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1.
Nucleic Acids Res ; 47(12): e68, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-30918971

RESUMO

We report an approach for visualizing DNA sequence and using these 'DNA barcodes' to search complex mixtures of genomic material for DNA molecules of interest. We demonstrate three applications of this methodology; identifying specific molecules of interest from a dataset containing gigabasepairs of genome; identification of a bacterium from such a dataset and, finally, by locating infecting virus molecules in a background of human genomic material. As a result of the dense fluorescent labelling of the DNA, individual barcodes of the order 40 kb pairs in length can be reliably identified. This means DNA can be prepared for imaging using standard handling and purification techniques. The recorded dataset provides stable physical and electronic records of the total genomic content of a sample that can be readily searched for a molecule or region of interest.


Assuntos
DNA/química , Genômica/métodos , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Bacteriófago lambda/genética , Sequência de Bases , Sistemas CRISPR-Cas , Simulação por Computador , DNA Bacteriano/química , DNA Viral/química , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Corantes Fluorescentes , Humanos , Klebsiella pneumoniae/genética
2.
Phys Chem Chem Phys ; 18(21): 14691-700, 2016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27186599

RESUMO

The fluorescence properties of dinucleotides incorporating 2-aminopurine (2AP) suggest that the simplest oligonucleotides adopt conformations similar to those found in duplex DNA. However, there is a lack of structural data for these systems. We report a density functional theory (DFT) study of the structures of 2AP-containing dinucleotides (deoxydinucleoside monophosphates), including full geometry optimisation of the sugar-phosphate backbone. Our DFT calculations employ the M06-2X functional for reliable treatment of dispersion interactions and include implicit aqueous solvation. Dinucleotides with 2AP in the 5'-position and each of the natural bases in the 3'-position are examined, together with the analogous 5'-adenine-containing systems. Computed structures are compared in detail with typical B-DNA base-step parameters, backbone torsional angles and sugar pucker, derived from crystallographic data. We find that 2AP-containing dinucleotides adopt structures that closely conform to B-DNA in all characteristic parameters. The structures of 2AP-containing dinucleotides closely resemble those of their adenine-containing counterparts, demonstrating the fidelity of 2AP as a mimic of the natural base. As a first step towards exploring the conformational heterogeneity of dinucleotides, we also characterise an imperfectly stacked conformation and one in which the bases are completely unstacked.


Assuntos
2-Aminopurina/química , DNA de Forma B/química , Oligonucleotídeos/química , Fosfatos de Dinucleosídeos/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Termodinâmica
3.
Biochemistry ; 52(10): 1677-85, 2013 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-23409782

RESUMO

A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.


Assuntos
Metilação de DNA/fisiologia , DNA-Citosina Metilases/química , DNA-Citosina Metilases/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico/genética , DNA-Citosina Metilases/genética , Cinética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Espectrometria de Fluorescência , Especificidade por Substrato
4.
J Bodyw Mov Ther ; 22(1): 13-17, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29332737

RESUMO

BACKGROUND: Mulligan's mobilisation-with-movement (MWM) techniques are proposed to achieve their clinical benefit via neurophysiological mechanisms. However, previous research has focussed on responses in the sympathetic nervous system only, and is not conclusive. An alternative measure of neurophysiological response to MWM is required to support or refute this mechanism of action. Recently, vibration threshold (VT) has been used to quantify changes in the sensory nervous system in patients experiencing musculoskeletal pain. OBJECTIVE: To investigate the effect of a lateral glide MWM of the hip joint on vibration threshold compared to a placebo and control condition in asymptomatic volunteers. METHODS: Fifteen asymptomatic volunteers participated in this single-blinded, randomised, within-subject, placebo, control design. Participants received each of three interventions in a randomised order; a lateral glide MWM of the hip joint into flexion, a placebo MWM, and a control intervention. Vibration threshold (VT) measures were taken at baseline and immediately after each intervention. Mean change in VT from baseline was calculated for each intervention and then analysed for between group differences using a one-way analysis of variance (ANOVA). RESULTS: A one-way ANOVA revealed no statistically significant differences between the three experimental conditions (P = 0.812). CONCLUSION: This small study found that a lateral glide MWM of the hip did not significantly change vibration threshold compared to a placebo and control intervention in an asymptomatic population. This study provides a method of using vibration threshold to investigate the potential neurophysiological effects of a manual therapy intervention that should be repeated in a larger, symptomatic population.


Assuntos
Articulação do Quadril/fisiologia , Manipulações Musculoesqueléticas/métodos , Vibração , Adulto , Feminino , Humanos , Masculino , Movimento , Método Simples-Cego
5.
Methods Appl Fluoresc ; 5(4): 042001, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29063861

RESUMO

We review various methods for analysing time-resolved fluorescence data acquired using the time-correlated single photon counting method in an attempt to evaluate their benefits and limitations. We have applied these methods to both experimental and simulated data. The relative merits of using deterministic approaches, such as the commonly used iterative reconvolution method, and probabilistic approaches, such as the smoothed exponential series method, the maximum entropy method and recently proposed basis pursuit denoising (compressed sensing) method, are outlined. In particular, we show the value of using multiple methods to arrive at the most appropriate choice of model. We show that the use of probabilistic analysis methods can indicate whether a discrete component or distribution analysis provides the better representation of the data.

6.
J Phys Chem B ; 116(34): 10290-3, 2012 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-22861666

RESUMO

We describe the engineering of reversible fluorescence photoswitching in DNA with high-density substitution, and its applications in advanced fluorescence microscopy methods. High-density labeling of DNA with cyanine dyes can be achieved by polymerase chain reaction using a modified DNA polymerase that has been evolved to efficiently incorporate Cy3- and Cy5-labeled cytosine base analogues into double-stranded DNA. The resulting biopolymer, "CyDNA", displays hundreds of fluorophores per DNA strand and is strongly colored and highly fluorescent, although previous observations suggest that fluorescence quenching at such high density might be a concern, especially for Cy5. Herein, we first investigate the mechanisms of fluorescence quenching in CyDNA and we suggest that two different mechanisms, aggregate formation and resonance energy transfer, are responsible for fluorescence quenching at high labeling densities. Moreover, we have been able to re-engineer CyDNA into a reversible fluorescence photoswitchable biopolymer by using the properties of the Cy3-Cy5 pair. This novel biopolymer constitutes a new class of photoactive DNA-based nanomaterial and is of great interest for advanced microscopy applications. We show that reversible fluorescence photoswitching in CyDNA can be exploited in optical lock-in detection imaging. It also lays the foundations for improved and sequence-specific super-resolution fluorescence microscopy of DNA.


Assuntos
DNA/química , Fluorescência , Citosina/análogos & derivados , Citosina/química , Modelos Moleculares , Tamanho da Partícula , Processos Fotoquímicos , Propriedades de Superfície
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