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1.
Phys Rev Lett ; 127(13): 138103, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34623840

RESUMO

Nanopores in 2D materials are highly desirable for DNA sequencing, yet achieving single-stranded DNA (ssDNA) transport through them is challenging. Using density functional theory calculations and molecular dynamics simulations we show that ssDNA transport through a pore in monolayer hexagonal boron nitride (h-BN) is marked by a basic nanomechanical conflict. It arises from the notably inhomogeneous flexural rigidity of ssDNA and causes high friction via transient DNA desorption costs exacerbated by solvation effects. For a similarly sized pore in bilayer h-BN, its self-passivated atomically smooth edge enables continuous ssDNA transport. Our findings shed light on the fundamental physics of biopolymer transport through pores in 2D materials.


Assuntos
Compostos de Boro/química , DNA de Cadeia Simples/química , Nanoestruturas/química , Fenômenos Biofísicos , Modelos Químicos , Simulação de Dinâmica Molecular , Nanoporos
2.
J Phys Chem Lett ; 12(37): 9132-9141, 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34523927

RESUMO

Different nucleotides generate specific ionic currents that discriminate between the nucleotides while they are passing through the nanopore constriction. MspA is a commonly used nanopore for DNA sequencing. However, the reasons of the current variation remain ambiguous. Our work unveils the microscopic mechanism of current variation for an ssDNA passing through the MspA nanopore by all-atom molecular dynamic simulations. Besides the physical rigidity and dimensions of the nucleotides, nucleotide orientation is observed to induce nonignorable current variation. Besides the generally considered MspA nanopore constriction, it is also found that the region below constriction could be used to detect and differentiate single nucleotides when the single-stranded DNA translocates in the form of base-constriction-base meshing and ratcheting across the nanopore constriction compared to other regions. The work provides a novel insight into facilitating the development of low-cost and high-throughput nanopore DNA sequencing.


Assuntos
Nanoporos , Porinas/química , Análise de Sequência de DNA/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Simulação de Dinâmica Molecular , Mycobacterium smegmatis/metabolismo , Porinas/metabolismo
3.
Nature ; 597(7876): 426-429, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471288

RESUMO

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Biológicos , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Homologia de Sequência do Ácido Nucleico , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Fatores de Tempo
4.
Chem Commun (Camb) ; 57(71): 8977-8980, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34486619

RESUMO

This study reports a photoelectrochemical biosensor for dopamine-loaded liposome-encoded magnetic beads cleaved by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas 12a system for the quantification of human papilloma virus (HPV)-related DNA using neodymium-doped BiOBr nanosheets (Nd-BiOBr) as a photoactive matrix. Magnetic beads and dopamine-loaded liposomes are covalently attached to the both ends of ssDNA to construct dumbbell-shaped dopamine-loaded liposome-encoded magnetic bead (DLL-MB) probes. When the guide RNA binds to the target HPV-16, the ssDNA will be cleaved by Cas12a, thereby degrading the double dumbbell probes. After magnetic separation, the dissolved DLLs are treated with Triton X-100 to release the dopamine (as an electron donor), which was then detected by an amplified photocurrent using the Nd-BiOBr-based photoelectrode.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , DNA Viral/análise , Endodesoxirribonucleases/química , DNA de Cadeia Simples/química , Dopamina/química , Técnicas Eletroquímicas/métodos , Fluoresceínas/química , Corantes Fluorescentes/química , Papillomavirus Humano 16/química , Lipossomos/química , Fenômenos Magnéticos , Processos Fotoquímicos
5.
Nat Commun ; 12(1): 5545, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545070

RESUMO

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Nucleotídeos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Homologia de Sequência de Aminoácidos , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , DNA de Cadeia Simples/metabolismo , Fluorescência , Interferometria , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Especificidade da Espécie
6.
J Chem Phys ; 155(9): 094305, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34496579

