טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMalik Omri
SubjectThe Mechano-Chemistry of Reverse Transcriptase
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor Professor Ariel Kaplan


Abstract

Reverse transcriptase (RT) catalyzes the conversion of retroviral RNA into an integration-competent double-stranded DNA , with a variety of enzymatic activities that include “strand displacement” DNA synthesis, i.e. the ability to unwind nucleic-acids duplexes concomitantly with polymerization. A comprehensive understanding of  strand displacement synthesis by RT and its potential regulation by secondary structure motifs is lacking, in part due to the limitations of traditional biochemical techniques. In our research, we designed and constructed a high-resolution optical-tweezers to study the polymerization activity of the Moloney Murine Leukemia Virus RT on a DNA hairpin template, at the single molecule level and on a wide range of chemical and mechanical conditions. Our results show that strand-displacement polymerization is frequently interrupted by inactive events, which include intrinsic pauses during which the enzyme remains bound to the template and events of enzyme dissociation followed by reinitiation. The pauses were shown to be modulated by the strength of the DNA duplex 8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Moreover, our results indicate that RT functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that RT is a relatively passive enzyme, able to polymerize on structured templates only by exploiting their thermal breathing. Taken together, our results elucidate the mechanism of polymerization by RT and emphasize the potential regulatory role of conserved structural motifs, which may provide useful information for the development of better inhibitors to function as anti-retroviral drugs. Finally, to further explore the process of reverse transcription, in particular the interplay between the enzyme’s function and the elongation complex structure, we designed and built a new and versatile instrument, Fleezers, that uses acousto-optic modulation  combineto combines high-resolution optical tweezers with different modalities of single-molecule fluorescence detection. As  a proof-of-concept of the integrated system, we used the Fleezers to stretch a DNA tether and applied epi-fluorescence imaging and confocal detection to monitor the fluorescence of a trapped fluorescently-labeled microspheres. These demonstrations have indicated the Fleezers feasibility to simultaneously study RT  function-structure relationships and its interaction with other viral or cellular factors by multipole single-molecule techniques.