DIRECTED EVOLUTION OF ENZYMES USING OPTICAL SORTING

DESCRIPTION: (Adapted from the Applicant's Abstract): Noninvasive methodologies for assaying enzymatic activity within cells are currently unable to differentiate between increased levels of protein expression and improved catalytic activity. In protein engineering, the former category mutants are often considered false positives and are usually the result of 'promoter up' mutations, rather than the desired physicochemical effect of improving enzyme efficiency. It is proposed to use the fluorescence signal from the green fluorescent protein (GFP) in fusion with enzymes to normalize for expression levels in single cells assayed by noninvasive optical techniques. In model studies, E-coli strains expressing a GFP-glucosidase fusion protein will be screened by quantitative imaging techniques. The chromogenic signal resulting from enzyme-catalyzed turnover of the substrate X-glu will be normalized by the fluorescence signal from the co-translated GFP tag, thus giving a measure of the specific activity of the enzyme. This optical screening procedure will be validated by constructing a series of GFP-glucosidase fusions in which the glucosidase gene product is either wild-type or impaired due to mutations affecting the rate of substrate turnover. Optical parameters (absorbance combined with fluorescence) will be measured on a cell sorter for selecting repetitively mutagenized bacteria to isolate enzymes with novel catalytic activities from very large mutagenized populations (>106 clones). $ = TOTAL AWARD AMTS & NOT LIMITED TO PORTION OF PROJECT RELATED TO SUBJECT OF SEARCH SUBPROJECT $ = TOTAL AWARD AMOUNT DIVIDED BY NUMBER OF SUBPROJECTS SOURCE: CRISP FORMAT F FY 97 LAST UPDATE 04-07-98 1QUERY 1536 ID SEARCH 06/01/98 PAGE 339 --PROJECT NUMBER......1 R43 GM56044-01 INVESTIGATOR NAME/ADDRESS FY 97 SCARINGE, STEPHEN A IRG/INTRAMURAL UNIT..ZRG3 DHARMACON RESEARCH, INC AWARD AMOUNT......... $99,714 3200 VALMONT RD #5 BOULDER, CO 80301 PERFORMING ORGANIZATION: DHARMACON RESEARCH, INC. TITLE CHEMICAL SYNTHESIS OF RNA VIA NOVEL STRATEGY ABSTRACT: The growing interest in RNA structural components, ribozymes and other RNA functions has generated a need for reliable methods of synthesizing small RNA oligonucleotides (less than 50 nucleotides). Although RNA synthesis can be accomplished for some applications by knowledgeable specialists via either biochemical methods, e.g. transcription, or chemically, e.g. 5'-dimethoxytrityl-2'-silyl chemistry, there is a definite need for improved RNA synthesis methods. RNA synthesis would be considerably more powerful if readily accessible to biologists, who play central roles in driving advances in health-related research. Final processing of synthesized RNA must be done under mild, sterile conditions with minimal handling. Our final processing is accomplished under mild aqueous conditions, pH 3, 55 angstroms C for 10 minutes. The purpose of this study is twofold: (1) establish that this chemistry can be made commercially available, i.e. research and develop the novel protecting group so that they are amenable to large scale economical synthesis, (2) establish the general utility of this chemistry by synthesizing a variety of RNA oligonucleotides to be evaluated by several collaborators. Accomplishment of these goals would sastify the market need in the research community for ready access to RNA oligonucleotides. $ = TOTAL AWARD AMTS & NOT LIMITED TO PORTION OF PROJECT RELATED TO SUBJECT OF SEARCH SUBPROJECT $ = TOTAL AWARD AMOUNT DIVIDED BY NUMBER OF SUBPROJECTS SOURCE: CRISP FORMAT F FY 97 LAST UPDATE 04-07-98 1QUERY 1536 ID SEARCH 06/01/98 PAGE 340 --PROJECT NUMBER......1 R43 GM56045-01 INVESTIGATOR NAME/ADDRESS FY 97 NAZARENKO, IRINA A IRG/INTRAMURAL UNIT..ZRG2 ONCOR, INC AWARD AMOUNT......... $99,499 209 PERRY PARKWAY GAITHERSBURG, MD 20877 PERFORMING ORGANIZATION: ONCOR, INC. TITLE SIMULTANEOUS AMPLIFICATION AND DETECTION OF DNA ABSTRACT: Although nucleic acid amplification is an important diagnostic tool, its use in clinical practice is limited by the complexity of protocols and the problem of carry-over contamination, which is a source of false- positive results. We suggest a new method for direct detection of amplification products in a closed system. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The primers are designed in such a way that the fluorescent signal is generated only when the primers are incorporated into the amplification product. The proposed technology can eliminate carry-over contamination, simplify the amplification assay, and open up new possibilities for cost-effective automated devices for amplification. The feasibility of this technique has been demonstrated in preliminary experiments. The objective of this Phase I project include: i) determination of optimal structures of oligonucleotide primers and locations of energy transfer labels giving highest signal to background ration along with the maximum amplification efficiency; ii) determination of the laboratory equipment for the simultaneous and quantitive detection for the large amount of reactions. iii) comparison of the sensitivity of the proposed method with traditional amplification/detection techniques. In Phase II study we will assess the clinical utility of this method.