RESUMO

DNA strands are polymeric ligands that both protect and tune molecular-sized silver cluster chromophores. We studied single-stranded DNA C4AC4TC3XT4 with X = guanosine and inosine that form a green fluorescent Ag10 6+ cluster, but these two hosts are distinguished by their binding sites and the brightness of their Ag10 6+ adducts. The nucleobase subunits in these oligomers collectively coordinate this cluster, and fs time-resolved infrared spectra previously identified one point of contact between the C2-NH2 of the X = guanosine, an interaction that is precluded for inosine. Furthermore, this single nucleobase controls the cluster fluorescence as the X = guanosine complex is ∼2.5× dimmer. We discuss the electronic relaxation in these two complexes using transient absorption spectroscopy in the time window 200 fs-400 µs. Three prominent features emerged: a ground state bleach, an excited state absorption, and a stimulated emission. Stimulated emission at the earliest delay time (200 fs) suggests that the emissive state is populated promptly following photoexcitation. Concurrently, the excited state decays and the ground state recovers, and these changes are ∼2× faster for the X = guanosine compared to the X = inosine cluster, paralleling their brightness difference. In contrast to similar radiative decay rates, the nonradiative decay rate is 7× higher with the X = guanosine vs inosine strand. A minor decay channel via a dark state is discussed. The possible correlation between the nonradiative decay and selective coordination with the X = guanosine/inosine suggests that specific nucleobase subunits within a DNA strand can modulate cluster-ligand interactions and, in turn, cluster brightness.


Assuntos
DNA de Cadeia Simples/química , Guanosina/química , Inosina/química , Prata/química , Sítios de Ligação , Fluorescência
7.
Talanta ; 235: 122749, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517617

RESUMO

Signal output mode is the important part of biosensor. In general, "signal on" and "signal off" are two common output modes. The development of dual signals-based ratio analysis as a powerful diagnostic tool has attracted widespread attention in the biosensor field in recent years. Dual signals ratio sensors with "signal on" and "signal off" are more favored because of their low background signal and better sensitivity and selectivity. In this study, inspired by the idea that EcoR V can cut specific sites of DNA to produce two corresponding fragments, and by using the capturing probe as guy wires, a reliable and sensitive method for EcoR V assay is developed based on the ratio of dual chemiluminescence (CL) signals for the first time. In particular, in the existence of the objective EcoR V, the substrate DNA would be degraded into two double stranded oligonucleotides with blunt ends which include the sequence I and the sequence II, then they can separately compete with two different corresponding capture probes on magnetic beads (MBs). One of capture probe hybridized with the sequence I containing more guanine (G) bases that reacted with the phenylglyoxal (PG) to produce chemical reaction which triggered a positive CL signal output I + CL as "signal-on"; another capture probe is priority to hybridize the sequence II, which triggered the weaker reporter DNA linked with horseradish peroxidase (HRP) probe to fall off the MBs, thereby outputting a negative CL signal I-CL as "signal-off". By comparing the linear relation and the correlation coefficient, the I-CL/I + CL ratio method has better linear relation (0.01-10 U/mL) and higher sensitivity (0.0045 U/mL). In addition, this developed strategy of high selectivity which can directly detect low concentration of target EcoR V in human serum, and thus this dual ratio biosensor might offer a promising detection approach for clinical diagnostics.


Assuntos
Técnicas Biossensoriais , DNA de Cadeia Simples , DNA/genética , DNA de Cadeia Simples/genética , Peroxidase do Rábano Silvestre , Humanos , Luminescência
8.
Talanta ; 235: 122763, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517624

RESUMO

The disease diagnosis by detecting single microRNAs (miRNAs) can produce high false positive rate. Herein, a novel fluorescence biosensor method for one-step simultaneous detection of multiple miRNAs was proposed by using single-stranded DNA (ssDNA) functionalized double quantum dots (QDs) and black hole quencher (BHQ)-decorated magnetic nanobeads (MNs). MNs were linked with two black hole quenchers (BHQ1 and BHQ3) via a complementary DNA (cDNA). The ssDNA/cDNA hybridization contributed to the fluorescence quenching of double QDs due to the fluorescence resonance energy transfer (FRET) between double QDs and BHQ. In the presence of target miRNA-33 (miR-33) and miRNA-125b (miR-125b), the ssDNA1 and ssDNA2 were respectively hybridized with miR-33 and miR-125b to form more stable duplexes. Thus, the double QDs were released into supernatant after the magnetic separation, leading to the fluorescence signals recovery at 537 nm and 647 nm. A wide linear range (0.5 nM-320 nM for miR-33 and 0.1 nM-250 nM for miR-125b) and low limits of detection (0.09 nM for miR-33 and 0.02 nM for miR-125b) were achieved. Moreover, our approach has been demonstrated to simultaneously detect miR-33 and miR-125b in cell extracts. With advantages of high sensitivity, strong specificity, low background and low cost, the strategies show great potentials for the detection of various targets in bioanalysis and disease diagnosis.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Pontos Quânticos , DNA de Cadeia Simples/genética , Transferência Ressonante de Energia de Fluorescência , MicroRNAs/genética , Hibridização de Ácido Nucleico
9.
Talanta ; 235: 122783, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517641

