ABSTRACT
Short hairpin RNAs (shRNAs) and short interfering RNAs (siRNAs) probably enter different stages of the microRNA (miRNA) pathway for depletion of mRNA and suppression of protein translation. Primary and secondary structural characteristics that are shared between endogenous primary miRNA transcripts (pri-miRNAs) may contribute toward efficient biogenesis and potent silencing. This study investigates known miRNA transcripts for characteristics that are conserved between miRNAs and that distinguish them from random hairpins with similar lengths. The primary structure is conserved, as demonstrated by a significant presence or absence of certain bases at specific positions in the miRNA precursors and their flanking regions. The secondary structure is also conserved between miRNAs, as internal loops and bulges commonly appear in specific positions in the miRNA stem. The conservation of base-pairing continues past the mature duplex and 13 bases into the primary stem, with no detectable conservation of secondary structure beyond this region. Based on these observations, we have designed a hairpin construct that incorporates the most important characteristics present in endogenous miRNAs. Preliminary experiments suggest that this construct may rescue the efficacy of shRNA triggers that cannot be used with a miR-30-based hairpin, and vice versa.
