Mechanistic analysis of gene-expression machinery and DNA nanodevices using single-molecule fluorescence spectroscopy

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This proposal describes the construction and further development of a special, ultra-sensitive light microscope along with proposals for using this microscope to study how tiny biological machines work and how DNA (the basis of genetic material) can be used to transport electricity or energy.The microscope, which we refer to as a single-molecule fluorescence microscope , is specifically designed to allow detection and monitoring of individual ( single ) fluorescent molecules present in a detection zone (as opposed to conventional microscopes that require thousands or millions of molecules to be present in a detection zone). An additional unique feature of the microscope rests on the fact that two or three lasers are used in rapid alternation (alternating much faster than the average time that one molecule spends in the detection zone) for the detection and the analysis of single molecules; the alternating-laser microscope format carries several advantages compared to earlier single-laser versions. This version of the single-molecule fluorescence microscope allows us to determine the number of parts that make up a biological machine, to measure how strong the parts bind to each other, how far apart the parts are spaced and at what orientation, and what are the movements of the parts when the tiny biological machine works.We will use this special microscope to understand processes that occur during gene expression, the path that leads from the genetic information (stored in DNA) to the manufacturing of proteins (the molecules that make up most of the machines and structures of living cells). Specifically, we will focus on the process of gene transcription, which uses tiny biological machines that read DNA and copy the information into a messenger molecule (messenger RNA). We will also study the process of gene silencing, which uses tiny biological machines that recognise and destroy messenger RNA. Finally, we will use our special microscope to develop strategies that will allow us to use DNA molecules as tiny wires that conduct electricity and energy; these DNA-based wires can be useful in building smaller and more efficient electronic circuits.Our results will help the scientific community to understand how gene expression works and why certain diseases arise when gene expression malfunctions; our work on gene expression will also aid in the development of new drugs that will improve health. Finally, our work on DNA-based wires will be helpful in developing the next generation of electronics.