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1.
J Chem Theory Comput ; 19(23): 8955-8966, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38014857

RESUMO

In addition to the well-characterized B-form of DNA, duplex DNA can adopt various conformations, such as A or Z-DNA. Though less common, these structures can be induced biologically through protein or ligand interactions or experimentally with niche environmental conditions, such as high salt concentrations or in mixed water-ethanol. Reproducing these alternate structures through molecular dynamics simulations in recent years has been quite challenging with the currently available force fields, simulation techniques, and time scales. In this study, the Drude polarizable force field is tested for its ability to facilitate transitions between A-DNA and B-DNA or maintain A-DNA. Though transitions away from B-DNA were observed in high concentrations of ethanol, the resulting structures had hybrid properties taken from both B-DNA and A-DNA structures. This was also true for A-DNA in ethanol, which lost some of the A-DNA properties that it was expected to maintain. When B-DNA was tested in high salt environments, the resulting B-DNA structures showed no distinguishable differences with the increasing salt concentrations tested. These results with the Drude FF and recent results with additive force fields suggest that at present the current additive and polarizable force fields do not facilitate a complete transition between B- to A-DNA conformations under the conditions simulated. At present, the Drude FF favors A-B DNA hybrid structures when simulated in nonphysiological conditions.


Assuntos
DNA Forma A , DNA de Forma B , DNA/química , Simulação de Dinâmica Molecular , Etanol
2.
Chem Commun (Camb) ; 59(32): 4726-4741, 2023 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-37000514

RESUMO

DNA origami nanostructures are self-assembled into almost arbitrary two- and three-dimensional shapes from a long, single-stranded viral scaffold strand and a set of short artificial oligonucleotides. Each DNA strand can be functionalized individually using well-established DNA chemistry, representing addressable sites that allow for the nanometre precise placement of various chemical entities such as proteins, molecular chromophores, nanoparticles, or simply DNA motifs. By means of microscopic and spectroscopic techniques, these entities can be visualized or detected, and either their mutual interaction or their interaction with external stimuli such as radiation can be studied. This gives rise to the Lab-on-a-DNA origami approach, which is introduced in this Feature Article, and the state-of-the-art is summarized with a focus on light-harvesting nanoantennas and DNA platforms for single-molecule analysis either by optical spectroscopy or atomic force microscopy (AFM). Light-harvesting antennas can be generated by the precise arrangement of chromophores to channel and direct excitation energy. At the same time, plasmonic nanoparticles represent a complementary approach to focus light on the nanoscale. Plasmonic nanoantennas also allow for the observation of single molecules either by Raman scattering or fluorescence spectroscopy and DNA origami platforms provide unique opportunities to arrange nanoparticles and molecules to be studied. Finally, the analysis of single DNA motifs by AFM allows for an investigation of radiation-induced processes in DNA with unprecedented detail and accuracy.


Assuntos
DNA Forma A , Nanoestruturas , Nanotecnologia/métodos , DNA/química , Nanoestruturas/química , Microscopia de Força Atômica/métodos , Conformação de Ácido Nucleico
3.
Redox Biol ; 62: 102674, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36989575

RESUMO

Renal fibrosis is the common histopathological feature of chronic kidney diseases (CKD), and there is increasing evidence that epigenetic regulation is involved in the occurrence and progression of renal fibrosis. N-myc downstream-regulated gene 2 (NDRG2) is significantly down-regulated in renal fibrosis, the mechanism of which remains unclear. Previous studies have confirmed that the inhibition of NDRG2 expression in tumor cells is related to hyper-methylation, mainly regulated by DNA methyltransferases (DNMTS). Herein, we explored the expression of NDRG2 and its epigenetic regulatory mechanism in renal fibrosis. The results showed that the expression of NDRG2 was significantly inhibited in vivo and in vitro, while the overexpression of NDRG2 effectively alleviated renal fibrosis. Meanwhile, we found that the expression of DNMT1/3A/3B was significantly increased in hypoxia-induced HK2 cells and Unilateral Ureteral Obstruction (UUO) mice accompanied by hyper-methylation of the NDGR2 promoter. Methyltransferase inhibitor (5-AZA-dC) corrected the abnormal expression of DNMT1/3A/3B, reduced the methylation level of NDRG2 promoter and restored the expression of NDRG2. The upstream events that mediate changes in NDRG2 methylation were further explored. Reactive oxygen species (ROS) are important epigenetic regulators and have been shown to play a key role in renal injury due to various causes. Accordingly, we further explored whether ROS could induce DNA-epigenetic changes of the expression of NDRG2 and then participated in the development of renal fibrosis. Our results showed that mitochondria-targeted antioxidants (Mito-TEMPO) could reverse the epigenetic inhibition of NDRG2 in a DNMT-sensitive manner, showing strong ability of DNA demethylation, exhibiting epigenetic regulation and anti-fibrosis effects similar to 5-AZA-dC. More importantly, the anti-fibrotic effects of 5-AZA-dC and Mito-TEMPO were eliminated in HK2 cells with NDRG2 knockdown. These findings highlight that targeting ROS-mediated hyper-methylation of NDRG2 promoter is a potentially effective therapeutic strategy for renal fibrosis, which will provide new insights into the treatment of CKD.


