Protein Mediated miRNA Detection and siRNA Enrichment Using p19

Abstract

p19 RNA binding protein from the Carnation Italian ringspot virus (CIRV) is an RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. We created a bifunctional p19 fusion protein with an N-terminal maltose binding protein (MBP), for protein purification, and a C-terminal chitin binding domain (CBD) to bind p19 to chitin magnetic beads. The fusion protein binds dsRNAs in the size range of 20-23 nucleotides, but does not bind ssRNA or dsDNA. Relative affinities of the p19 fusion protein for different-length RNA and DNA substrates were determined. Binding specificity of the p19 fusion protein for small dsRNA allows detection of miRNA:RNA probe duplexes. Using radioactive RNA probes, we were able to detect low levels of miRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Detection is linear over three logs. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. Rules for designing optimal RNA probes for p19 detection of miRNAs were determined by in vitro binding of 18 different dsRNA oligos to p19. These studies demonstrate the potential of p19 fusion protein to detect miRNAs and isolate endogenous siRNAs.

pH‐sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

“…13,14 Over the past few years, p19 has been used in several systems to suppress RNAi [15][16][17][18][19][20][21][22] and has also emerged as a valuable tool for characterizing small RNAs. 8,9,[23][24][25][26] Furthermore, as environmental factors (e.g., acidity/alkalinity, salt concentration, water levels, etc.) can vary significantly across different plant hosts, it has become increasingly important to understand how the protein environment can affect the function of p19.…”

Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions

“…The p19 siRNA binding protein (10 U/μL) used here was a commercially produced double-fusion protein, with a maltose binding protein fused to the N-terminus and a chitin-binding domain fused to the C-terminus (New England BioLabs). The binding properties of this double-fusion version of p19 have been previously described [32]. Twelve-microliter binding reactions were prepared with 20 ng of double-stranded RNA in 1× Binding Buffer (NEB) with varying protein concentrations.…”

Bioengineered non-coding RNA agent (BERA) in action

“…A 19 kD siRNA-binding protein, p19, [23][24][25] was employed to bind to recombinant siRNA to form a siRNA-p19 complex and then purified by nickel affinity chromatography and followed by anion exchange high performance liquid chromatography (HPLC). 22 While this method allows the production of fully-processed, ready-to-use siRNAs or miRNAs, the overall yield is very low and it unlikely provides a much desired large quantity of target siRNA agents.…”