Photoclickable MicroRNA for the Intracellular Target Identification of MicroRNAs.

Abstract

MicroRNAs (miRNAs) are important gene regulators that bind with target genes and repress target gene expression at the post-transcriptional level. The identification of target genes associated with miRNAs inside different cells is a major challenge in miRNA chemical biology due to the lack of functional miRNAs bearing appropriate tags. Here we report photoclickable miRNAs as appropriately pretagged miRNAs that keep the intracellular function of miRNAs and allow the addition of molecular handles through photoclick reaction. The photoclickable miRNAs upon transfection inside cells were able to form functional complexes with target genes and repress target gene expression. Target genes associated with the photoclickable miRNAs in the complexes were then tagged with the molecular handle through photoclick reaction for pull-down and identification. Using photoclickable miR-106a, miR-27, and miR-122, we first verified that their intracellular function was comparable to that of intact miRNAs, which showed obvious advantage over corresponding biotinylated miRNAs. After attaching the biotin handle to the associated complexes containing the photoclickable miRNAs through the tetrazole-ene photoclick reaction, target genes previously bound with these miRNAs inside cells were successfully pulled town and analyzed. The application of this strategy was demonstrated by the identification of several new target genes of miR-122, followed by revealing a novel regulatory pathway in HepG2 cells with regard to the role of PEG10 in miR-122-promoted cell apoptosis.