Investigation of potential Z-DNA binding proteins in Halobacter salinarum & Preparation and characterization of DNA samples

Abstract

Halophiles require high salt concentrations, from 3.5 up to 5 M, in their environments to function and survive. The adaptations that allow halophiles such as Halobacter salinarum to survive in high saline conditions are well studied. However, the differences in proteins produced by halophiles as compared to organisms inhabiting low salinity environments are not well understood. In this project, oligonucleotides will be developed to study potential Z-DNA binding proteins. Z-DNA forming d(CG)n repeats will be synthesized with a linker tail attached to a non-cleavable solid support, allowing the sequences to be retrieved from H. salinarum lysate together with potential Z-DNA binding proteins. The synthesis of d(CG)18(Spacer9)10 was successful with a 98% detritylation yield. Addition of DBCO phosphoramidites to d(CG)18(Spacer9)10 was unsuccessful. Mass spectrometric analysis of purified products suggested failure in the synthesis. DNA is a stable compound when stored as a dried stock, however, there has been recent interest in using DNA as barcodes to identify oil and natural gas sources underground. For this application, the underground temperature and pH vary, and the effects of these conditions on the stability on DNA have not been studied. In this project, the degradation of a 21 nucleotide DNA sequence will be studied. The DNA sequence was found to be stable at pH above 6 at 30-90°C, but quickly underwent degradation at pH below 5.