Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics

Abstract

MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (K), a photolabile nucleoside analog. We demonstrate that biotin-tagged, K-containing miR-29b (K-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering K-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including, , and, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and K-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest thatK-containing small RNA mimics may find applicability in other experimental models.

miRNAs secreted by human blastocyst could be potential gene expression regulators during implantation

“…Previous studies have shown that downregulated miR-29b is related to upregulated HOXA10 [45]. Other studies have the same result that increasing the expression of miR-29b could reduce the expression of hOXA10 [46].…”

Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities: Application to Biotin-mediated RNA-Pulldown

“…49,50 Indeed, CNV chemistry was recently applied to the target identification of miR-29b targets, but the possibility of reverting the formed crosslink after pull-down was not explored. 51 Moreover, CNV is reported to selectively crosslink to thymine, and, secondly, to cytosine placed at the N+1 position on the target strand. 52 We here reported an approach that has a greater tolerance, given its ability to target both cytosine and adenine in good yields and guanine to a minor extent, placed at the 3'-flanking region of the RNA target.…”

Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities: Application to Biotin-mediated RNA-Pulldown

“…48,49 Indeed, CNV chemistry was recently applied to the target identification of miR-29b targets, but the possibility of reverting the formed crosslink after pull-down was not explored. 50 Moreover, CNV is reported to selectively crosslink to thymine, and, secondly, to cytosine placed at the N+1 position on the target strand. 51 We here reported an approach that has a greater tolerance, given its ability to target both cytosine and adenine in good yields and guanine to a minor extent, placed at the 3'-flanking region of the RNA target.…”