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1.
Phys Chem Chem Phys ; 22(5): 2999-3007, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957771

RESUMO

Infrared multiple photon dissociation (IRMPD) spectroscopy has been used to probe the structures of the three protonated base-pair mismatches containing 9-ethylguanine (9eG) in the gas phase. Computational chemistry has been used to determine the relative energies and compute the infrared spectra of these complexes. By comparing the IRMPD spectra with the computed spectra, in all cases, it was possible to deduce that what was observed experimentally were the lowest energy computed structures. The protonated complex between 9eG and 1-methylthymine (1mT) is protonated at N7 of 9eG-the most basic site of all three bases in this study-and bound in a Hoogsteen type structure with two hydrogen bonds. The experimental IRMPD spectrum for the protonated complex between 9eG and 9-methyladenine (9mA) is described as arising from a combination of the two lowest energy structures, both which are protonated at N1 of adenine and each containing two hydrogen bonds with 9eG being the acceptor of both. The protonated dimer of 9eG is protonated at N7 with an N7-H+-N7 ionic hydrogen bond. It also contains an interaction between a C-H of protonated guanine and the O6 carbonyl of neutral guanine which is manifested in a slight red shift of that carbonyl stretch. The protonated 9eG/9mA structures have been previously identified by X-ray crystallography in RNA and are contained within the protein database.


Assuntos
Gases/química , Guanina/análogos & derivados , Espectrofotometria Infravermelho , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Pareamento Incorreto de Bases , Cristalografia por Raios X , Guanina/química , Guanina/metabolismo , Ligações de Hidrogênio , Modelos Moleculares , Fótons , Timina/análogos & derivados , Timina/química , Timina/metabolismo
2.
Ann Lab Med ; 40(1): 27-32, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432636

RESUMO

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase ß-subunit gene sequence analysis. RESULTS: PCR amplification of gyrA and parC was successful for all A. baumannii strains. In 11 FQ -susceptible strains, the gyrA and parC PCR products were digested by the selected restriction enzymes at the site containing gyrA (codons 83 and 87) and parC (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in gyrA and parC were not digested by the restriction enzymes at the site containing the mutation. As for the non-baumannii Acinetobacter strains, although amplification products for gyrA were obtained for 28 strains, no parC amplification product was obtained for any strain. CONCLUSIONS: This assay specifically amplified gyrA and parC from A. baumannii and detected A. baumannii gyrA and parC mutations with FQ resistance.


Assuntos
Acinetobacter baumannii/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Reação em Cadeia da Polimerase/métodos , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Pareamento Incorreto de Bases , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Fluoroquinolonas/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA
3.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 10): 652-656, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31584014

RESUMO

The structure of a 22-base-pair RNA helix with mismatched pyrimidine base pairs is reported. The helix contains two symmetry-related CUG sequences: a triplet-repeat motif implicated in myotonic dystrophy type 1. The CUG repeat contains a U-U mismatch sandwiched between Watson-Crick pairs. Additionally, the center of the helix contains a dimerized UUCG motif with tandem pyrimidine (U-C/C-U) mismatches flanked by U-G wobble pairs. This region of the structure is significantly different from previously observed structures that share the same sequence and neighboring base pairs. The tandem pyrimidine mismatches are unusual and display sheared, cross-strand stacking geometries that locally constrict the helical width, a type of stacking previously associated with purines in internal loops. Thus, pyrimidine-rich regions of RNA have a high degree of structural diversity.


Assuntos
Pareamento Incorreto de Bases , Pirimidinas/química , RNA/química , Cristalografia por Raios X , Ligações de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico
4.
Chem Commun (Camb) ; 55(77): 11615-11618, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31501837

RESUMO

To date, implementation of renewable DNA circuits remains challenging due to issues including reactant depletion and waste accumulation. Herein we simultaneously addressed both issues through nicking enzyme-assisted waste-to-reactant transformation. As a proof-of-concept, a renewable entropy-driven catalytic DNA circuit was implemented, exhibiting a good renewability when replenishing fuel.


