Super-Resolution Force Spectroscopy for Revealing Sub-nucleotide Motion in Ribosomal Frameshifting and DNA Methylation

Programmed ribosomal frameshifting plays an important role for many viruses in their protein synthesis, with well-known cases including HIV and SARS-CoV-2. Aiming for an approach to defend against such viruses, techniques including X-ray crystallography, cryo-electron microscopy (Cryo-EM) and fluorescent resonance energy transfer (FRET) have been utilized to study the translocation mechanism. However, it remains challenging to directly resolve the different reading frames. This dissertation focuses on applying super-resolution force spectroscopy (SURFS) as a unique tool to resolve frameshifting products and intermediate states, with unprecedented resolution. Two different forms of forces have been studied, centrifugal force and acoustic radiation force. Using centrifugal force with 2 pN resolution that is sufficient to resolve a single nucleotide difference, we were able to reveal the efficiency of SufB2-suppressed frameshifting. Combined with collaborators’ kinetic measurements, our results identified twice exploration of +1 frameshifting in an elongation cycle, one during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the second during occupancy in the P site. Using acoustic radiation force, the force resolution was improved to 0.5 pN, sufficient to resolve a single hydrogen bond difference. Taking the advantage of the precision, we observed a new intermediate state in frameshifting induced by the GA7G slippery motif beside its original “-1” and “-2” frameshifting complexes in the presence of fusidic acid. We termed this new state as “-1*”, whose dissociation force was shifted by approximately half a nucleotide compared to the normal “-1” reading frame at the 5’-end of the mRNA. With this unprecedented force resolution, we were also able distinguished DNA duplexes with 1 and 2 methylated cytosine compared with the nonmethylated duplexes. Moreover, interactions between d-/l-THP and DNA duplexes were studied. We found that single-methylation only partly limits the interaction between l-THP and DNA, when leaving d-THP – DNA interaction changed. We have utilized SURFS as a uniquely capable tool in those biophysical systems with small stability differences, where no other force techniques have sufficient resolution to deal with. Broader applications of SURFS are expected in both fundamental and applied biological research.