Nonenzymatic DNA ligation in Escherichia coli cells.
We report on a new fluorescence reporting strategy in which dabsyl, a well-known quencher, activates a hydroxyl group in a probe to convert it to a leaving group. When a nucleophilic phosphorothioate probe binds adjacent to a dabsyl quenched probe, autoligation occurs, releasing the quencher, and lighting up the probes. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in E. coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.
Sando
Shinsuke
S
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606-8501, Japan.
Kool
Eric T
ET
D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S