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1.
Sci Rep ; 2: 246, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22355758

RESUMO

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5' ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism - single crossing CPA - and provide details on alternative mechanisms.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , DNA Bacteriano/genética , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Moldes Genéticos
2.
Biochemistry ; 49(12): 2732-40, 2010 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-20175561

RESUMO

A class of RNA aptamers that demonstrates a high affinity for a large variety of C(2)H(2) zinc finger proteins was isolated from a library of random RNA sequences by the zinc finger protein TFIIIA. These aptamers have one or more copies of the consensus sequence GGGUGGG, which is part of a putative hairpin loop in the proposed structure of the most abundant aptamer, RNA1. Binding of zinc finger proteins to RNA1 relies upon zinc-dependent folding of the protein, the affinity of an individual protein for RNA1 being determined by the number of tandem zinc finger motifs. The properties of RNA1 were compared to the properties of two other aptamers from the same selection experiment: RNA21, which binds to some but not all zinc finger proteins tested, and RNA22, which binds only to the 5S rRNA binding zinc finger proteins TFIIIA and p43. The binding of three different zinc finger proteins to RNA1 was compared, and the results indicate that the RNA1-protein interaction occurs by several distinct mechanisms. Mutagenesis of RNA1 confirmed that the GGGUGGG consensus sequence presented in a hairpin conformation is required for high-affinity binding of zinc finger proteins.


Assuntos
Aptâmeros de Nucleotídeos/química , Dedos de Zinco , Animais , Aptâmeros de Nucleotídeos/metabolismo , Sítios de Ligação/genética , DNA Fúngico/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Ligação Proteica/genética , RNA Ribossômico 5S/química , Fator de Transcrição TFIIIA/química , Fator de Transcrição TFIIIA/metabolismo , Xenopus
3.
Biochemistry ; 49(8): 1755-65, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20095591

RESUMO

The Xenopus zinc finger proteins TFIIIA and p43 bind to 5S RNA in immature oocytes to form 7S and 42S ribonucleoprotein storage particles. To probe the similarities and differences in the RNA binding domains of these two proteins, a library of random RNA molecules was enriched using TFIIIA as the bait protein. One of the abundant aptamers isolated, RNA22, bound to both TFIIIA and p43 derived zinc finger peptides with high affinity and specificity even though the predicted secondary structure of the RNA was unrelated to that of 5S RNA. The interactions of TFIIIA and p43 peptides with RNA22 were compared to their interactions with 5S RNA by characterizing the effects of assay conditions, mutations in RNA22, and mutations in the zinc finger proteins. The similarities and differences in the mechanisms by which these two zinc finger proteins interact with 5S RNA compared to RNA22 suggest they share a common platform for RNA binding with enough flexibility to form specific interactions with both RNAs.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Fator de Transcrição TFIIIA/genética , Proteínas de Xenopus/genética , Xenopus laevis
4.
Biochemistry ; 48(1): 148-55, 2009 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-19123921

RESUMO

In addition to binding to DNA, the zinc finger protein WT1 can also bind specifically to RNA. To determine the role of individual zinc fingers of the protein in this RNA binding activity, deletion and substitution mutants of the WT1 zinc finger domain were constructed. The effects of the various mutations on the binding of WT1 to the RNA aptamers RNA22 and RNA38 were determined using a quantitative equilibrium binding assay. The results indicate that zinc fingers 2 and 3 of WT1 are essential for the binding of the protein to the RNA aptamers. For both of these fingers, the arginine residue immediately preceding the alpha-helix makes a significant contribution to RNA binding. For zinc finger 2, a second arginine residue within the alpha-helix is also critical for RNA binding, while several alpha-helical residues in zinc finger 3 contribute to the overall affinity of WT1 for RNA. Investigating the effects of the same point mutations on DNA binding indicates that there are similarities and differences in the contributions of zinc fingers 2 and 3 to the DNA and RNA binding activities of WT1.


Assuntos
Aminoácidos/química , Aptâmeros de Nucleotídeos/química , RNA/química , Proteínas WT1/química , DNA/química , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas WT1/genética , Dedos de Zinco
5.
Biochemistry ; 47(32): 8398-405, 2008 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-18636752

RESUMO

Xenopus zinc finger protein p43 binds to 5S RNA in immature oocytes to form a 42S ribonucleoprotein storage particle. To determine the role of individual zinc fingers of the protein in this RNA binding activity, a series of deletion and substitution mutants of p43 were constructed. The effects of the various mutations on the RNA binding activity of p43 were determined using a quantitative equilibrium binding assay. The results indicate that zinc fingers 1 and 4 of p43 are essential for the binding of the protein to 5S RNA. In the case of finger 1, four amino acids key to RNA binding are found on the same face of the alpha-helix, while in the case of finger 4, two key residues are clustered at the start of the alpha-helix. The similarities and differences in the mechanisms by which fingers 1 and 4 of p43 interact with 5S RNA are compared to the interaction of the zinc fingers of Xenopus transcription factor IIIA with 5S RNA.


Assuntos
Aminoácidos/metabolismo , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Xenopus/metabolismo , Dedos de Zinco/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/genética , Animais , Sítios de Ligação , Feminino , Dados de Sequência Molecular , RNA Ribossômico 5S/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis , Dedos de Zinco/genética
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