Assuntos
DNA Forma A , Insuficiência Renal Crônica , Animais , Camundongos , Epigênese Genética , Espécies Reativas de Oxigênio , Metiltransferases/genética , Metilação de DNA , Fibrose , Insuficiência Renal Crônica/patologia , Azacitidina/uso terapêutico
4.
Biosensors (Basel) ; 12(9)2022 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-36140109

RESUMO

Because microRNAs (miRNAs) are biological indicators for the diagnosis, treatment, and monitoring of tumors, cancers, and other diseases, it is significant to develop a rapid, sensitive, and reliable miRNA detection platform. In this study, based on miRNA-21 detection, DNA-a with a 3' end overhang and Texas Red fluorophore-labeled 5' end was designed, which reacts with miRNA-21 and hybridizes with exonuclease III (Exo III), where the part connected to miRNA-21 is hydrolyzed, leaving a-DNA. At the same time, miRNA-21 is released to participate in the following reaction, to achieve cyclic amplification. a-DNA reacts with DNA-b conjugated to gold nanoparticles to achieve fluorescence quenching, with the quenching value denoted as F; additionally, after adding DNA-d and linked streptavidin immunomagnetic beads (SIBs), fluorescence recovery was achieved using DNA-c, with the recovered fluorescence recorded as F0. By comparing the difference in the fluorescence (F0 - F) between the two experiments, the amount of DNA-a hydrolyzed to produce a-DNA was established to determine the target miRNA-21 content. Under optimized conditions, by comparing the changes in the fluorescence signal, the developed strategy shows good sensitivity and repeatability, with a detection limit of 18 pM, good discriminative ability and selectivity, and promise for the early diagnosis of breast and intestinal cancers.


Assuntos
Técnicas Biossensoriais , DNA Forma A , Nanopartículas Metálicas , MicroRNAs , DNA , Ouro , Limite de Detecção , Estreptavidina
5.
Biophys Chem ; 288: 106845, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35785647

RESUMO

The structure of A-DNA with biological activity is double helix. Researches of A-DNA structure have wide application on the development of DNA drug. In aqueous solution, the structure of A-DNA will have a free transformation over time and be finally stabilized in the B-form. In this work, molecular dynamics simulations have been performed on the A-DNA with sequence of CCCGGCCGGG, to elucidate the influence of ionic valence and temperature on the structural stability of A-DNA in salt solution. The results show that the structure of A-DNA is more stable in AlCl3 solution compared with MgCl2 and NaCl solution. To further discuss the influence of ionic valency, two kinds of virtual ions (monovalent X+ and trivalent Z3+) are created based on the properties of magnesium ions. From the simulations, the stability of A-DNA structure is better in high valent cationic salt solution. This has something to do with electrostatic interaction between metal ions and DNA chains. The electrostatic interaction force is gradually improved with the increasing of ionic valency, which is conducive to the formation of more compact DNA structure. Also, in the condition of same concentration and ionc valency, low temperature has a contribution to the stability of A-DNA structure.


Assuntos
DNA Forma A , Simulação de Dinâmica Molecular , Cátions , DNA/química , Cloreto de Sódio
6.
Acta Crystallogr D Struct Biol ; 78(Pt 6): 709-715, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35647918

RESUMO

The ease with which racemic mixtures crystallize compared with the equivalent chiral systems is routinely taken advantage of to produce crystals of small molecules. However, biological macromolecules such as DNA and proteins are naturally chiral, and thus the limited range of chiral space groups available hampers the crystallization of such molecules. Inspiring work over the past 15 years has shown that racemic mixtures of proteins, which were made possible by impressive advances in protein chemical synthesis, can indeed improve the success rate of protein crystallization experiments. More recently, the racemic crystallization approach was extended to include nucleic acids as a possible aid in the determination of enantiopure DNA crystal structures. Here, findings are reported that suggest that the benefits may extend beyond this. Two racemic crystal structures of the DNA sequence d(CCCGGG) are described which were found to fold into A-form DNA. This form differs from the Z-form DNA conformation adopted by the chiral equivalent in the solid state, suggesting that the use of racemates may also favour the emergence of new conformations. Importantly, the racemic mixture forms interactions in the solid state that differ from the chiral equivalent (including the formation of racemic pseudo-helices), suggesting that the use of racemic DNA mixtures could provide new possibilities for the design of precise self-assembled nanomaterials and nanostructures.


