Studies of a viral suppressor of RNA silencing p19-CFP fusion protein: a FRET-based probe for sensing double-stranded fluorophore tagged small RNAs.
Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) p19-CFP fusion protein and Cy3-labeled siRNA. The CIRV p19 fusion protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET. [corrected]
Koukiekolo
Roger
R
Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Canada. Roger.Koukiekolo@nrc.ca
Jakubek
Zygmunt J
ZJ
Cheng
Jenny
J
Sagan
Selena M
SM
Pezacki
John Paul
JP
D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D015347:RNA Probes / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000032:analysis* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; C493885:P19 protein, tomato bushy stunt virus; D015347:RNA Probes; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins