Factors affecting the stability of nucleic acids

Bijapur, Jeevan (1999) Factors affecting the stability of nucleic acids. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Knowledge of the thermodynamic strength of nucleic acid duplexes and triplexes has many important applications, in particular for use in the antisense and antigene strategies. The work described here is divided into three main parts:-

1. DNA/RNA hybrids. The effect of sequence on the stability of phosphorothioate modified DNA/RNA hybrids was studied by ultraviolet melting for use in antisense therapy. It has been found that it is more favourable to target purine rich or mixed sequence RNA in preference to pyrimidine rich RNA in order to form a stable complex. Several modifications have also been made to the DNA strand of the DNA/RNA hybrid in order to assess their relative effect on duplex stability. The 5-propyne pyrimidine modification gave the largest increase in stability while the methyl phosphonate modified backbone gave the largest decrease in stability.

2. Crystallography. In order to elucidate the structural reason for the large increase in duplex stability through propyne addition, X-ray diffraction studies were employed. Propyne modified oligonucleotides were synthesised and conditions for crystal growth have been optimised. In addition, the conditions for crystallisation of an RNA sequence were optimised. This will help to study the relationship between RNA structure and stability.

3. Triple helical DNA. The effect of DNA modifications on triplex stability have been studied. Chemical synthesis of modified oligonucleotides, followed by ultraviolet melting and DNase1 footprinting studies indicated a large and pH dependent increase in triplex stability on 5-propargylamino dU addition, and 5-bisamino dU addition. In contrast, addition of amino groups to the 5 position of deoxycytidine has only a minor effect. A method for the synthesis of the promising 5-propynylguanidinium dU modification, is also reported.

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