Alternate strand DNA triple helix formation

Washbrook, Elinor Mary (1996) Alternate strand DNA triple helix formation. University of Southampton, Doctoral Thesis.

Abstract

Oligonucleotides can bind to duplex DNA in a sequence specific manner, forming triple standard DNA. These triplex forming oligonulceotides present a method for targeting discrete sequences in DNA that could be exploitedto control gene expression, for example by repression of oncogenes or inactivation of viral genes. Bases in the third strand form hydrogen bonds with purines in double stranded DNA, and therefore only form at homopurine sequences. These are two types of triplex, which are dependent on the composition and orientation of the third strand. Third strands composed prodominantly of pyrimidines possess a parallel orientation relative to the purine strand of the duplex, whilst purine-rich third strands run antiparallel to the duplex purines.

One method for extending the range of sequences that can be targeted by triplex formation is to use oligonucleotides designed to form complexes which contain both triplex motifs. In this strategy purines on opposite duplex strands are recognised by different motifs. I have examined the formation of such triplexes at (purine)_n(pyrimidine)_m(RY) and (pyrimidine)_n(purine)_m(YR) junctions. For example, G₅T₅·A₅C₅ can be targeted with T₅G₅. The G-containing half of the duplex is targeted by purines, using the antiparallel motif, while the A-containing half is recognised by pyrimidines using the parallel motif. The two halves of the third strand possess the same orientation, and can therefore form a continuous triplex that crosses from one strand of the duplex to the other, called an alternate strand triplex. Since the third strand crosses from one side of the duplex to the other, at the junctions, the structures of RY and YR complexes are not the same. At the RY junction the third strand is thought to skip one or two duplex base pairs, whereas the YR junction requires one or two additional bases to bridge the duplex.

I have studied the factors which affect the formation and stability of alternate strand triplexes. Initial experiments used simple target sites with sequences A₆C₆·G₆T₆ (RY) and C₆A₆·T₆G₆ (YR), which were targeted with short acridine-linked oligonucleotide, Acr-T₅G₅ and Acr-G₅T₅ respectively. The target sites were cloned into longer DNA fragments, and triplex formation was assessed by DNase I footprinting. As expected triplex formation at the RY junction was more stable than at the YR junction. These experiments were extended using longer sequences, A₁₁(TC)₆·(GA)₆T₁₁ and T₁₁(AG)₆A₁₁, generating triplexes with unmodified oligonucleotides, such as T₁₁(AG)₆ and (GA)₆T₁₁. By using these target sites I have examined how the formation and stability of complexes at the two types of target sequences are affected by the by different base triplets, buffers conatining different divalent cations, and mismatches between the bases of the third strand and the duplex. Since the third strand is thought to skip over one or two base pairs at the RY junction, I have investigated the structure of triplexes formed at the A₁₁(TC)₆·(GA)₆T₁₁ sequence. I have also cloned target sites containing two extra base pairs at the RY junction, with general sequence A₁₁NN(TC)₆·(GA)₆NNT₁₁, to determinewhether different bases at the junction would affect the formation of the complexes. Finally, I have used several different cleavage agents which are sensitive to different features of DNA structure, in an attempt to further characterise the nature of the central RY junction of these alternate strand triplexes.

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