Assuntos
DNA Forma A , Cristalização , DNA/química , Modelos Moleculares , Proteínas , Estereoisomerismo
7.
Phys Rev E ; 103(4-1): 042409, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34005973

RESUMO

Double stranded DNA can adopt different forms, the so-called A-, B-, and Z-DNA, which play different biological roles. In this work, the thermodynamic and the kinetic parameters for the base-pair closing and opening in A-DNA and B-DNA were calculated by all-atom molecular dynamics simulations at different temperatures. The thermodynamic parameters of the base pair in B-DNA were in good agreement with the experimental results. The free energy barrier of breaking a single base stack results from the enthalpy increase ΔH caused by the disruption of hydrogen bonding and base-stacking interactions, as well as water and base interactions. The free energy barrier of base pair closing comes from the unfavorable entropy loss ΔS caused by the restriction of torsional angles and hydration. It was found that the enthalpy change ΔH and the entropy change ΔS for the base pair in A-DNA are much larger than those in B-DNA, and the transition rates between the opening and the closing state for the base pair in A-DNA are much slower than those in B-DNA. The large difference of the enthalpy and entropy change for forming the base pair in A-DNA and B-DNA results from different hydration in A-DNA and B-DNA. The hydration pattern observed around DNA is an accompanying process for forming the base pair, rather than a follow-up of the conformation.


Assuntos
DNA Forma A , DNA de Forma B , Pareamento de Bases , Simulação de Dinâmica Molecular , Termodinâmica
8.
Nucleic Acids Res ; 49(16): 9526-9538, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-33836081

RESUMO

The use of a small molecule compound to reduce toxic repeat RNA transcripts or their translated aberrant proteins to target repeat-expanded RNA/DNA with a G4C2 motif is a promising strategy to treat C9orf72-linked disorders. In this study, the crystal structures of DNA and RNA-DNA hybrid duplexes with the -GGGCCG- region as a G4C2 repeat motif were solved. Unusual groove widening and sharper bending of the G4C2 DNA duplex A-DNA conformation with B-form characteristics inside was observed. The G4C2 RNA-DNA hybrid duplex adopts a more typical rigid A form structure. Detailed structural analysis revealed that the G4C2 repeat motif of the DNA duplex exhibits a hydration shell and greater flexibility and serves as a 'hot-spot' for binding of the anthracene-based nickel complex, NiII(Chro)2 (Chro = Chromomycin A3). In addition to the original GGCC recognition site, NiII(Chro)2 has extended specificity and binds the flanked G:C base pairs of the GGCC core, resulting in minor groove contraction and straightening of the DNA backbone. We have also shown that Chro-metal complexes inhibit neuronal toxicity and suppresses locomotor deficits in a Drosophila model of C9orf72-associated ALS. The approach represents a new direction for drug discovery against ALS and FTD diseases by targeting G4C2 repeat motif DNA.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Proteína C9orf72/genética , DNA Forma A/ultraestrutura , Demência Frontotemporal/tratamento farmacológico , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Antracenos/química , Antracenos/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , DNA/efeitos dos fármacos , DNA/ultraestrutura , DNA Forma A/efeitos dos fármacos , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Conformação de Ácido Nucleico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia
9.
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
10.
J Phys Chem B ; 124(1): 38-49, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31805230

RESUMO

Accurate parametrization of force fields (FFs) is of ultimate importance for computer simulations to be reliable and to possess a predictive power. In this work, we analyzed, in multi-microsecond simulations of a 40-base-pair DNA fragment, the performance of four force fields, namely, the two recent major updates of CHARMM and two from the AMBER family. We focused on a description of double-helix DNA flexibility and dynamics both at atomistic and at mesoscale level in coarse-grained (CG) simulations. In addition to the traditional analysis of different base-pair and base-step parameters, we extended our analysis to investigate the ability of the force field to parametrize a CG DNA model by structure-based bottom-up coarse-graining, computing DNA persistence length as a function of ionic strength. Our simulations unambiguously showed that the CHARMM36 force field is unable to preserve DNA's structural stability at over-microsecond time scale. Both versions of the AMBER FF, parmbsc0 and parmbsc1, showed good agreement with experiment, with some bias of parmbsc0 parameters for intermediate A/B form DNA structures. The CHARMM27 force field provides stable atomistic trajectories and overall (among the considered force fields) the best fit to experimentally determined DNA flexibility parameters both at atomistic and at mesoscale level.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Cloreto de Sódio , Sequência de Bases , DNA Forma A/química , DNA de Forma B/química , Conformação de Ácido Nucleico , Cloreto de Sódio/química
11.
Nat Struct Mol Biol ; 26(11): 1013-1022, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659330

RESUMO

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.


Assuntos
Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Guanosina Trifosfato/química , Transposases/química , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Forma A/química , DNA de Forma B/química , Drosophila melanogaster/genética , Modelos Moleculares , Conformação Proteica
12.
Proc Natl Acad Sci U S A ; 116(45): 22591-22597, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636205

RESUMO

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.


Assuntos
Vírus de Archaea/fisiologia , Vírus de DNA/fisiologia , DNA Forma A/genética , DNA Viral/genética , Montagem de Vírus , Vírus de Archaea/genética , Vírus de Archaea/ultraestrutura , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Vírus de DNA/genética , Vírus de DNA/ultraestrutura , DNA Forma A/metabolismo , DNA Viral/metabolismo , Sulfolobus/virologia
13.
Nat Commun ; 10(1): 4818, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645548

RESUMO

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-HgII-T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-HgII-(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-HgII-(N4)C base pairs and greater A-form characteristics. Ten-fold different 1J coupling constants (15N,199Hg) are observed for (N3)C-HgII (114 Hz) versus (N4)C-HgII (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-HgII-T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding HgII, demonstrating that C-HgII-T has unique switching potential in DNA-based materials and devices.


Assuntos
DNA Forma A/ultraestrutura , DNA de Forma B/ultraestrutura , Mercúrio/química , Pareamento de Bases , Citosina , DNA/química , DNA/ultraestrutura , DNA Forma A/química , DNA de Forma B/química , Metais , Modelos Moleculares , Conformação de Ácido Nucleico , Espectroscopia de Prótons por Ressonância Magnética , Soluções , Timina
14.
J Chem Inf Model ; 59(5): 2324-2330, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-30767527

RESUMO

B- to A-DNA transition is known to be sensitive to the macroscopic properties of the solution, such as salt and ethanol concentrations. Microenvironmental effects on DNA conformational transition have been broadly studied. Providing an intuitive picture of how DNA responds to environmental changes is, however, still needed. Analyzing the chemical equilibrium of B-to-A DNA transition at critical concentrations, employing explicit-solvent simulations, is envisioned to help understand such microenvironmental effects. In the present study, free-energy calculations characterizing the B- to A-DNA transition and the distribution of cations were carried out in solvents with different ethanol concentrations. With the addition of ethanol, the most stable structure of DNA changes from the B- to A-form, in agreement with previous experimental observation. In 60% ethanol, a chemical equilibrium is found, showing reversible transition between B- and A-DNA. Analysis of the microenvironment around DNA suggests that with the increase of ethanol concentration, the cations exhibit a significant tendency to move toward the backbone, and mobility of water molecules around the major groove and backbone decreases gradually, leading eventually to a B-to-A transition. The present results provide a free-energy view of DNA microenvironment and of the role of cation motion in the conformational transition.


Assuntos
DNA Forma A/química , DNA de Forma B/química , Modelos Moleculares , Relação Dose-Resposta a Droga , Etanol/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos
15.
Methods ; 169: 11-20, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776405

RESUMO

Apart from being storage devices for genetic information, nucleic acids can provide regulatory structures through evolutionarily optimized sequences. The interaction of proteins binding specifically to such sequences and resulting secondary structures, or the exposure of single-stranded DNA add a versatile regulatory framework for cells. Biochemical and structural biology experiments have revealed important underlying concepts of protein-DNA interactions but are often limited by ensemble averaging or static information. To decipher the dynamics of conformations adopted by protein-DNA complexes, single-molecule approaches have become a powerful resource over the past two decades. In particular single-molecule FRET (smFRET), which allows a read-out of DNA or protein conformations, became widely used. Here, we illustrate how to implement the technique and exemplarily describe how smFRET yields insights into conformational changes of DNA secondary structures induced by the single-stranded DNA binding protein SSB. We further explain how we use smFRET to study mechanisms of the replication initiator DnaA and the competition of DnaA and SSB for single-stranded DNA. We anticipate that smFRET will further develop into a particularly useful technique to study dynamic competitions of proteins for the same DNA substrate.


