Bimolecular DNA Triplexes:  Duplex Extensions Show Implications for H-Form DNA Stability

Abstract

H-form DNA has recently been shown to be biologically relevant by its involvement in the process of homologous recombination [Kohwi, Y. , and Panchenko, Y. (1993) Genes Dev. 7, 1766-1778]. A bimolecular DNA triple-stranded structure (triplex) is central to the formation of H-form DNA. Understanding the formation and factors governing the stability of such bimolecular triplexes is necessary to fully elucidate the structure/function relationship of H-form DNA. In this study, we extend known information on bimolecular triplexes by examining the effect of a variable CNC base triad (where N = A, C, T, or G) on a 10 base triad triplex that mimics the triplex motif in H-form DNA. We also examine the effect that a duplex extension of four base pairs has on triplex stability and selectivity for the base N. Results from thermal denaturation experiments indicate that the fully complementary triplex is more stable than its duplex counterpart (DeltaTm = 13 degrees C) and is resistant to degradation by bovine spleen phosphodiesterase for at least 24 h at 10 degrees C. A single-base mismatch in the purine strand of the triplex structure is destabilizing (DeltaTm = approximately 20 degrees C), and all structures containing a mismatch were readily degraded by bovine spleen phosphodiesterase. An extension of four duplex base pairs onto the triplex structure affects the stability of the DNA complex and may have implications relevant to H-form DNA.

Targeting of single‐stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides

“…The approach of targeting single‐stranded nucleic acids through the formation of triple helices by clamp or circular oligonucleotides [4–15] is based on oligonucleotides containing two domains. One domain is complementary in the Watson–Crick sense and creates a duplex with the target strand, and the other is complementary in the Hoogsteen sense and constitutes the ‘third strand’ of the triple helix.…”

“…One domain is complementary in the Watson–Crick sense and creates a duplex with the target strand, and the other is complementary in the Hoogsteen sense and constitutes the ‘third strand’ of the triple helix. The two domains of the oligonucleotide are attached to each other by nucleotidic [5–10] or non‐nucleotidic [4,11] linking groups. A variety of structures, including clamp [5,10] and circular oligonucleotides [6–9], have been used to form such bimolecular triplexes.…”

“…The two domains of the oligonucleotide are attached to each other by nucleotidic [5–10] or non‐nucleotidic [4,11] linking groups. A variety of structures, including clamp [5,10] and circular oligonucleotides [6–9], have been used to form such bimolecular triplexes. The advantage of the triple helix approach in improving binding affinity and selectivity towards the single‐stranded target, as opposed to standard Watson–Crick recognition, stems from three aspects.…”

“…The targeting of single‐stranded purine tracts by triple‐helix formation has been studied extensively [5–11]. Pyrimidine tracts, however, can also be aimed at forming bimolecular triplexes [13,14].…”

“…All bimolecular triplexes exhibit melting temperature curves with a single transition, signifying that both Watson±Crick and Hoogsteen hydrogen bonds dissociate in the same temperature range. It is important to note that triplexes formed between purine single strands and pyrimidinecontaining circular or clamp oligonucleotides are stable at pH 7 [8,10]. Both DNA [6,7,10,12] and RNA [5,9,14] can be used as single-stranded targets.…”