A method is presented for acquiring sequence data from single nucleic acid molecules. The approach involves a fluorescence resonance energy transfer assay (FRET) based on molecules involved in protein biosynthesis. The fluorescence signal is acquired from single molecules using a fluorescence correlation spectroscope in several configurations, including measurements in solution, on surfaces and in nanocavities (zero-mode waveguides). The project's specific aims are to: (i) perform site-directed labeling with a fluorescent dye and quencher;(ii) optimize the FRET assay;(iii) construct a synthetic template and demonstrate the performance of the system on this template;(iv) investigate nanostructures capable of enhancing fluorescence, (v) study the behavior of single molecules in the system;and (vi) demonstrate the capability of the system to acquire high volumes of sequence data. We will also explore the use of nanoparticles as fluorescent tags and quenchers. The method, once fully developed, will allow fast analyses of many types of nucleic acids, including whole genomes, and will lead to ultra-low cost genome sequencing, in accordance with the NIH's $1,000 genome program. The method can be used for nucleic acid diagnostics in vitro and in drug discovery.
| Liu, Wei; Kavaliauskas, Darius; Schrader, Jared M et al. (2014) Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation. ACS Chem Biol 9:2421-31 |
| Chudaev, Maxim; Poruri, Kiran; Goldman, Emanuel et al. (2013) Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation. Protein Eng Des Sel 26:347-57 |
| Gryczynski, Ignacy; Luchowski, Rafal; Matveeva, Evgenia G et al. (2012) Metal-enhanced immunoassays. Methods Mol Biol 875:217-29 |
| Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin et al. (2011) Enhancement of single-molecule fluorescence signals by colloidal silver nanoparticles in studies of protein translation. ACS Nano 5:399-407 |
| Perla-Kajan, Joanna; Lin, Xin; Cooperman, Barry S et al. (2010) Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules. Protein Eng Des Sel 23:129-36 |
| Luchowski, Rafal; Sabnis, Sushant; Szabelski, Mariusz et al. (2010) Self-quenching of uranin: Instrument response function for color sensitive photo-detectors. J Lumin 130:2446-2451 |
| Sørensen, Thomas J; Laursen, Bo W; Luchowski, Rafal et al. (2009) Enhanced Fluorescence Emission of Me-ADOTA by Self-Assembled Silver Nanoparticles on a Gold Film. Chem Phys Lett 476:46-50 |
| Matveeva, Evgenia G; Terpetschnig, Ewald A; Stevens, Megan et al. (2009) Near-infrared squaraine dyes for fluorescence enhanced surface assay. Dyes Pigm 80:41-46 |
| Matveeva, Evgenia G; Shtoyko, Tanya; Gryczynski, Ignacy et al. (2008) Fluorescence Quenching/Enhancement Surface Assays: Signal Manipulation Using Silver-coated Gold Nanoparticles. Chem Phys Lett 454:85-90 |
| Shtoyko, Tanya; Matveeva, Evgenia G; Chang, I-Fen et al. (2008) Enhanced fluorescent immunoassays on silver fractal-like structures. Anal Chem 80:1962-6 |
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