Toehold-mediated Strand Displacement to Measure Released Product from Self-cleaving Ribozymes

Overview

Journal RNA

Specialty Molecular Biology

Date 2021 Dec 4

PMID 34862273

Authors

Jay Bhakti Kapadia

Nawwaf Kharma

Alen Nellikulam Davis

Nicolas Kamel

Jonathan Perreault

Affiliations

Soon will be listed here.

Abstract

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labeled with a fluorophore at its 5'-end, while the other strand is labeled with a quencher at its 3'-end. These two DNA strands are perfectly complementary, but with a 3'-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of fragments, resulting from that cleavage reaction.

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