Final Report Summary - BIOPHYSRETRAIN (Biophysics of Reverse Transcription and its Inhibition)
The process of reverse-transcription, in which a single-stranded RNA is copied into a double-stranded DNA, is an obligatory step in the life cycle of retroviruses, and is catalyzed by the enzyme Reverse Transcriptase (RT). Of particular importance is the RT of the Human Immunodeficiency Virus (HIV), responsible for the Acquired Immunodeficiency Syndrome (AIDS). As a major target for therapy, HIV RT has been the subject of extensive research. However, despite an ever-increasing amount of information from traditional studies, the detailed mechanisms of RT’s action are still not completely understood. This is due, partially, to the stochastic nature of individual enzymes, which makes it impossible to follow more than one or two individual steps of their activity using classical, ensemble-based, methods. Single-molecule methods are well suited to overcome this synchronization problem.
In the framework of this project, we have conducted a mechanistic study of HIV RT, by developing a high-resolution optical tweezers, capable of following the polymerization and translocation of RT, at the single-molecule level, and at a broad range of forces and nucleotides concentrations. The project represents a truly interdisciplinary approach to the life sciences: On one side, we have developed an instrument capable of applying mechanical forces on biological molecules and complexes, and directly measure molecular movements as small as Angstroms. On the other side, we have used this instrument to study the process of synthesis of DNA by the Reverse Transcriptase of the HIV virus. Achieving these goals required development in a number of areas covering a wide spectrum from biology to physics.
Our results can have a fundamental as well as a practical impact: First, our experiments may contribute to a mechanistic understanding of RT’s function, and in general the shared mechanisms of nucleic-acid polymerases. Next, by systematically studying the physical limitations in our instruments, we are developing new designs and methodologies to measure enzymatic movements at increasing detail. Finally, from a practical point of view, our results can provide a fresh look into mechanisms of RT inhibition by existing drugs, as well as the resistance to them developed by certain mutations, and by that may contribute to the design of new and better drugs.
As this was the first and leading project in my lab, it has helped me to establish myself as an independent investigator, and to integrate in the Technion community as a teacher and researcher. My research group is now fully functional, and we have established a number of promising collaborations, in my university and beyond.
In the framework of this project, we have conducted a mechanistic study of HIV RT, by developing a high-resolution optical tweezers, capable of following the polymerization and translocation of RT, at the single-molecule level, and at a broad range of forces and nucleotides concentrations. The project represents a truly interdisciplinary approach to the life sciences: On one side, we have developed an instrument capable of applying mechanical forces on biological molecules and complexes, and directly measure molecular movements as small as Angstroms. On the other side, we have used this instrument to study the process of synthesis of DNA by the Reverse Transcriptase of the HIV virus. Achieving these goals required development in a number of areas covering a wide spectrum from biology to physics.
Our results can have a fundamental as well as a practical impact: First, our experiments may contribute to a mechanistic understanding of RT’s function, and in general the shared mechanisms of nucleic-acid polymerases. Next, by systematically studying the physical limitations in our instruments, we are developing new designs and methodologies to measure enzymatic movements at increasing detail. Finally, from a practical point of view, our results can provide a fresh look into mechanisms of RT inhibition by existing drugs, as well as the resistance to them developed by certain mutations, and by that may contribute to the design of new and better drugs.
As this was the first and leading project in my lab, it has helped me to establish myself as an independent investigator, and to integrate in the Technion community as a teacher and researcher. My research group is now fully functional, and we have established a number of promising collaborations, in my university and beyond.