RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

Hirokazu Takahashi,Tsunehiro Aki,Kyohei Horio,Masahiko Ohkawachi,Yukihiko Matsumura,Toshiro Kobori,Yoshiko Okamura,Yutaka Nakashimada

doi:10.1038/s41598-018-26132-x

Abstract

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3′-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Highlights

Many recent studies have focused on rolling circle amplification (RCA) for detecting biological molecules[1,2]
Specific mRNA detection via ribonuclease H (RNase H)-assisted RNA-primed rolling circle amplification (RPRCA) with a pre-circularized probe only works in the absence of non-target RNA molecules in the reaction mixture, presumably because pre-circularized probes can hybridize with non-target RNAs and initiate DNA synthesis upon RNA digestion by RNase H
H-assisted RPRCA using a padlocked probe, which consists of five reaction steps as follows: (i) the padlock probe is hybridized to a sequence of interest in the target mRNA molecule; (ii) the hybridized padlock probe is sealed by SplintR ligase to create a circular DNA probe; (iii) RNase H produces a nick site in the hybridized mRNA; (iv) phi[29] DNA polymerase catalyzes DNA synthesis at the nick site of RNA with strand displacement; and (v) RPRCA synthesizes long single-stranded DNA (ssDNA), which has a periodic sequence complementary to the padlock probe

Summary

Introduction

Many recent studies have focused on rolling circle amplification (RCA) for detecting biological molecules[1,2]. We previously reported RNA-primed rolling circle amplification (RPRCA) using phi[29] DNA polymerase, circularized probes, and SYBR Green II for the real-time detection of mRNA from living microbes[3]. Conventional RPRCA requires the 3′-terminal sequence of target RNA as a primer for DNA synthesis, available sequence database information on the 3′-terminal sequences of prokaryotic mRNAs is limited at present. For the simple detection of most types of RNA molecules in prokaryotes and eukaryotes, it is necessary to modify the RPRCA procedure to work without full-length and/or 3′-terminal sequences of the target RNA molecule. Specific mRNA detection via RNase H-assisted RPRCA with a pre-circularized probe only works in the absence of non-target RNA molecules in the reaction mixture, presumably because pre-circularized probes can hybridize with non-target RNAs and initiate DNA synthesis upon RNA digestion by RNase H. The specificity of RNase H-assisted RPRCA should be improved to make RNA detection simple and practical

Methods

Results

Conclusion