Part I: Detection of RNA in cells with quenched autoligation probes. Part II: Probing active site tightness in nucleic acid replication enzymes.

摘要

This thesis concerns two research projects. In the first project, the application of Quenched Autoligation (QUAL) probes toward detection of RNA in bacteria is described. QUAL probes are a class of self-reacting nucleic acid probes that generate strong fluorescence signal only in the presence of fully complementary RNAs and are highly selective against single nucleotide mismatches. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored by microscopy and flow cytometry without any washing steps. Discrimination of the closely related bacteria species E. coli, Salmonella enterica, and Pseudomonas putida was achieved, based on single nucleotide differences in their 16S rRNA. A FRET-based QUAL system was developed to allow multiple colors to be visualized under a single fluorescence filter set, thereby simplifying detection protocols. The utility of this approach was demonstrated in an application with clinically important pathogens of the Shigella genus. The results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.;In the second project, a series of nonpolar thymidine and uracil base mimics, varying in size over a 1.0-A range, were used to probe the active site sterics of nucleic acid replication enzymes. The nonpolar base analogs for thymidine and uracil were dihalotoluene and dihalobenzene C-nucleosides, respectively. The synthesis of these compounds and their incorporation into DNA and RNA is reported. Single nucleotide incorporation and extension kinetic studies on human immunodeficiency virus type-1 reverse transcriptase (HIV-RT) were performed. Results showed a large size dependence for the DNA polymerase activity of HIV-RT, but a much smaller size dependence for the reverse transcriptase activity. Kinetics were also performed on E. coli Polymerases II and IV, two SOS-induced replication enzymes. Results for these enzymes showed generally poor incorporation and extension past unnatural base pairs, but in the case of Pol II, very good incorporation of the nonpolar dNTP analogs in a size-dependent manner.