Cis-regulatory hairpin-shaped mRNA encoding a reporter protein: Catalytic sensing of nucleic acid sequence at single nucleotide resolution

Abstract

DNA sensing at a single nucleotide resolution is achieved using a hairpin-shaped, unmodified (unlabeled) RNA probe or the precursor double-stranded DNA (dsDNA) in a prokaryotic cell-free translation medium. The molecular-beacon-like probe consists of a loop region that is complementary to the target sequence and a stem composed of a ribosome-binding site (RBS) and its docking domain; the RBS is followed by the gene for a reporter protein such as luciferase or β-galactosidase. Target binding at the loop opens the hairpin to make RBS accessible by the ribosome to start translation of the reporter protein. This sensing system is signal amplifying by virtue of catalytic DNA-to-RNA transcription when using a dsDNA probe, catalytic RNA-to-protein translation, catalytic signal transduction by the enzymatic reaction of the translated reporter protein and, in the presence of RNase H, catalytic or even irreversible translation-activation of the target-probe heteroduplex. Preparation of a probe takes 1-3 d and gene sensing using the probe takes 1-3 h.