Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

Abstract

We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 39/59 RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted to inner RNA sequences and their peculiarities can be analyzed directly. We demonstrate that the exoribonucleolytic activity of Phi29 DNA polymerase can be successfully applied in vitro and in situ. These findings expand the potential for detection and analysis of RNA sequences distanced from 39-end.

Cleavage-based signal amplification of RNA

“…Thermal cycling of the PCR technique imposes instrumental constraints, limiting the technique to a laboratory setting, and dual-labelled fluorescent probes, such as Taqman probes 12 , are usually needed to determine the specificity of amplification. Therefore, isothermal amplification of RNA, such as nucleic acid sequence-based amplification [13][14][15] , rolling-cycle amplification 16,17 and loopmediated isothermal amplification 18,19 have emerged as alternative amplification techniques. As the above mentioned reactions can be preceded at a constant temperature, there is no need of specialized instruments for RNA detection, and in addition, they have potential for 'on-site' testing.…”

“…Two kinds of catalytic DNA sequences, RNA-cleaving DNAzyme (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) 26,27 and G-quadruplex horseradish peroxidase-mimicking DNAzyme (PW17) [28][29][30] were applied in our research. The 10-23 RNAcleaving DNAzyme has been reported to cleave any purinepyrimidine (RY) junction under simulated physiological conditions 26 ; hence, it was applied in our strategy for the cleaving of target RNA molecule to initiate the following exponential amplification.…”

Expanding Possibilities of Rolling Circle Amplification as a Biosensing Platform

“…This type of RCA is called RNA-primed RCA (RPRCA) and, indeed, RPRCA is used for microRNA and small RNA detection without reverse transcription. 21,40,[42][43][44][45][46][47][48] When several circular ssDNA probes are tested in detecting in vitro transcribed GFP messenger RNA (mRNA) as a standard RPRCA procedure, the RPRCA reaction is observed under a certain ssDNA probe (Fig. 4).…”

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

“…RT-qPCR remains the gold standard for RNA quantification; however, this technique is limited by time and equipment constraints and can be prone to contamination. To minimize these limitations, isothermal RNA amplification techniques have been developed [7][8][9][10][11][12][13][14], but these still remain dependent on nucleic acid replication and are therefore hindered by polymerase speed and fidelity. Recent RNA detection methods that have employed duplex specific nuclease (DSN) isolated from the Paralithodes camtschaticus crab [15,16], remain limited to microRNA [17] and are thus unsuitable for longer RNA templates like virus genomic or mRNA targets.…”