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.
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.
Merkiene
Egle
E
Fermentas UAB, Graiciuno 8, Vilnius LT-02241, Lithuania.
Gaidamaviciute
Edita
E
Riauba
Laurynas
L
Janulaitis
Arvydas
A
Lagunavicius
Arunas
A
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics; D043244:Ribonuclease III / Q000235:genetics
D017931:DNA Primers; D004277:DNA, Single-Stranded; C034477:RNA primers; D012330:RNA, Double-Stranded; D012313:RNA; D004259:DNA-Directed DNA Polymerase; D043244:Ribonuclease III