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

Scientific Reports
Hirokazu TakahashiYoshiko Okamura

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 potentia...Continue Reading

References

Sep 1, 1970·Proceedings of the National Academy of Sciences of the United States of America·K KleppeH G Khorana
Jan 1, 1981·CRC Critical Reviews in Biochemistry·G Brawerman
Nov 4, 1998·Nucleic Acids Research·J BanérU Landegren
Nov 11, 1999·Leukemia·R A PaduaC Chomienne
Jul 11, 2000·Nature Biotechnology·M NilssonU Landegren
Jan 5, 2001·Nucleic Acids Research·M NilssonU Landegren
Sep 14, 2002·Proceedings of the National Academy of Sciences of the United States of America·C Kiong Ho, Stewart Shuman
Apr 16, 2004·The Journal of Biological Chemistry·Jayakrishnan NandakumarStewart Shuman
Oct 21, 2004·Molecular Cell·Jayakrishnan Nandakumar, Stewart Shuman
Nov 16, 2005·Proceedings of the National Academy of Sciences of the United States of America·Clyde A HutchisonJ Craig Venter
May 5, 2006·The Biochemical Journal·Desmond R Bullard, Richard P Bowater
Dec 17, 2009·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Toshiro KoboriShigeru Sugiyama
Apr 13, 2010·Nature Methods·Chatarina LarssonMats Nilsson
Sep 20, 2011·Integrative Biology : Quantitative Biosciences From Nano to Macro·Magnus StougaardBirgitta R Knudsen
Nov 10, 2013·Nucleic Acids Research·Gregory J S LohmanThomas C Evans
Jan 15, 2014·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Toshiro Kobori, Hirokazu Takahashi
May 8, 2016·Nucleic Acids Research·Jingmin JinLarry A McReynolds
Jul 13, 2016·BioTechniques·Hirokazu TakahashiToshiro Kobori

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Citations

Jul 2, 2020·The Analyst·Huan-Huan SunBang-Ce Ye
Nov 21, 2020·PLoS Biology·Alexandros SountoulidisChristos Samakovlis
Feb 5, 2021·Chemical Society Reviews·Thomas JetValérie Taly
Nov 8, 2020·Journal of Microbiological Methods·Dana M NieuwkerkValerie J Harwood

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Methods Mentioned

BETA
RNA-seq
electrophoresis
in vitro transcription
PCR
Assay

Software Mentioned

CircLigase
ImageJ
NIH Image
OligoAnalyzer

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