Assuntos
DNA Catalítico/química , Endonucleases/química , Redes Reguladoras de Genes , Pareamento Incorreto de Bases , Catálise , Entropia , Corantes Fluorescentes/química , Cinética , Conformação de Ácido Nucleico , Estudo de Prova de Conceito , Espectrometria de Fluorescência
5.
Nanoscale ; 11(37): 17206-17210, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31535117

RESUMO

Atomic force microscopy-based single-molecule-force spectroscopy is limited by low throughput. We introduce addressable DNA origami to study multiple target molecules. Six target DNAs that differed by only a single base-pair mismatch were clearly differentiated a rupture force of only 4 pN.


Assuntos
Pareamento Incorreto de Bases , DNA/química , DNA/genética , Microscopia de Força Atômica
6.
Chem Commun (Camb) ; 55(80): 12052-12055, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31535680

RESUMO

In this paper we report the kinetics based detection of single nucleotide variation (SNV) at room temperature by allele specific extension with different concentrations and types of crowding agents. In general, the crowding conditions enhanced the specificity in the detection of SNV.


Assuntos
DNA/genética , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , Bovinos , Dextranos/química , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Cinética , Polietilenoglicóis/química , Soroalbumina Bovina/química , Temperatura Ambiente
7.
J Phys Chem Lett ; 10(20): 6208-6212, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31560209

RESUMO

The Cas9 nuclease binds and cleaves DNA through its large-scale structural rearrangements. However, its unique property of not releasing the cleaved DNA has forbidden spectroscopic detection of the cleavage event. Here, we employ a novel fluorescence probe based on pyrene excimer emission to detect a minute structural change not detectable by other methods and demonstrate its applicability to spectroscopic tracking of the Cas9 nuclease activity in time. We show that the intensity of excimer emission depends sensitively on a subtle change in the structural environment of the target nucleic acid, which enables discrimination between cleaved and uncleaved nucleic acids within the DNA/Cas9/gRNA ternary complex. Kinetic parameters were obtained from the temporal evolution of the excimer emission, which revealed that DNA binding is hardly affected by PAM-distal mismatches, whereas the rate of cleavage by Cas9 decreases dramatically even with a 1-bp mismatch. Spectroscopic studies using the pyrene-based probe should be promising for biomolecular systems affected by subnm structural changes.


Assuntos
Proteína 9 Associada à CRISPR/química , DNA/química , Corantes Fluorescentes/química , Pirenos/química , Pareamento Incorreto de Bases , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Catálise , Clivagem do DNA , Fluorescência , RNA Guia/química , RNA Guia/genética
8.
Int J Mol Sci ; 20(17)2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31480444

RESUMO

The mismatch repair (MMR) pathway maintains genome integrity by correcting errors such as mismatched base pairs formed during DNA replication. In MMR, Msh2-Msh6, a heterodimeric protein, targets single base mismatches and small insertion/deletion loops for repair. By incorporating the fluorescent nucleoside base analog 6-methylisoxanthopterin (6-MI) at or adjacent to a mismatch site to probe the structural and dynamic elements of the mismatch, we address how Msh2-Msh6 recognizes these mismatches for repair within the context of matched DNA. Fluorescence quantum yield and rotational correlation time measurements indicate that local base dynamics linearly correlate with Saccharomyces cerevisiae Msh2-Msh6 binding affinity where the protein exhibits a higher affinity (KD ≤ 25 nM) for mismatches that have a significant amount of dynamic motion. Energy transfer measurements measuring global DNA bending find that mismatches that are both well and poorly recognized by Msh2-Msh6 experience the same amount of protein-induced bending. Finally, base-specific dynamics coupled with protein-induced blue shifts in peak emission strongly support the crystallographic model of directional binding, in which Phe 432 of Msh6 intercalates 3' of the mismatch. These results imply an important role for local base dynamics in the initial recognition step of MMR.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Pareamento Incorreto de Bases , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Modelos Moleculares , Proteína 2 Homóloga a MutS/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química
9.
Genes Dev ; 33(17-18): 1191-1207, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371435

RESUMO

The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.


