Probing RNA structure and RNA-protein interactions using nanopore force spectroscopy.

摘要

Since the first demonstration of electrophoretically threading of single-stranded nucleic acid through the alpha-Hemolysin (alpha-HL) protein pore in 1996, the use of nanopores for the study of various biological systems has grown rapidly, leading to new and exciting applications in both biophysics and nano-biotechnology. One of the most prominent applications of nanopores is a method called 'Nanopore Force Spectroscopy' (NFS), which has enabled studies of DNA structures and DNA-protein interactions, at the single-molecule level. Much less work has been reported on RNA, despite its vast biological relevance. The objective of this thesis is to further extend NFS to probe the secondary structure of RNA and RNA-protein interactions, with an emphasis on revealing the dynamical properties of these bio-complexes' formation at the single molecule level.;In this thesis, I present three studies related to specific processes involving RNA secondary structures and its interaction with relevant proteins. (1) The kinetics of polyadenylic acid (poly(A)) base stacking-unstacking, which plays a role in the translational control at the 3' end of messenger RNA. The confinement of an RNA molecule inside a protein channel slows its kinetics by three orders of magnitude as compared with bulk measurement of free poly(A) in solution. A phenomenological model describing our results is also described. (2) The unzipping kinetics of double-stranded RNA molecules. Our study reveals clear differences between RNA and DNA duplexes with same sequence, highlighting the nanopore's ability to probe subtle differences in nucleic acids' free energy and their interactions with the pore. (3) Finally, the sensitivity of the nanopore system is used to examine the subtle interaction between poly(A) and the poly(A) binding protein (PABP), which is part of the molecular pathway associated with translation initiation control. For the first time the cooperative and non-cooperative binding modes of multiple PABPs on the same poly(A) strand are directly observed at the single molecule level. The decreased cooperative binding events of C-terminus truncated PABP supports that C-terminus is the PABP-PABP interaction domain, in agreement with previous bulk biochemical studies.