Continuous fluorescence-based method for assessing dicer cleavage efficiency reveals 3' overhang nucleotide preference.
The cleavage of double-stranded RNA (dsRNA) molecules by Dicer is a critical step in silencing genes by the RNA induced silencing complex (RISC). The development of Dicer substrates as nucleic acid-based therapeutics brings about a need to rapidly evaluate chemically modified RNA molecules for their ability to be processed by Dicer. This study outlines a quantitative fluorescence quencher-based assay for studying the ability of Dicer substrates to be processed by Dicer. By using a dsRNA probe labeled with Cy5-Iowa Black RQ, a panel of unlabeled test substrates can be rapidly assessed in heterologous competition assays without the need for electrophoresis or radiolabeling. This assay was piloted by evaluation of 196 unlabeled 27-mer Dicer substrates with various overhang structures in a purified Dicer enzyme assay system. Results indicate that Dicer has no preference for the sequence of RNA in the main double-stranded region of the substrate. However, a preference for Dicer substrate RNAs (D-siRNAs) containing purine/purine 3' overhang nucleotides was observed. These results demonstrate that the method is useful for studying the effects of modified nucleic acids in addition to rapidly accessing the influence of potential regulatory factors on Dicer processing.
DiNitto
Jonathan P
JP
Pfizer Research Technology Center, Cambridge, MA, USA. jonathan.dinitto@biogenidec.com
Wang
Leyu
L
Wu
Joe C
JC
D001483:Base Sequence; D001667:Binding, Competitive; D002232:Carbocyanines / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002232:Carbocyanines; D005456:Fluorescent Dyes; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; C085321:cyanine dye 5; D043244:Ribonuclease III