P19-mediated enrichment and detection of siRNAs.

Abstract

p19 is an RNA binding protein originally isolated from the Carnation Italian ring-spot virus (CIRV). It has been shown that p19 is a plant RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. A bifunctional p19 fusion protein, with an N-terminal maltose binding protein (MBP) and a C-terminal chitin binding domain (CBD) allows protein purification and binding of p19 to chitin magnetic beads via the chitin binding domain. The fusion p19 protein recognizes and binds double-stranded RNAs (dsRNA) in the size range of 20-23 nucleotides, but does not bind single strand RNA (ssRNA) or dsDNA. Furthermore, p19 can also bind mRNA, if there is a 19 bp blunt RNA duplex at the exact end of the RNA. Binding specificity of the p19 fusion protein for small dsRNA allows for detection of siRNAs derived either from exogenous or endogenous long dsRNA or microRNAs when hybridized to a complementary RNA. Here we describe a robust method using p19 and radioactive RNA probes to detect siRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. This chapter describes the potential of p19 fusion protein to detect miRNAs, isolate exogenous or endogenous siRNAs, and purify longer RNAs that contain a 19-bp terminal RNA duplex.