Assuntos
DNA Forma A/química , DNA de Cadeia Simples/química , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Sequências Repetidas Invertidas , Cadeias de Markov , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Conformação Proteica
16.
Nucleic Acids Res ; 47(2): 546-558, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30517736

RESUMO

CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar-phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA 'seed' region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2'-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2'-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2'-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , RNA Bacteriano/química , Clivagem do DNA , DNA Forma A/química , RNA Bacteriano/metabolismo , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Relação Estrutura-Atividade
17.
J Phys Chem B ; 123(2): 457-467, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30576133

RESUMO

Using several computational tools such as density functional theory analysis, docking, and MD simulations, we performed a study on cis, trans-[Ru(II)(dmso)4Cl2] complexes, which have therapeutic potential as antimetastatic agents, and their association with DNA. Kohn-Sham energy decomposition analysis reveals that dmso ligands have much smaller interaction energies compared to the chlorido ligands, and their substitution by aquo ligands induces an extra stabilization of the other metal-ligand bonds. Once the complex is hydrolyzed, the aquo ligands have the weakest interactions to the metallic center and therefore are more labile for substitution by a DNA atom. Molecular docking and molecular dynamics were employed to understand the complex preassociation to DNA, pointing to a higher affinity of the hydrolyzed complexes, as well as showing spontaneous binding events during the simulations. Our results are consistent with the experimentally available data that suggest a mechanism in which the complexes are quickly hydrolyzed in solution, before forming cross-links with the DNA molecule. We present a set of methods that could be used to optimize these complexes computationally, aiding in the development of new drugs based on transition metals.


Assuntos
Complexos de Coordenação/metabolismo , DNA Forma A/metabolismo , Complexos de Coordenação/química , DNA Forma A/química , Teoria da Densidade Funcional , Hidrólise , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Rutênio/química , Estereoisomerismo , Termodinâmica
18.
Structure ; 26(12): 1645-1650.e3, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30244969

RESUMO

Nucleic acids can fold into well-defined 3D structures that help determine their function. Knowing precise nucleic acid structures can also be used for the design of nucleic acid-based therapeutics. However, locations of hydrogen atoms, which are key players of nucleic acid function, are normally not determined with X-ray crystallography. Accurate determination of hydrogen atom positions can provide indispensable information on protonation states, hydrogen bonding, and water architecture in nucleic acids. Here, we used neutron crystallography in combination with X-ray diffraction to obtain joint X-ray/neutron structures at both room and cryo temperatures of a self-complementary A-DNA oligonucleotide d[GTGG(CSe)CAC]2 containing 2'-SeCH3 modification on Cyt5 (CSe) at pH 5.6. We directly observed protonation of a backbone phosphate oxygen of Ade7 at room temperature. The proton is replaced with hydrated Mg2+ upon cooling the crystal to 100 K, indicating that metal binding is favored at low temperature, whereas proton binding is dominant at room temperature.


Assuntos
DNA Forma A/química , Fosfatos/metabolismo , Cristalografia por Raios X , DNA Forma A/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Difração de Nêutrons , Conformação de Ácido Nucleico , Prótons , Temperatura
19.
Langmuir ; 34(49): 15021-15027, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30160973

RESUMO

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.


Assuntos
DNA Forma A/química , Oligodesoxirribonucleotídeos/química , Estreptavidina/química , Animais , Biotina/química , Bovinos , DNA Forma A/genética , Vidro/química , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Proteínas Imobilizadas/química , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Soroalbumina Bovina/química , Propriedades de Superfície
20.
J Phys Chem B ; 122(33): 7990-7996, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30067905

RESUMO

In ordinary aqueous solution, B-DNA is the major structural form of DNA. After the addition of ethanol, DNA is thought to be aggregated/condensed in the A-form structure. However, there is uncertainty as to whether the B-to-A conformational change is connected to the aggregation/condensation steps. In this study, we performed all-atom molecular dynamics simulations and calculated the free-energy surface involved in the A/B conformational transition for isolated and aggregated Dickerson-Drew dodecamers (DDDs) in water and 85% ethanol environments. We found in the case of an isolated DDD, the overall free-energy profile is entirely downhill to give the B-DNA conformation in both water and 85% ethanol. However, in the aggregated state and 85% ethanol environment, there is a free-energy minimum associated with the A-DNA region in addition to the global B-DNA minimum, and there is a ∼3 kcal/mol free-energy barrier to the A-to-B conformational change. The molecular dynamics results suggest that aggregation of DNA is essential for forming A-DNA.


Assuntos
DNA Forma A/química , DNA de Forma B/química , Etanol/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Transição de Fase , Termodinâmica , Água/química
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