Base Analogues and ligands for stabilising triple Helical Nucleic acids

Darby, Richard Anthony James (2002) Base Analogues and ligands for stabilising triple Helical Nucleic acids. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

DNase I footprinting has demonstrated that the thymidine analogues 5-propargylamino dU, 2'-aminoethoxy T, 5-propynyl dU, and 5-aminopropyl dU, do not significantly increase triplex stability when incorporated at only two positions within a 9-mer oligonucleotide. More stable triplexes are generated using oligonucleotides substituted at four positions and examined at pH 5. However oligonucleotides substituted with the bis-modified thymidine analogue 2'-aminoethoxy-5-propargylamino U form stable triplexes at both pH 5 and 7.5, with only three substitutions. Furthermore 5-propargylamino dU cannot be used to stabilise GT containing triplexes formed in either the parallel or antiparallel orientation. Similarly the cytidine analogue 5-aminohexyl dC does not appear to enhance parallel triplex formation.

The requirement for recognition of an uninterrupted polypurine tract has been examined using two charged abasic analogues, 1'-β-hexylaminoribose, and 1'-methoxy, 2'-aminoethoxyribose. However, these do not increase the stability of parallel triplexes across a central pyrimidine base compared to the abasic analogue 1'2-H-dideoxyribose.

Triplex stability may also be enhanced by the use of triplex binding ligands. It is demonstrated that some proflavine derivatives stabilise triplexes containing adjacent T^.AT triplets. Furthermore it is shown that the alpha anomer of naphthoflavone stabilises parallel triplexes independent of the DNA sequence. It appears that positively charged ligands are selective for T^.AT triplets, while neutral ligands bind to both T^.AT and C^+.GC.

This thesis also describes a novel method for measuring DNA thermal denaturation profiles. This technique uses synthetic oligonucleotides containing a fluorophore and quencher which are positioned so that folded complexes have weak fluorescence, which is greatly enhanced upon denaturation. The fluorescent melting profiles are measured using a Roche LightCycler. This technique has been successfully employed for examining the stabilising effect of base analogues and triplex stabilising ligands.

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