RESUMO

As the light-harvesting "antenna", G-rich oligonucleotides (such as the G-quadruplex) can interact with lanthanide (III) to bring a luminescent enhancement response. In this study, phenomenon of luminescent enhancement of G-triplex/terbium (III) (G3/Tb3+) and interaction between G3 and Tb3+ were first reported and characterized. Based on G3/Tb3+ luminescence, a label-free aptasensor for the detection of ofloxacin (OFL) residues in the food was developed. The OFL triggered the action of rolling circle amplification (RCA) allowed for the amplification product of G3-forming sequences in the single-stranded DNA, which promoted the conformational transition of the G3/Tb3+ complexes once the addition of Tb3+. Under the optimal conditions, the logarithmic correlation between the G3/Tb3+ luminescence intensity and the concentration of OFL was found to be linear in the range of 5-1000 pmol L-1 (R2 = 0.9949). The limit of detection was 0.18 pmol L-1 (3σ/slope). Additionally, the good recoveries of 90.19-108.89 % and the relative standard deviations values of 0.59-5.87 % were obtained in the application of the aptasensor detecting OFL in the practical samples. These results confirmed that the present aptasensor has a good analytical performance and bright prospect for detecting ofloxacin residues in food.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA de Cadeia Simples , Limite de Detecção , Luminescência , Ofloxacino , Térbio
10.
Biomolecules ; 11(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34439828

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.


Assuntos
Doenças Autoimunes/terapia , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Metilação de DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Engenharia Genética/métodos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Guia/genética , RNA Guia/metabolismo
11.
J Phys Chem B ; 125(33): 9426-9440, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34379430

RESUMO

Thermally driven conformational fluctuations (or "breathing") of DNA play important roles in the function and regulation of the "macromolecular machinery of genome expression." Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here, we use microsecond-resolved single-molecule Förster resonance energy transfer (smFRET) experiments to investigate the backbone fluctuations of short [oligo(dT)n] templates within DNA constructs that also serve as models for ss-dsDNA junctions. Such junctions, together with the attached ssDNA sequences, are involved in interactions with the ssDNA binding (ssb) proteins that control and integrate the functions of DNA replication complexes. We analyze these data using a chemical network model based on multiorder time-correlation functions and probability distribution functions that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs interconvert, on submillisecond time scales, between three macrostates with distinctly different end-to-end distances. These are (i) a "compact" macrostate that represents the dominant species at equilibrium; (ii) a "partially extended" macrostate that exists as minority species; and (iii) a "highly extended" macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA-associating proteins.


Assuntos
DNA de Cadeia Simples , Transferência Ressonante de Energia de Fluorescência , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica
12.
Biosens Bioelectron ; 194: 113587, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34455224

RESUMO

To realize the full potential of the CRISPR/Cas system and expand its applicability up to the detection of molecular interactions, we herein describe a novel method to identify protein/small molecule interactions by utilizing the CRISPR/Cas12a collateral cleavage activity. This technique employs a single-stranded activator DNA modified with a specific small molecule, which would switch on the CRISPR/Cas12a collateral cleavage activity upon binding to crRNA within the CRISPR/Cas12a system. When the target protein binds to the small molecule on the activator DNA, the bound protein sterically hinders the access of the activator DNA to crRNA, thereby promoting less collateral cleavage activity of CRISPR/Cas12a. As a consequence, fewer reporter probes nearby are cleaved to produce accordingly reduced fluorescence signals in response to target protein. Based on this unique design principle, the two model protein/small molecule interactions, streptavidin/biotin and anti-digoxigenin/digoxigenin, were successfully determined down to 0.03 nM and 0.09 nM, respectively, with a fast and simple detection workflow (11 min). The practical applicability of this method was also verified by reliably detecting target streptavidin spiked in heterogeneous human serum. This work would provide great insight to construct novel strategies to identify protein/small molecule interaction by making the most of the CRISPR/Cas12a system beyond its superior capabilities in genome editing and molecular diagnostics.


Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Clivagem do DNA , DNA de Cadeia Simples , Edição de Genes , Humanos
13.
Anal Chim Acta ; 1176: 338755, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34399899

RESUMO

The extraordinary genome-editing tool CRISPR/Cas12a has also been utilized as a powerful sensing technology owing to its highly-specificity and isothermal signal amplification. Nevertheless, the widespread application of Cas12a-based sensing methods in nucleic acid detection is limited by the targeting range and high undesired background. Herein, we established a universal Cas12a-based nucleic acid sensing strategy by using proximity extension and transcription-unleashed self-suppling of crRNA. The target was recognized and bound to a pair of adjacent probes, and then triggered the proximity-induced primer extension and transcription amplification to produce numerous crRNAs. The amplified abundant crRNAs assembled with Cas12a and dsDNA activators containing PAM to form a ternary complex, which trans-cleaved ssDNA-FQ reporters continuously to generate a strong fluorescent signal. Thus, the cascade enzymatic amplification was performed and subsequently applied for detecting target DNA down to 41.7 amol with a low nonspecific background. The application of this strategy in RNA detection has also been demonstrated, and it is expected to provide a universal and sensitive sensing platform for molecular diagnosis applications.


Assuntos
Sistemas CRISPR-Cas , Ácidos Nucleicos , DNA/genética , DNA de Cadeia Simples
14.
Nat Commun ; 12(1): 4843, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376693

RESUMO

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Repetições de Microssatélites/genética , Proteínas de Ligação a Telômeros/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Reparo do DNA por Junção de Extremidades , DNA Polimerase I/genética , DNA Primase/genética , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
15.
Nucleic Acids Res ; 49(14): 8110-8119, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34255844

RESUMO

Shelterin is a six-protein complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1 and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Schizosaccharomyces pombe/genética , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Cromossomos/genética , DNA/genética , DNA de Cadeia Simples/genética , Humanos , Cinética , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Mutação , Schizosaccharomyces/genética , Telômero/genética , Proteínas de Ligação a Telômeros/ultraestrutura
16.
Nucleic Acids Res ; 49(14): 7870-7883, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34283224

RESUMO

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.


Assuntos
Compostos Azo/metabolismo , Atrofia Muscular Espinal/genética , Pirimidinas/metabolismo , Precursores de RNA/genética , Splicing de RNA , Compostos Azo/química , Compostos Azo/uso terapêutico , Sequência de Bases , Sítios de Ligação/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Éxons/genética , Cinética , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Estrutura Molecular , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/metabolismo , Mutação , Fármacos Neuromusculares/química , Fármacos Neuromusculares/metabolismo , Fármacos Neuromusculares/uso terapêutico , Conformação de Ácido Nucleico , Pirimidinas/química , Pirimidinas/uso terapêutico , Precursores de RNA/química , Precursores de RNA/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética
17.
Molecules ; 26(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299520

RESUMO

Layers formed from single-stranded DNA on nanostructured plasmonic metals can be applied as "working elements" in surface-enhanced Raman scattering (SERS) sensors used to sensitively and accurately identify specific DNA fragments in various biological samples (for example, in samples of blood). Therefore, the proper formation of the desired DNA layers on SERS substrates is of great practical importance, and many research groups are working to improve the process in forming such structures. In this work, we propose two modifications of a standard method used for depositing DNA with an attached linking thiol moiety on certain SERS-active structures; the modifications yield DNA layers that generate a stronger SERS signal. We propose: (i) freezing the sample when forming DNA layers on the nanoparticles, and (ii) when forming DNA layers on SERS-active macroscopic silver substrates, using ω-substituted alkanethiols with very short alkane chains (such as cysteamine or mercaptopropionic acid) to backfill the empty spaces on the metal surface unoccupied by DNA. When 6-mercapto-1-hexanol is used to fill the unoccupied places on a silver surface (as in experiments on standard gold substrates), a quick detachment of chemisorbed DNA from the silver surface is observed. Whereas, using ω-substituted alkanethiols with a shorter alkane chain makes it possible to easily form mixed DNA/backfilling thiol monolayers. Probably, the significantly lower desorption rate of the thiolated DNA induced by alkanethiols with shorter chains is due to the lower stabilization energy in monolayers formed from such compounds.


