Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing

Abstract

A large number of miRNAs have recently been discovered in plants and animals. Development of reverse genetic approaches that act to inhibit microRNA function would facilitate the study of this new class of noncoding RNA. Here we show that 2'-O-methyl oligoribonucleotides, but not 2'-deoxyoligonucleotides specifically inactivate the RNAi activity associated with miRNA-protein complexes in human cell extracts as well as in cultured human cells.

FIGURE 1.

Antisense 2′-O-methyl oligoribonucleotide specifically inhibit miR-21 guided cleavage activity in HeLa cell S100 cytoplasmic extracts. The black bar to the left of the RNase T1 ladder represents the region of the target RNA complementary to miR-21. Oligonucleotides complementary to miR-21 were preincubated in S100 extracts prior to the addition of ³²P-cap-labeled cleavage substrate. Cleavage bands and T1 hydrolysis bands appear as doublets after a 1-nt slipping of the T7 RNA polymerase near the middle of the transcript indicated by the asterisk.

FIGURE 2.

Antisense 2′-O-methyl oligoribonucleotides block EGFP-targeting RISC in EGFP stably expressing HeLa cells. (A) EGFP expressing HeLa cells were cotransfected with EGFP siRNA duplex and the indicated single-stranded oligonucleotides. EGFP fluorescence was recorded 48 h past transfection. The antisense 2′-OMe oligoribonucleotide is complementary to the guide siRNA strand. The reversed sequence (not the reverse complement) of antisense 2′-OMe oligoribonucleotide was used as control. The guide siRNA is complementary to a segment of the EGFP mRNA. The top row shows fluorescence microscopy images recording the EGFP fluorescence; the bottom row shows phase contrast images. (B) EGFP expressing HeLa cells were first transfected with EGFP siRNA duplex followed by transfection of and the indicated single-stranded 2′-OMe oligoribonucleotides after 24 h.

FIGURE 3.

Antisense 2′-O-methyl oligoribonucleotides interfere with endogenous miR-21 RNP cleavage in HeLa cells. HeLa cells were transfected with pHcRed and pEGFP or its derivatives, with or without inhibitory or control oligonucleotides. EGFP and HcRed protein fluorescence were excited and recorded individually by fluorescence microscopy 24 h after transfection. Coexpression of cotransfected reporter plasmids was documented by superimposing of the fluorescence images in the right panel.