bases , DNA/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Processamento Pós-Transcricional do RNA , Uridina/química , Uridina/genética , Uridina/metabolismo
3.
Curr Opin Struct Biol ; 70: 16-25, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33836446

RESUMO

Nucleic acids do not fold into a single conformation, and dynamic ensembles are needed to describe their propensities to cycle between different conformations when performing cellular functions. We review recent advances in solution-state nuclear magnetic resonance (NMR) methods and their integration with computational techniques that are improving the ability to probe the dynamic ensembles of DNA and RNA. These include computational approaches for predicting chemical shifts from structure and generating conformational libraries from sequence, measurements of exact nuclear Overhauser effects, development of new probes to study chemical exchange using relaxation dispersion, faster and more sensitive real-time NMR techniques, and new NMR approaches to tackle large nucleic acid assemblies. We discuss how these advances are leading to new mechanistic insights into gene expression and regulation.


Assuntos
Ácidos Nucleicos , DNA , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , RNA
4.
RNA ; 27(1): 12-26, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33028652

RESUMO

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.


Assuntos
Aminoglicosídeos/farmacologia , Genes env/efeitos dos fármacos , Repetição Terminal Longa de HIV/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Bioensaio , Descoberta de Drogas , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação de Hidrogênio , Isoquinolinas/química , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Conformação de Ácido Nucleico , Pentamidina/química , Pentamidina/metabolismo , Pentamidina/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Eletricidade Estática , Ativação Transcricional/efeitos dos fármacos , Ioimbina/química , Ioimbina/metabolismo , Ioimbina/farmacologia
5.
Nat Commun ; 11(1): 5531, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139729

RESUMO

Biomolecules form dynamic ensembles of many inter-converting conformations which are key for understanding how they fold and function. However, determining ensembles is challenging because the information required to specify atomic structures for thousands of conformations far exceeds that of experimental measurements. We addressed this data gap and dramatically simplified and accelerated RNA ensemble determination by using structure prediction tools that leverage the growing database of RNA structures to generate a conformation library. Refinement of this library with NMR residual dipolar couplings provided an atomistic ensemble model for HIV-1 TAR, and the model accuracy was independently supported by comparisons to quantum-mechanical calculations of NMR chemical shifts, comparison to a crystal structure of a substate, and through designed ensemble redistribution via atomic mutagenesis. Applications to TAR bulge variants and more complex tertiary RNAs support the generality of this approach and the potential to make the determination of atomic-resolution RNA ensembles routine.


Assuntos
Quimioinformática/métodos , HIV-1/química , Dobramento de RNA , RNA Viral/ultraestrutura , Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/ultraestrutura , Modelos Químicos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , RNA Viral/química , RNA Viral/genética
6.
Nature ; 587(7833): 291-296, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33087930

RESUMO

Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.


Assuntos
Proteínas de Ligação a DNA/química , Conformação Molecular , Ácidos Nucleicos Heteroduplexes/química , Proteínas de Arabidopsis/química , Pareamento de Bases , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica , Fatores de Transcrição/química
7.
Nucleic Acids Res ; 48(21): 12365-12379, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33104789

RESUMO

2'-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3'-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.


Assuntos
Repetição Terminal Longa de HIV , Magnésio/química , Processamento Pós-Transcricional do RNA , RNA Viral/química , Pareamento de Bases , Cátions Bivalentes , Teoria da Densidade Funcional , HIV-1/química , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/genética , RNA Viral/metabolismo , Termodinâmica
8.
J Biol Chem ; 295(47): 15933-15947, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-32913127

RESUMO

As the Watson-Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson-Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.


Assuntos
Pareamento de Bases , Metilação de DNA , DNA/química , Ésteres do Ácido Sulfúrico/química
9.
J Biomol NMR ; 74(8-9): 457-471, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32789613

RESUMO

NMR off-resonance R1ρ relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson-Crick-like conformations. As Watson-Crick-like G·T mismatches have base pairing geometries similar to Watson-Crick base pairs, we hypothesized that they would mimic Watson-Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R1ρ measurements targeting the sugar C3' and C4' nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson-Crick conformation and that transitions toward tautomeric and anionic Watson-Crick-like G·T mismatches restore the canonical Watson-Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson-Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T-, and also establish sugar carbons as new non-exchangeable probes of this exchange process.


Assuntos
Pareamento Incorreto de Bases , Carbono/química , DNA/química , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Açúcares/química , Pareamento de Bases , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Timina
10.
Nat Struct Mol Biol ; 27(6): 604, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32376863

