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.

Wavelength Dependence of Light-Induced Cycloadditions

“…Their applications include surface functionalization, 27 the synthesis of sequence defined macromolecules, 28,29 l-orthogonal ligation, 30,31 light-induced in situ selfassembly of polymers, 32 photoresists design for 3D laser lithography, 33 synthesis of hydrogels 34 and functionalization of biomolecules such as labeling of proteins [35][36][37][38] or microRNA. 39 In addition, nitrile imines are susceptible to reactions with nucleophiles, which was exploited for site selective surface patterning 40 as well as single chain nanoparticle synthesis. 41 The bioorthogonality of the nitrile imine mediated tetrazole ene cycloaddition -even though limited in some instances -is of high value for biological applications.…”

Current experimental strategies for intracellular target identification of microRNA

“…To establish a universal strategy for tagging miRNAs without affecting their intracellular function and efficient capture of miRNA-associated targets, we recently developed a novel strategy for miRNA target identification based on photo-click chemistry (Fig. 6) [47]. In comparison with 3'-biotinylated miRNAs, photo-clickable miRNA with tetrazole modification at the 3'-end of several miRNAs showed intact biological function inside cells, which were also comparable to unmodified miRNA mimics.…”

“…Recent results revealed 3'-biotinylation greatly hampered the association of miRNA with their targets in RISC [22,45], indicating direct biotinylation is not suitable for miRNA target identification. To address this issue, we recently developed photo-clickable miRNA that pre-tagged miRNA with tetrazole groups on 3'-miRNAs without affecting their function, which allowed further attachment of affinity tags onto miRNA-mRNA complexes post their binding [47]. Moreover, combination of other bio-orthogonal reactions, such as click reaction and tetrazine reaction [46], should further improve the accuracy and efficiency of miRNA target identification through miRNA probes bearing bio-orthogonal groups and should allow simultaneous target identification for different miRNAs in same biological environment.…”

PEG10 as an oncogene: expression regulatory mechanisms and role in tumor progression

“…Several miRNAs have also been proven to regulate the expression level of PEG10. In HCC, miR-122 repressed the translation level of PEG10 via directly binding to sites 2310 and 2403 in PEG10 3′-UTR [ 37 , 38 ]. Additionally, miR-491 has also been confirmed to negatively regulate the expression of PEG10 directly in colorectal cancer [ 24 ].…”