DNA recognition by triple helix formation

Abstract

The work described in this thesis uses DNase I footprinting and thermal melting experiments to examine the triplex-forming properties of several novel nucleoside analogues.

The simplest strategy to alleviate the charge repulsion problem is to incorporate positively charged moieties into the TFO by either modifying the backbone, the sugar or the base. Triplexes containing three or more substitutions with the bis-amino modified thymidine analogue 2’-aminoethoxy,5-propargylamino-U (BAU) were shown to be stable at pH 7.0, even though they contain several C.GC base triplets. In contrast, the unmodified TFO product a stable triplex only at pH 5.0 and in the presence of magnesium. These modified TFOs retain exquisite sequence specificity, with enhanced discrimination against YR base pairs (especially CG) and a requirement for binding in the parallel motif.

To overcome the pH dependency of triplex formation 2’-aminoethoxy derivatives of N7G and 6-oxo-C were examined for their ability to recognise GC base pairs at different pH values. Both derivatives produced less stable complexes than C at pH 5.0 but were of a similar stability at pH 6.0. A better approach was achieved by combining the analogue 3-methyl,2-aminopyridine (^MeP) with BAU. Oligonucleotides containing three substrates of each analogue extended triplex formation to pH 7.5 with a nanomolar binding affinity. Oligonucleotides in which these analogues were evenly distributed throughout the third strand bound much better than those in which they were clustered.

The recognition of pyrimidine bases within a target oligopurine tract was also investigated using modified nucleosides. Of these, a substituted pyrrolopyrimidone-2one analogue (^APP) selectively recognised CG with the highest affinity. To improve the stability of the G.TA triplet several deoxyguanosine derivatives were also studied. The addition of either a propargylamino chain to the base or 2’-aminoethoxy group to the sugar enhanced the affinity of this base for GC and not TA.

By using triplex-forming oligonucleotides that contain four of such modified nucleosides it was possible to achieve recognition of each of the four base pairs by triple helix formation at physiological pH. Fluorescence melting and DNase I footprinting demonstrated successful triplex formation at a polypurine.polypyrimidine target site that contained two CG and two TA interruptions. The complexes were pH dependent but were still stable at pH 7.5. Three of the four analogues used retained considerable selectivity (BAU, ^MeP, ^APP) and single base changes opposite these residues cause a large reduction in affinity. In contrast, S was less selective and tolerated CG base pairs as well as TA.

Lastly, the formation of DNA triple helices at target sites that contain mismatches in the duplex target was investigated.

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