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Anal Biochem ; 543: 108-115, 2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29233680


Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings.

Nat Chem ; 5(5): 383-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23609088


The recent synthesis of pyrimidine ribonucleoside-2',3'-cyclic phosphates under prebiotically plausible conditions has strengthened the case for the involvement of ribonucleic acid (RNA) at an early stage in the origin of life. However, a prebiotic conversion of these weakly activated monomers, and their purine counterparts, to the 3',5'-linked RNA polymers of extant biochemistry has been lacking (previous attempts led only to short oligomers with mixed linkages). Here we show that the 2'-hydroxyl group of oligoribonucleotide-3'-phosphates can be chemoselectively acetylated in water under prebiotically credible conditions, which allows rapid and efficient template-directed ligation. The 2'-O-acetyl group at the ligation junction of the product RNA strand can be removed under conditions that leave the internucleotide bonds intact. Remarkably, acetylation of mixed oligomers that possess either 2'- or 3'-terminal phosphates is selective for the 2'-hydroxyl group of the latter. This newly discovered chemistry thus suggests a prebiotic route from ribonucleoside-2',3'-cyclic phosphates to predominantly 3',5'-linked RNA via partially 2'-O-acetylated RNA.

Prebióticos , RNA/química , Acetilação , Biopolímeros/química , Ressonância Magnética Nuclear Biomolecular
Blood ; 121(6): 1028-38, 2013 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-23190533


The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3'-5' exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3' end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2', 3' cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream.We show that the 3' ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3' oligo(A) tails that are characteristic of nuclear RNA surveillance targets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3'-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN.

Mutação , Neutropenia/genética , Diester Fosfórico Hidrolases/genética , RNA Nuclear Pequeno/genética , Anormalidades da Pele/genética , Regiões 3' não Traduzidas/genética , Nucleotídeos de Adenina/genética , Nucleotídeos de Adenina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Teste de Complementação Genética , Humanos , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Neutropenia/metabolismo , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Nuclear Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Anormalidades da Pele/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Uridina/genética , Uridina/metabolismo