Jianxun Lin - Ph.D Final Oral Examination

When: July 29, 2011 (Fri), 02:00PM to 03:00PM (add to my calendar)
Location: SCI 352

This event is part of the PhD Final Oral Exams.

Title of Dissertation:  Probing RNA Structure and RNA-Protein Interations Using Nanopore Force Spectroscopy

Examining Committee:  Amit Meller, Kenneth Rothschild, Ophelia Tsui, Sidney Redner, Robert Carey

ABSTRACT
Since the first demonstration of electrophoretically threading of single-stranded nucleic acid through the alpha-Hemolysin (-HL) protein pore in 1996, the use of nanopores for the study of various biological systems has rapidly grown, leading to new and exciting applications in both biophysics and nano-biotechnology. One of the most prominent applications of nanopore 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 protein. (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 a 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 similar bulk thermodynamic properties, highlighting the nanopore 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.