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1.
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
2.
J Phys Chem B ; 125(28): 7613-7627, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34236202

RESUMO

Measuring the strength of the hydrogen bonds between DNA base pairs is of vital importance for understanding how our genetic code is physically accessed and recognized in cells, particularly during replication and transcription. Therefore, it is important to develop probes for these key hydrogen bonds (H-bonds) that dictate events critical to cellular function, such as the localized melting of DNA. The vibrations of carbonyl bonds are well-known probes of their H-bonding environment, and their signals can be observed with infrared (IR) spectroscopy. Yet, pinpointing a single bond of interest in the complex IR spectrum of DNA is challenging due to the large number of carbonyl signals that overlap with each other. Here, we develop a method using isotope editing and infrared (IR) spectroscopy to isolate IR signals from the thymine (T) C2═O carbonyl. We use solvatochromatic studies to show that the TC2═O signal's position in the IR spectrum is sensitive to the H-bonding capacity of the solvent. Our results indicate that C2═O of a single T base within DNA duplexes experiences weak H-bonding interactions. This finding is consistent with the existence of a third, noncanonical CH···O H-bond between adenine and thymine in both Watson-Crick and Hoogsteen base pairs in DNA.


Assuntos
DNA , Isótopos , Hidrogênio , Ligação de Hidrogênio , Análise Espectral
3.
Curr Opin Struct Biol ; 70: 16-25, 2021 Apr 06.
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.

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.

10.
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)
11.
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
12.
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
13.
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
14.
Nucleic Acids Res ; 46(20): 11099-11114, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30285154

RESUMO

A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson-Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2'-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5-4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3-4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.


Assuntos
Pareamento de Bases/fisiologia , DNA de Forma B/química , Conformação de Ácido Nucleico , Purinas/química , RNA/química , DNA/química , Metabolismo Energético , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pirimidinas/química , Termodinâmica
15.
RNA ; 24(10): 1363-1376, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30012568

RESUMO

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1-4, 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+, helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg2+, the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg2+ NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+ We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated interhelical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.


Assuntos
Conformação de Ácido Nucleico , Polirribonucleotídeos/química , Polirribonucleotídeos/genética , Pirimidinas , RNA Viral/química , RNA Viral/genética , HIV-1/genética , Humanos , Magnésio/química , Espectroscopia de Ressonância Magnética , Elementos de Resposta , Relação Estrutura-Atividade
16.
J Biomol NMR ; 70(4): 229-244, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29675775

RESUMO

NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3' and C4' spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A-T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A-T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.


Assuntos
Adenina/química , Pareamento de Bases , Ressonância Magnética Nuclear Biomolecular/métodos , Timina/química , DNA/química , Ligação de Hidrogênio , Estrutura Molecular , Mutagênese , Conformação de Ácido Nucleico
17.
Nucleic Acids Res ; 45(9): 5586-5601, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28369571

RESUMO

In the canonical DNA double helix, Watson-Crick (WC) base pairs (bps) exist in dynamic equilibrium with sparsely populated (∼0.02-0.4%) and short-lived (lifetimes ∼0.2-2.5 ms) Hoogsteen (HG) bps. To gain insights into transient HG bps, we used solution-state nuclear magnetic resonance spectroscopy, including measurements of residual dipolar couplings and molecular dynamics simulations, to examine how a single HG bp trapped using the N1-methylated adenine (m1A) lesion affects the structural and dynamic properties of two duplexes. The solution structure and dynamic ensembles of the duplexes reveals that in both cases, m1A forms a m1A•T HG bp, which is accompanied by local and global structural and dynamic perturbations in the double helix. These include a bias toward the BI backbone conformation; sugar repuckering, major-groove directed kinking (∼9°); and local melting of neighboring WC bps. These results provide atomic insights into WC/HG breathing dynamics in unmodified DNA duplexes as well as identify structural and dynamic signatures that could play roles in m1A recognition and repair.


Assuntos
Adenina/química , Pareamento de Bases , Reparo do DNA , DNA/química , Conformação de Ácido Nucleico , Espectroscopia de Ressonância Magnética , Metilação , Soluções , Termodinâmica , Fatores de Tempo
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