Development and Modification of Pre-miRNAs with a FRET Dye Pair for the Intracellular Visualization of Processing Intermediates That Are Generated in Cells.

microRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs), which regulate gene expression via the RNA interference (RNAi) system. miRNAs have attracted enormous interest because of their biological significance and disease relationship. In cell systems, the generation of miRNA is regulated by multiple steps: the transfer of primary miRNA from the nucleus to the cytosol, the generation of the precursor-miRNA (pre-miRNA), the production of double-stranded RNA from pre-miRNA by the Dicer, the interaction with protein argonaute-2 (AGO2), and the subsequent release of one strand to form miRISC with AGO2. In this study, we attempt to visualize the intermediates that were generated in the miRNA-maturation step in the cells to acquire a detailed understanding of the maturation process of miRNA. To achieve this, we developed pre-miRNAs labeling with a Dicer- or AGO2-responsible fluorescence resonance energy transfer (FRET) dye pair. We observed that modifications with the dye at suitable positions did not interfere with the biological activities of pre-miRNAs. Further, imaging analyses employing these pre-miRNAs demonstrated that the processing of pre-miRNA promoted the accumulation of miRNA at the specific foci in the cytosol. The FRET-labeled pre-miRNA would further elucidate the mechanisms of the RNAi process and provide the basis for development of nucleic acid drugs working in the RNAi system.

Introduction of 2,6-Diaminopurines into Serinol Nucleic Acid Improves Anti-miRNA Performance.

MicroRNAs (miRNAs) are endogenous small RNAs that regulate gene expression at the post-transcriptional level by sequence-specific hybridisation. Anti-miRNA oligonucleotides (AMOs) are inhibitors of miRNA activity. Chemical modification of AMOs is required to increase binding affinity and stability in serum and cells. In this study, we synthesised AMOs with our original acyclic nucleic acid, serinol nucleic acid (SNA), backbone and with the artificial nucleobase 2,6-diaminopurine. The AMO composed of only SNA had strong nuclease resistance and blocked endogenous miRNA activity. A significant improvement in anti-miRNA activity of the AMO was achieved by introduction of a 2,6-diaminopurine residues into the SNA backbone. In addition, we found that the enhancement in AMO activity depended on the position of the 2,6-diaminopurine residue in the sequence. The high potency of the SNA-AMOs suggests that these oligomers will be useful as therapeutic reagents for control of miRNA function in patients and as tools for investigating the roles of microRNAs in cells.