RESUMO
Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplification. Unfortunately, in comparison with their applications, their catalytic mechanism studies have relatively lagged behind due to a paucity of crystal structure information. Nt.BstNBI is one of those widely used NEs. However, many aspects of its catalytic mechanism still remained to be explored. Herein, we employed only rolling circle amplification (RCA) assay as a major analytic tool and succeeded in identifying the potential binding positions and regions of the DNA substrate based on locked nucleic acid modification, DNA duplex length of substrate, and substrate mismatch designs. Based on these data, we, for the first time, revealed that Nt.BstNBI was likely to recognize six adjacent positions of the recognition sequence (G1rt, A2rt, G3rt, A2rb, C3rb, and T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, and N7cb) in the minor groove of DNA duplex for nicking. Moreover, this work also demonstrated the unexpected efficiency of RCA to study the macromolecular interaction for certain kind of nucleases in an easy and high-throughput way.
Assuntos
Quebras de DNA de Cadeia Simples , Replicação do DNA , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , CatáliseRESUMO
Oligonucleotides were screened for strongly silver-stained repetitive sequences. An 'AG'-clustered purine sequence showed strong staining, and the staining density can be compromised by disrupting the continuity of the 'AG'-clustered sequence. The staining-favored sequence was then employed in rolling circle amplification (RCA) for its product detection. A tube-staining method was developed for convenient and visual RCA assay. Moreover, by introducing GalR into RCA, d-galactose was detected by RCA tube-staining with naked eyes without any equipment. About 10 mM d-galactose can be easily identified, and the detection of d-galactose was specific in comparison with that of several other monosaccharides.
Assuntos
Galactose/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Oligonucleotídeos/análise , Coloração pela Prata/métodos , Sequência de Bases , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Eletroforese em Gel de Poliacrilamida , Galactose/química , Galactose/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/genética , Reprodutibilidade dos TestesRESUMO
Transcription factor (TF)-based metabolite detection mainly depends on TF-regulated gene expression in cells. From TF activation to gene transcription and translation, the signal travels a relatively long way before it is received. Here, we propose a TF-splinting duplex DNA nanoswitch to detect metabolites. We show its feasibility using tryptophan repressor (TrpR) to detect l-tryptophan as a model. The assay has been optimized and characterized after obtaining a proof of concept, and the detection of l-tryptophan in complex biological samples is feasible. Unlike an equivalent gene expression approach, the whole process is a single-step, enzyme-free, and signal-on method. It can be completed within 20 min. This proposed TF-splinting duplex has the potential to be applied to the quick and convenient detection of other metabolites or even TFs.