Assuntos
Aminoácidos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Recombinases/química , Recombinases/metabolismo , Recombinação Genética/genética , Aminoácidos/genética , Animais , Pareamento Incorreto de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Mutação , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Anal Chim Acta ; 1079: 139-145, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387704

RESUMO

A short G-rich sequence with three G-tracts can form a G-triplex (G3), a recently identified noncanonical DNA structure. Until now, very limited functional study and application of G3 is reported. Herein, we integrated G3 with isothermal exponential amplification reaction (EXPAR) for achieving simple and sensitive biosensing strategy. In this strategy, the cascade EXPAR cycles produces larger numbers of short G-rich sequences, which self-assemble into G3 structure and then bind hemin to form G3/hemin DNAzyme. This G3/hemin DNAzyme-based EXPAR strategy could detect as low as 4.7 fM target DNA with colorimetric detection, and the sensitivity of G3/hemin DNAzyme-based EXPAR strategy was much higher than that of the conventional G-quadruplex/hemin DNAzyme-based EXPAR strategy. We explored the reason for higher sensitivity of G3/hemin DNAzyme-based EXPAR strategy. The experimental results demonstrated that G3/hemin DNAzyme is an ideal signal generator for EXPAR-based biosensing platform. This work opens a new avenue to develop effective signal amplification strategy for ultrasensitive biosensing. This work is also helpful for a deeper understanding of G3 structure and the future application of G3.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , DNA Catalítico/química , DNA Viral/análise , DNA/química , Hemina/química , Pareamento Incorreto de Bases , Benzotiazóis/química , DNA/genética , DNA Catalítico/genética , DNA Viral/genética , HIV/genética , Peróxido de Hidrogênio/química , Indicadores e Reagentes/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ácidos Sulfônicos/química
11.
Chem Commun (Camb) ; 55(69): 10300-10303, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31397452

RESUMO

Shorter DNA probes provide better specificity for hybridization, but they may not form stable duplexes at room temperature. In this study, we used thiazole orange to follow DNA hybridization upon freezing and achieved stable 5-mer duplex DNA. Using multiple short probes in tandem, long DNA could also be studied. This study provides insights into DNA hybridization in the frozen state and expands the application of freezing for nucleic acid chemistry.


Assuntos
DNA/química , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Pareamento Incorreto de Bases , Sequência de Bases , Benzotiazóis/análise , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/análise , Congelamento , Oligonucleotídeos/genética , Quinolinas/análise , Espectrometria de Fluorescência
12.
Phys Rev Lett ; 122(21): 218101, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31283336

RESUMO

Base-pair mismatch can relieve mechanical stress in highly strained DNA molecules, but how it affects their kinetic stability is not known. Using single-molecule fluorescence resonance energy transfer, we measured the lifetimes of tightly bent DNA loops with and without base-pair mismatch. Surprisingly, for loops captured by stackable sticky ends which leave single-stranded DNA breaks (or nicks) upon annealing, the mismatch decreased the loop lifetime despite reducing the overall bending stress, and the decrease was largest when the mismatch was placed at the DNA midpoint. These findings suggest that base-pair mismatch increases bending stress at the opposite side of the loop through an allosteric mechanism known as cooperative kinking. Based on this mechanism, we present a three-state model that explains the apparent dichotomy between thermodynamic and kinetic stability.


Assuntos
Pareamento Incorreto de Bases , DNA/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Químicos , Conformação de Ácido Nucleico , Termodinâmica
13.
Nucleic Acids Res ; 47(16): 8899-8912, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31361900

RESUMO

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.


