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1.
Nucleic Acids Res ; 48(22): e132, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33152076

RESUMO

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.


Assuntos
DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/métodos , Imagem Individual de Molécula , Composição de Bases/genética , Humanos , Nanotecnologia , Nucleotídeos/genética
2.
Nucleic Acids Res ; 47(17): e101, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31318971

RESUMO

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.


Assuntos
Desoxirribonucleotídeos/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleosídeos/química , Desoxirribonucleotídeos/química , Limite de Detecção , Microscopia de Fluorescência , Oligodesoxirribonucleotídeos/biossíntese , Oligodesoxirribonucleotídeos/química , Sensibilidade e Especificidade
3.
Biochemistry ; 52(51): 9269-74, 2013 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-24358934

RESUMO

RNA-protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA-protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5'-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by ¹H NMR spectroscopy.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , RNA Líder para Processamento/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Ribonucleoproteínas/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/química , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Células HeLa , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Concentração Osmolar , Compostos de Quinolínio/efeitos adversos , Compostos de Quinolínio/química , Compostos de Quinolínio/farmacologia , RNA Viral/química , RNA Viral/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Bases de Schiff/efeitos adversos , Bases de Schiff/química , Bases de Schiff/farmacologia , Bibliotecas de Moléculas Pequenas , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
4.
Biochemistry ; 51(15): 3162-9, 2012 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-22448757

RESUMO

The major RNA binding region of the HIV-1 Gag polyprotein is the nucleocapsid (NC) domain, which is responsible for the specific capture of the genomic RNA genome during viral assembly. The Gag polyprotein has other RNA chaperone functions, which are mirrored by the isolated NC protein after physiological cleavage from Gag. Gag, however, is suggested to have superior nucleic acid chaperone activity. Here we investigate the interaction of Gag and NC with the core RNA structure of the HIV-1 packaging signal (Ψ), using 2-aminopurine substitution to create a series of modified RNAs based on the Ψ helix loop structure. The effects of 2-aminopurine substitution on the physical and structural properties of the viral Ψ were characterized. The fluorescence properties of the 2-aminopurine substitutions showed features consistent with the native GNAR tetraloop. Dissociation constants (K(d)) of the two viral proteins, measured by fluorescence polarization (FP), were similar, and both NC and Gag affected the 2-aminopurine fluorescence of bases close to the loop binding region in a similar fashion. However, the influence of Gag on the fluorescence of the 2-aminopurine nucleotides at the base of the helix implied a much more potent helix destabilizing action on the RNA stem loop (SL) versus that seen with NC. This was further supported when the viral Ψ SL was tagged with a 5' fluorophore and 3' quencher. In the absence of any viral protein, minimal fluorescence was detected; addition of NC yielded a slight increase in fluorescence, while addition of the Gag protein yielded a large change in fluorescence, further suggesting that, compared to NC, the Gag protein has a greater propensity to affect RNA structure and that Ψ helix unwinding may be an intrinsic step in RNA encapsidation.


Assuntos
HIV-1/metabolismo , Proteínas do Nucleocapsídeo/química , RNA Viral/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Sítios de Ligação , Cinética , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
7.
Chem Commun (Camb) ; 50(14): 1704-7, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24394582

RESUMO

We report here on the screening of a fragment library against a G-quadruplex element in the human c-MYC promoter. The ten fragment hits had significant concordance between a biophysical assay, in silico modelling and c-MYC expression inhibition, highlighting the feasibility of applying a fragment-based approach to the targeting of a quadruplex nucleic acid.


Assuntos
Quadruplex G , Proteínas Proto-Oncogênicas c-myc/genética , Simulação por Computador , Humanos , Modelos Moleculares , Regiões Promotoras Genéticas/genética
8.
Trends Microbiol ; 21(3): 136-44, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23266279

RESUMO

Over the past several decades, extensive research into the Gag polyprotein, the main structural protein of HIV-1 and all other retroviruses, has changed the way that we describe Gag's role within viral lifecycles. Initially thought of as a simple scaffold protein forming the viral core, Gag has demonstrated the ability to specifically recognize genomic RNA and both viral and host proteins as it traffics to the cell membrane. There, Gag forms higher ordered structures required for the correct assembly, budding, and maturation of new infectious particles.


Assuntos
Produtos do Gene gag/metabolismo , HIV-1/fisiologia , Montagem de Vírus , Ligação Proteica
9.
Nat Protoc ; 8(10): 1841-51, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24008380

RESUMO

To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.


Assuntos
5-Metilcitosina/química , Citosina/análogos & derivados , Metilação de DNA , Análise de Sequência de DNA/métodos , Citosina/química , Oxirredução
10.
Org Biomol Chem ; 6(1): 92-103, 2008 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-18075653

RESUMO

Pyrrolidine-amide oligonucleotide mimics (POMs) can cross-pair strongly with complementary parallel and antiparallel DNA and RNA targets in a sequence-specific fashion. As a result POMs have significant potential for applications including in vivo gene silencing, diagnostics and bioanalysis. To further modulate the DNA- and RNA-recognition properties and fine-tune the physiochemical properties of POMs for nucleic acid targeting, backbone-extended pyrrolidine-amide oligonucleotide mimics (bePOM I and II) were introduced. The bePOMs differ from the original POMs through the insertion of an additional methylene group into the backbone units, which increases the flexibility of the oligomers. bePOM I and II oligomers were synthesised using solid-phase peptide chemistry. Interestingly, UV thermal denaturation and circular dichroism studies reveals bePOM I and II can hybridise with complementary RNA, but not DNA.


Assuntos
Amidas/química , DNA Complementar/química , Oligonucleotídeos/química , Oligonucleotídeos/síntese química , Pirrolidinas/química , RNA Complementar/química , Dicroísmo Circular , Desnaturação de Ácido Nucleico/efeitos da radiação , Oligonucleotídeos/efeitos da radiação , Especificidade por Substrato , Termodinâmica , Raios Ultravioleta
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