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

11.
J Am Chem Soc ; 142(25): 11183-11191, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32459476

RESUMO

DNA bases can adopt energetically unfavorable tautomeric forms that enable the formation of Watson-Crick-like (WC-like) mispairs, which have been proposed to give rise to spontaneous mutations in DNA and misincorporation errors in DNA replication and translation. Previous NMR and computational studies have indicated that the population of WC-like guanine-thymine (G-T) mispairs depends on the environment, such as the local nucleic acid sequence and solvation. To investigate these environmental effects, herein G-T mispair tautomerization processes are studied computationally in aqueous solution, in A-form and B-form DNA duplexes, and within the active site of a DNA polymerase λ variant. The wobble G-T (wG-T), WC-like G-T*, and WC-like G*-T forms are considered, where * indicates the enol tautomer of the base. The minimum free energy paths for the tautomerization from the wG-T to the WC-like G-T* and from the WC-like G-T* to the WC-like G*-T are computed with mixed quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The reaction free energies and free energy barriers are found to be significantly influenced by the environment. The wG-T→G-T* tautomerization is predicted to be endoergic in aqueous solution and the DNA duplexes but slightly exoergic in the polymerase, with Arg517 and Asn513 providing electrostatic stabilization of G-T*. The G-T*→G*-T tautomerization is also predicted to be slightly more thermodynamically favorable in the polymerase relative to these DNA duplexes. These simulations are consistent with an experimentally driven kinetic misincorporation model suggesting that G-T mispair tautomerization occurs in the ajar polymerase conformation or concertedly with the transition from the ajar to the closed polymerase conformation. Furthermore, the order of the associated two proton transfer reactions is predicted to be different in the polymerase than in aqueous solution and the DNA duplexes. These studies highlight the impact of the environment on the thermodynamics, kinetics, and fundamental mechanisms of G-T mispair tautomerization, which plays a role in a wide range of biochemically important processes.


Assuntos
DNA Forma A/química , DNA de Forma B/química , Pareamento Incorreto de Bases , Pareamento de Bases , Domínio Catalítico , DNA Polimerase beta/química , DNA Forma A/genética , DNA de Forma B/genética , Guanina/química , Isomerismo , Modelos Moleculares , Teoria Quântica , Termodinâmica , Timina/química
12.
Angew Chem Int Ed Engl ; 59(28): 11262-11266, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32168407

RESUMO

Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr-Purcell-Meiboom-Gill (CPMG), chemical exchange saturation transfer (CEST), and R1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1ρ experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high-power radio-frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high-power fields in CEST, this should make it easier to characterize micro-to-millisecond dynamics in biomolecules.


Assuntos
DNA/química , Espectroscopia de Ressonância Magnética/métodos , Ondas de Rádio , Limite de Detecção , Movimento (Física)
13.
Cell Rep ; 30(8): 2472-2480.e4, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32101729

RESUMO

Low-abundance short-lived non-native conformations referred to as excited states (ESs) are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs. We developed an approach for assessing the relative abundance of RNA ESs within the functional cellular context. Nuclear magnetic resonance (NMR) spectroscopy was used to estimate the degree to which substitution mutations bias conformational equilibria toward the inactive ES in vitro. The cellular activity of the ES-stabilizing mutants was used as an indirect measure of the conformational equilibria within the functional cellular context. Compensatory mutations that restore the ground-state conformation were used to control for changes in sequence. Using this approach, we show that the ESs of two regulatory RNAs from HIV-1, the transactivation response element (TAR) and the Rev response element (RRE), likely form in cells with abundances comparable to those measured in vitro, and their targeted stabilization may provide an avenue for developing anti-HIV therapeutics.


Assuntos
Células/metabolismo , Conformação de Ácido Nucleico , Microambiente Celular , Genes env , Células HEK293 , Células HeLa , Humanos , Estabilidade de RNA
14.
J Am Chem Soc ; 142(2): 907-921, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31815464

RESUMO

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 study 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 and relaxation dispersion 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 ≈ 0.2 mM), it binds the prefolded conformation with a Kd 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.


Assuntos
Conformação de Ácido Nucleico , RNA/química , Sítios de Ligação , HIV-1/genética , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular , RNA Viral/química
15.
J Mol Biol ; 432(4): 1297-1304, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31863746

RESUMO

Many promising RNA drug targets have functions that require the formation of RNA-protein complexes, but inhibiting RNA-protein interactions can prove difficult using small molecules. Regulatory RNAs have been shown to transiently form excited conformational states (ESs) that remodel local aspects of secondary structure. In some cases, the ES conformation has been shown to be inactive and to be poorly recognized by protein binding partners. In these cases, specifically targeting and stabilizing the RNA ES using a small molecule provides a rational structure-based strategy for inhibiting RNA activity. However, this requires that a small molecule discriminates between two conformations of the same RNA to preferentially bind and stabilize the short-lived low-abundance ES relative to the long-lived more abundant ground state (GS). Here, we tested the feasibility of this approach by designing a mutant that inverts the conformational equilibrium of the HIV-1 transactivation response element (TAR) RNA, such that the native GS conformation becomes a low-abundance ES. Using this mutant and NMR chemical shift mapping experiments, we show that argininamide, a ligand mimic of TAR's cognate protein binding partner Tat, is able to restore a native-like conformation by preferentially binding and stabilizing the transient and low-populated ES. A synthetic small molecule optimized to bind the TAR GS also partially stabilized the ES, whereas an aminoglycoside molecule that binds RNAs nonspecifically did not preferentially stabilize the ES to a similar extent. These results support the feasibility of inhibiting RNA activity using small molecules that preferentially bind and stabilize the ES.