Assuntos
Antibióticos Antineoplásicos/química , Cromomicina A3/química , DNA Forma Z/química , Dactinomicina/química , Substâncias Intercalantes/química , Oligodesoxirribonucleotídeos/química , Antibióticos Antineoplásicos/metabolismo , Pareamento Incorreto de Bases , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cromomicina A3/metabolismo , Cristalização , Cristalografia por Raios X , DNA Forma Z/metabolismo , Dactinomicina/metabolismo , Humanos , Substâncias Intercalantes/metabolismo , Modelos Moleculares , Oligodesoxirribonucleotídeos/síntese química , Soluções
14.
Chem Commun (Camb) ; 55(52): 7514-7517, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31187833

RESUMO

A stem-loop clutch probe (SLCP)-based strategy has been explored to guide sequence-specific double stranded DNA (dsDNA) analysis with enhanced single-base mismatch selectivity. This assay method can also modulate the dynamic range by employing natural processes relying on distal-site mutation inhibition and allosteric activation.


Assuntos
Sondas de DNA/metabolismo , DNA/análise , Pareamento Incorreto de Bases , Técnicas Biossensoriais , DNA/química , DNA/metabolismo , Sondas de DNA/química , Corantes Fluorescentes/química , Limite de Detecção , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico
15.
World J Microbiol Biotechnol ; 35(6): 95, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187258

RESUMO

Recombinase polymerase amplification (RPA) is an isothermal amplification technique. Because of its short detection cycle and high specificity, it has been applied in various fields. However, the design of probe on the efficiency of RPA is not well understood and the effect of sequence mismatches of oligonucleotides on the performance of RPA is rarely discussed. In this study, we found that different primers with the same probe have a slight effect on the efficiency of fluorescent RPA, and different probes with the same amplified region have a great influence on the efficiency of fluorescent RPA. We summarized the design rules of probes suitable for fluorescent RPA by analyzing the experimental data. The rule is that the best distance between fluorescent groups in the probe is 1-2 bases, and the G content should be reduced as far as possible. In addition, we verified this rule by designing a series of probes. Furthermore, we found the base mismatches of the probe had a significant effect on RPA, which can lead to false positives and can change the amplification efficiency. However, 1-3 mismatches covering the center of the primer sequence only affect the amplification efficiency of RPA, not its specificity. And with an increase in the number of primer mismatches, the efficiency of RPA will decrease accordingly. This study suggests that the efficiency of fluorescent RPA is closely related to the probe. We recommend that when designing a fluorescent probe, one must consider the presence of closely related non-targets and specific bases.


Assuntos
Pareamento Incorreto de Bases , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases , Bactérias , Primers do DNA/genética , Sensibilidade e Especificidade
16.
J Vet Sci ; 20(3): e23, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31161741

RESUMO

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Ácidos Nucleicos Heteroduplexes/genética , RNA Guia/genética , Animais , Pareamento Incorreto de Bases/genética , Linhagem Celular , Edição de Genes/normas , Genes erbB-1/genética , Ácidos Nucleicos Heteroduplexes/química , RNA Guia/química , Suínos
17.
Colloids Surf B Biointerfaces ; 181: 333-340, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31154144

RESUMO

Rapid and sensitive single nucleotide polymorphisms (SNPs) genotyping is of particular important for early diagnosis, prevention, and treatment of specific human diseases. A simple and low-cost SNP detection method would be valuable for routine analysis in resource-limited settings. Here, we demonstrated a novel and convenient gold nanoparticle (AuNPs) based colorimetric approach for efficient screening of SNPs at room temperature without instrumentation. SNP detection is performed in a single tube with one set of unmodified AuNPs, a label-free peptide nucleic acid (PNA) probe, a single exonuclease (S1 nuclease), and the target to be tested. S1 nuclease could digest DNAs in DNA/PNA duplexes involving a mismatch into small fragments, while DNAs in the fully-matched DNA/PNA duplexes can be effectively protected by PNA from enzymatic degradation. This difference could be easily discriminated by color changes associated with gold aggregation. PNA oligomers can induce immediate AuNP aggregation even in the presence of nucleoside monophosphates (dNMPs), the digestion products of DNA. Whereas PNA/DNA duplexes can effectively stabilize unmodified AuNPs, and the stabilization effect of PNA/DNA is better than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Without the need of precise temperature control and extra salt addition, SNPs are detected with a detection limit of 2.3 nM in cell lysate. Moreover, this system can effectively discriminate a range of different mismatches even in spiked cell lysate, demonstrate the potential use of this biosensor for biological samples.


