Recent groundbreaking discoveries have changed our view on RNA from that of a passive information carrier to an important regulatory element. MicroRNA is a small regulatory RNA that controls nearly all mRNAs in eukaryotic cells. Since this regulation process (termed RNA interference/RNAi) occurs in a sequence-specific manner, we can manipulate gene expression using custom-designed small RNAs. This remarkable discovery introduced the possibility of RNA-based gene therapy and triggered intensive research on the RNA-induced silencing complex (RISC), the core machinery of RNAi. The molecular mechanism of RISC is, however, poorly understood due to the limited spatial and temporal resolution of traditional tools, which has deterred development of an RNAi assay applicable to medical sciences.I will use single-molecule fluorescence to investigate the entire process of RISC action with high spatio-temporal resolution. From ‘RISC assembly’ through ‘target mRNA search’ to ‘target mRNA degradation,’ it requires the cooperative action of multiple RISC components. As the protein-protein and protein-RNA interactions are dynamic processes, it is challenging to study them in bulk where the interactions are diffusion-limited and subsequent processes are masked from observation. With single-molecule microscopy, I will observe all the processes in real time and quantitatively examine the kinetics. In addition, I will dissect the complex processes of RISC action by observing multiple RISC components simultaneously, using multicolor FRET that I have developed. Furthermore, to elucidate the complex nature of RNAi, I will reconstitute protein complexes with a single-molecule immunoprecipitation technique that I have recently innovated.This first single-molecule study on RISC will enable us to reveal novel molecular mechanisms of RNAi. The fruitful outcome will aid the development of RNAi free from off-target interactions, which will lead to RNAi-based gene therapy in the near future.