Visible Sensing of Nucleic Acid Sequences with a Genetically Encodable Unmodified RNA Probe

Abstract

DNA‐Nachweis mit RNA: Ein einfacher und empfindlicher DNA‐Nachweis gelang mit einer RNA‐Sonde aus einer einem „molekularen Leuchtfeuer“ ähnlichen cis‐Repressorsequenz und einem Gen für das Reporterprotein. Das an die RNase‐H‐Aktivität gekoppelte Leuchtfeuer‐mRNA‐System ermöglichte den optischen Nachweis der Ziel‐Nucleotidsequenz in einer zellfreien Translationslösung (siehe Bild).

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

“…It thus irreversibly digests the target-bound loop region of the probe to release the anti-RBS domain from the mRNA body, thus allowing catalytic use of target DNA (RNase H-coupled MB-mRNA; Fig. 1c) with improved selectivity and enhanced sensitivity 25 , although, given the nature of RNase H, it can be used only in vitro for DNA targets and not for in-cell gene sensing, where the targets should be mRNAs.…”

“…1420) for luciferase/galactosidase assay . 25-ml reagent reservoir (VistaLab) for multichannel pipetting . Surgical stainless steel blades and handles (Feather Safety Razor) for gel cutting REAGENT SETUP 1% Agarose gel Weigh 0.5 g agarose, and dissolve in 50 ml MilliQ-grade deionized hot water.…”