Assuntos
Técnicas Biossensoriais , Colorimetria , DNA/química , Ouro/química , Nanopartículas Metálicas/química , Ácidos Nucleicos Peptídicos/química , Pareamento Incorreto de Bases , Linhagem Celular Tumoral , DNA/genética , Humanos , Ácidos Nucleicos Peptídicos/genética , Polimorfismo de Nucleotídeo Único/genética
18.
Phys Chem Chem Phys ; 21(27): 14923-14940, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31233058

RESUMO

Base flipping is widely observed in a number of important biological processes. The genetic codes deposited inside the DNA duplex become accessible to external agents upon base flipping. The sulfur substitution of guanine leads to thioguanine, which alters the thermodynamic stability of the GC base pairs and the GT mismatches. Experimental studies conclude that the sulfur substitution decreases the lifetime of the GC base pair. In this work, under three AMBER force fields for nucleotide systems, we firstly performed equilibrium and nonequilibrium free energy simulations to investigate the variation of the thermodynamic profiles in base flipping upon sulfur substitution. It is found that the bsc0 modification, the bsc1 modification and the OL15 modification of AMBER force fields are able to qualitatively describe the sulfur-substitution dependent behavior of the thermodynamics. However, only the two last-generation AMBER force fields are able to provide quantitatively correct predictions. The second computational study on the sulfur substitutions focused on the relative stability of the S6G-C base pair and the S6G-T mismatch. Two conflicting experimental observations were reported by the same authors. One suggested that the S6G-C base pair was more stable, while the other concludes that the S6G-T mismatch was more stable. We answered this question by constructing the free energy profiles along the base flipping pathway computationally.


Assuntos
Pareamento Incorreto de Bases , DNA/química , Enxofre/química , Pareamento de Bases , Termodinâmica
19.
Talanta ; 201: 358-363, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122435

RESUMO

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5'-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.


Assuntos
Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , Citidina Monofosfato/sangue , Sondas de DNA/química , DNA/química , Sequência de Bases , Citidina Monofosfato/genética , DNA/genética , Sondas de DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico
20.
Methods Appl Fluoresc ; 7(3): 035006, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31042679

RESUMO

Due to the concern over food safety, it is important to detect the pesticides residues in agricultural products. Here, a highly sensitive and low background fluorescent strategy for the detection of pesticides residues has been developed. The fluorescence intensity of N-methyl mesoporphyrin IX (NMM) binding G-quadruplex could be turn off because of inhibiting effect of the pesticides on the acetylcholinesterase (AChE) activity. For that, four single-stranded DNAs (named linker, trigger, H1 and H2, respectively) are rational designed and T-Hg-T mismatches duplex DNAs as a recognizer combined with the separation of magnetic beads. The design of hybridization chain reaction (HCR) amplification strategy assisted by magnetic separation has been adopted to improve the detection sensitivity. In the presence of pesticides, the amount of the thiol group generated by hydrolysis reaction of acetylcholine (ACh) is reduced, lead to release of less trigger DNA. Therefor subsequent HCR process is retarded with decreased fluorescence intensity. The reduced fluorescence intensity has a quantitative relationship with the pesticide concentration. The limit of detection of chlorpyrifos was estimated to be 2.0 ng ml-1. It has been applied to detect the pesticides residues in real samples.


Assuntos
Contaminação de Alimentos/análise , Resíduos de Praguicidas/análise , Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , Clorpirifos/análise , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Quadruplex G , Gengibre/química , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Fenômenos Magnéticos , Malus/química , Mercúrio/química , Mesoporfirinas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodos
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