Assuntos
HIV-1/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas Virais/metabolismo , Repetição Terminal Longa de HIV/genética , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteínas Virais/química
16.
J Am Chem Soc ; 141(51): 19988-19993, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31826614

RESUMO

N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. 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. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by ∼1-fold but significantly slows the rate of duplex annealing, decreasing kon by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.


Assuntos
Adenosina/análogos & derivados , Ressonância Magnética Nuclear Biomolecular , RNA/química , Adenosina/análise , Adenosina/metabolismo , RNA/metabolismo
17.
PLoS One ; 14(12): e0224850, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31825959

RESUMO

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. 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 HIV-1 Rev response element (RRE) stem IIB, 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. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.


Assuntos
Adenosina/análogos & derivados , RNA Viral/química , RNA Viral/metabolismo , Elementos de Resposta , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Adenosina/química , Pareamento de Bases , Sequência de Bases , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética
18.
Prog Nucl Magn Reson Spectrosc ; 112-113: 55-102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31481159

RESUMO

This review describes off-resonance R1ρ relaxation dispersion NMR methods for characterizing microsecond-to-millisecond chemical exchange in uniformly 13C/15N labeled nucleic acids in solution. The review opens with a historical account of key developments that formed the basis for modern R1ρ techniques used to study chemical exchange in biomolecules. A vector model is then used to describe the R1ρ relaxation dispersion experiment, and how the exchange contribution to relaxation varies with the amplitude and frequency offset of an applied spin-locking field, as well as the population, exchange rate, and differences in chemical shifts of two exchanging species. Mathematical treatment of chemical exchange based on the Bloch-McConnell equations is then presented and used to examine relaxation dispersion profiles for more complex exchange scenarios including three-state exchange. Pulse sequences that employ selective Hartmann-Hahn cross-polarization transfers to excite individual 13C or 15N spins are then described for measuring off-resonance R1ρ(13C) and R1ρ(15N) in uniformly 13C/15N labeled DNA and RNA samples prepared using commercially available 13C/15N labeled nucleotide triphosphates. Approaches for analyzing R1ρ data measured at a single static magnetic field to extract a full set of exchange parameters are then presented that rely on numerical integration of the Bloch-McConnell equations or the use of algebraic expressions. Methods for determining structures of nucleic acid excited states are then reviewed that rely on mutations and chemical modifications to bias conformational equilibria, as well as structure-based approaches to calculate chemical shifts. Applications of the methodology to the study of DNA and RNA conformational dynamics are reviewed and the biological significance of the exchange processes is briefly discussed.


Assuntos
DNA/química , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular
19.
J Magn Reson ; 308: 106589, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31539864

RESUMO

NMR relaxation dispersion studies have shown that Watson-Crick G-C and A-T base pairs in duplex DNA exist in dynamic equilibrium with their Hoogsteen counterparts. Hoogsteen base pairs form through concurrent rotation of the purine base about the glycosidic bond from an anti to a syn conformation and constriction of the C1'-C1' distance across the base pair by ∼2 Što allow Hoogsteen type hydrogen bonding. Owing to their unique structure, Hoogsteen base pairs can play important roles in DNA recognition, the accommodation, recognition, and repair of DNA damage, and in DNA replication. NMR relaxation dispersion experiments targeting imino nitrogen and protonated base and sugar carbons have provided insights into many structural features of transient Hoogsteen base pairs, including one of two predicted hydrogen bonds involving (G)N7···H-N3(C)+ and (A)N7···H-N3(T). Here, through measurement of cytosine amino (N4) R1ρ relaxation dispersion, we provide direct evidence for the second (G)O6···H2-N4(C)+ hydrogen bond in G(syn)-C+ transient Hoogsteen base pairs. The utility of cytosine N4 R1ρ relaxation dispersion as a new sensitive probe of transient Hoogsteen base pairs, and cytosine dynamics in general, is further demonstrated by measuring G(syn)-C+ Hoogsteen exchange near neutral pH and in the context of the naturally occurring DNA modification 5-methyl cytosine (m5C), in DNA samples prepared using chemical synthesis and a 15N labeled m5C phosphoramidite.


Assuntos
Pareamento de Bases , Citosina/química , DNA/química , Ligação de Hidrogênio , Nitrogênio/química , Adenosina/química , Teoria da Densidade Funcional , Epigênese Genética , Guanina/química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Oligonucleotídeos/química , Timina/química
20.
Angew Chem Int Ed Engl ; 58(35): 12010-12013, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31268220

RESUMO

Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson-Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X-ray structures of protein-DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson-Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X-ray crystallography due to ambiguous electron density and by solution-state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution-state observation of a Hoogsteen (G-C+ ) bp in a DNA:protein complex under solution conditions with specific application to DNA-bound TATA-box binding protein. These results support a previous assignment of a G-C+ Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes.


Assuntos
Citosina/química , DNA/metabolismo , Guanina/química , Proteína de Ligação a TATA-Box/metabolismo , Pareamento de Bases , Cristalografia por Raios X , DNA/química , Conformação de Ácido Nucleico , Espectrofotometria Infravermelho , Proteína de Ligação a TATA-Box/química
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