Effects of high pressure on DNA and its components : a thesis presented in partial fulfilment of the requirements for the degree of PhD in Bio Physics at Massey University, Manawatū, New Zealand

Abstract

There have been many speculations for the environment in which life originated but it has still yet to be determined what environmental chemical and physical conditions were necessary for the evolution of self-replicating chemical systems. While it has been determined that DNA, RNA and their components are chemically unstable at high temperatures, there has currently been only a small number of studies into the role of high pressures on the chemical and physical stabilities. High-pressure NMR spectroscopy has been used here to study the effects of high temperatures/pressures on the chemical stability of DNA and its components. This has been done with the use of a specialised commercial high-pressure NMR cell capable of withstanding pressures up to 250 MPa. In addition to this, a custom safe handling apparatus and pump system was developed for the operation of this cell. Studies into the effects of high pressures on the rate of hydrolysis of cytosine and cytidine at 100 °C were performed by measuring the rates of hydrolysis with time under various pressure conditions. These results have shown that the rates of hydrolysis of cytosine and cytidine increase considerably with pressure. The effects of high pressure on the physical stability of DNA were determined by performing dissociation (melting) experiments on several different DNA sequences under multiple pressure conditions. It was found that the melting point of a small DNA hexamer decreased slightly with pressure whereas the melting points of larger dodecamers increased overall with pressure. It was also found that the melting point of an i-motif structure decreased with increasing pressure. The effects of high pressure on the chemical stability of cytosine were again studied, this time for cytosine residues within both single- and double-stranded DNA. DNA samples for bacteriophage FX174 were incubated under various temperature and pressure conditions. Results for these studies have yet to be determined as the incubated DNA is yet to be sequenced. It has been discovered that high pressures have a negative effect on the chemical stability of DNA constituents while having an overall small positive effect on the physical stability of DNA.