Revisiting the Glass Treatment for Single-Molecule Analysis of ncRNA Function

  • Methods Mol Biol. 2022;2509:209-231. doi: 10.1007/978-1-0716-2380-0_13.
Shuting Shen   #  1 ,  Masahiro Naganuma   #  2  3 ,  Yukihide Tomari  2  4 ,  Hisashi Tadakuma  5  6
Affiliations
  • 1. School of Life Science and Technology & Gene Editing Center, ShanghaiTech University, Shanghai, China.
  • 2. Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan.
  • 3. RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan.
  • 4. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan.
  • 5. School of Life Science and Technology & Gene Editing Center, ShanghaiTech University, Shanghai, China. [email protected].
  • 6. Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan. [email protected].
  • # Contributed equally.
PMID: 35796966 DOI: 10.1007/978-1-0716-2380-0_13
Abstract

Single-molecule imaging is a powerful method for unveiling precise molecular mechanisms. Particularly, single-molecule analysis with total internal reflection fluorescence (TIRF ) microscopy has been successfully applied to the characterization of molecular mechanisms in ncRNA studies. Tracing interactions at the single-molecule level have elucidated the intermediate states of the reaction, which are hidden by ensemble averaging in combinational biochemical approaches, and clarified the key steps of the interaction. However, applying a single-molecule technique to ncRNA analysis still remains a challenge, requiring laborious trial and error to identify a suitable glass surface passivation method. In this chapter, we revisit the major glass surface passivation methods using polyethylene glycol (PEG) treatment and summarize a detailed protocol for single-molecule analysis of the dicing process of Dcr-2, which may apply piRNA studies in the future.

Keywords
Amino-silanization; Dicer 2; Halo ligand; Non-coding RNA (ncRNA); Polyethylene glycol (PEG); SNAP ligand; Single-molecule imaging; Surface passivation; Total internal reflection fluorescence (TIRF) microscopy.
Products