Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3'-end

• Published in: RNA (Cambridge) Vol. 16; no. 8; pp. 1508 - 1515
• Main Authors: Merkiene, Egle, Gaidamaviciute, Edita, Riauba, Laurynas, Janulaitis, Arvydas, Lagunavicius, Arunas
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.08.2010
• Subjects: Base Sequence; DNA Primers - genetics; DNA, Single-Stranded - genetics; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Ribonuclease III - genetics; RNA - genetics; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - genetics
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.2068510

We improved the target RNA-primed RCA technique for direct detection and analysis of RNA in vitro and in situ. Previously we showed that the 3' --> 5' single-stranded RNA exonucleolytic activity of Phi29 DNA polymerase converts the target RNA into a primer and uses it for RCA initiation. However, in some cases, the single-stranded RNA exoribonucleolytic activity of the polymerase is hindered by strong double-stranded structures at the 3'-end of target RNAs. We demonstrate that in such hampered cases, the double-stranded RNA-specific Escherichia coli RNase III efficiently assists Phi29 DNA polymerase in converting the target RNA into a primer. These observations extend the target RNA-primed RCA possibilities to test RNA sequences distanced far from the 3'-end and customize this technique for the inner RNA sequence analysis.