Selection of RNA Oligonucleotides That Can Modulate Human Dicer ActivityIn Vitro

Abstract

Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.

A New Short Oligonucleotide-Based Strategy for the Precursor-Specific Regulation of microRNA Processing by Dicer

“…2) selectively and permanently repressed the excision of miR-210 from its precursor. Our preliminary observations suggested that ATD_13.6 should be classified as allosteric inhibitor and that ATD_15.52 should be considered a competitive inhibitor [32]. Unfortunately, our earlier experiments did not reveal any molecular determinants of inhibitor specificity.…”

“…In a pool of hDicer-binding RNA oligomers identified by SELEX (using approximately 56-nt long oligoribonucleotides), we found molecules that inhibited pre-miRNA processing by acting as: (i) competitive inhibitors – these oligomers were cut by hDicer; and (ii) allosteric inhibitors – these molecules were not digested by hDicer [32]. Interestingly, among the selected oligomers, ATD_13.6 (Fig.…”

“…This oligonucleotide forms a stable complex with hDicer (Fig. S1A), but it is not cut by the enzyme [32]. To gain a better understanding of the nature of ATD_13.6:Dicer interactions, we performed a standard competitive displacement assay.…”

“…Recently, we demonstrated that recombinant human Dicer (hDicer) activity can be modulated by the binding of RNA molecules to this enzyme [32]. In addition, we identified RNA oligomers that differentially affected the processing of various pre-miRNAs.…”

Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer

“…We used an oligomer RNA [26] (Supplementary Table S1) which had a great affinity for Dicer (but not cleaved by Dicer) and added it to an antisense RNA targeting MALAT-1 RNA to form an artificial small RNA (asRNA) (Supplementary Table S2) (Figure 1). The plasmid pGPU6-GFP-NEO was used to express the asRNA after the cDNA sequence of asRNA was inserted into it.…”