Assuntos
DNA de Cadeia Simples/química , Ouro/química , Prata/química , Hexanóis/química , Nanopartículas Metálicas/química , Análise Espectral Raman , Compostos de Sulfidrila/química , Propriedades de Superfície
18.
Arch Virol ; 166(10): 2911-2926, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34331585

RESUMO

The family Genomoviridae (phylum Cressdnaviricota, class Repensiviricetes, order Geplafuvirales) includes viruses with circular single-stranded DNA genomes encoding two proteins, the capsid protein and the rolling-circle replication initiation protein. The genomes of the vast majority of members in this family have been sequenced directly from diverse environmental or animal- and plant-associated samples, but two genomoviruses have been identified infecting fungi. Since the last taxonomic update of the Genomoviridae, a number of new members of this family have been sequenced. Here, we report on the most recent taxonomic update, including the creation of one new genus, Gemytripvirus, and classification of ~420 new genomoviruses into 164 new species. We also announce the adoption of the "Genus + freeform epithet" binomial system for the naming of all 236 officially recognized species in the family Genomoviridae. The updated taxonomy presented in this article has been accepted by the International Committee on Taxonomy of Viruses (ICTV).


Assuntos
Vírus de DNA/classificação , Animais , Sequência de Bases , Vírus de DNA/genética , Vírus de DNA/fisiologia , DNA Circular , DNA de Cadeia Simples , Genoma Viral/genética , Especificidade de Hospedeiro , Filogenia , Terminologia como Assunto , Proteínas Virais/genética
19.
Chem Commun (Camb) ; 57(65): 8039-8042, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34291259

RESUMO

Two-dimensional (2D) hexagonal boron nitride nanosheet (h-BNNS) is proposed as an effective nanoquencher for fluorescence detection of biocompatible microRNA. Compared with bulk hexagonal boron nitride (h-BN), the exfoliated ultrathin nanosheet has a narrow band gap and increased conductivity, thus enabling fast electron transfer with this electron acceptor for more effective fluorescence detection of microRNA. Remarkably, using the nanoprobe consisting of h-BNNS and FAM dye-labeled ssDNA, a low detection limit of 2.39 nM is achieved and a rapid fluorescence response is observed compared with previously reported fluorescence sensing materials. More importantly, this sensing system could also distinguish base-mismatched microRNA, suggesting that the proposed sensing platform held excellent selectivity and great promise for application in the detection of nucleotide polymorphism. This work will benefit microRNA-related fundamental research and disease diagnosis.


Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , MicroRNAs/análise , Nanoestruturas/química , Pareamento Incorreto de Bases , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Fluoresceínas/química , Fluorescência , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência
20.
Molecules ; 26(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34279383

RESUMO

Silver has a long history of antibacterial effectiveness. The combination of atomically precise metal nanoclusters with the field of nucleic acid nanotechnology has given rise to DNA-templated silver nanoclusters (DNA-AgNCs) which can be engineered with reproducible and unique fluorescent properties and antibacterial activity. Furthermore, cytosine-rich single-stranded DNA oligonucleotides designed to fold into hairpin structures improve the stability of AgNCs and additionally modulate their antibacterial properties and the quality of observed fluorescent signals. In this work, we characterize the sequence-specific fluorescence and composition of four representative DNA-AgNCs, compare their corresponding antibacterial effectiveness at different pH, and assess cytotoxicity to several mammalian cell lines.


Assuntos
Antibacterianos/química , DNA de Cadeia Simples/química , Nanopartículas Metálicas/química , Prata/química , Antibacterianos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fluorescência , Humanos , Nanopartículas Metálicas/toxicidade , Células THP-1
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