Abstract
The stability of a triple helix formed between a DNA duplex and an incoming oligonucleotide strand strongly depends on the solvent conditions and on intrinsic chemical and conformational factors. Attempts to increase triple helix stability in the past included chemical modification of the backbone, sugar ring, and bases in the third strand. However, the predictive power of such modifications is still rather poor. We therefore developed a method that allows for rapid screening of conformationally diverse third strand oligonucleotides for triplex stability in the parallel pairing motif to a given DNA double helix sequence. Combinatorial libraries of oligonucleotides of the requisite (fixed) base composition and length that vary in their sugar unit (ribose or deoxyribose) at each position were generated. After affinity chromatography against their corresponding immobilized DNA target duplex, utilizing a temperature gradient as the selection criterion, the oligonucleotides forming the most stable triple helices were selected and characterized by physicochemical methods. Thus, a series of oligonucleotides were identified that allowed us to define basic rules for triple helix stability in this conformationally diverse system. It was found that ribocytidines in the third strand increase triplex stability relative to deoxyribocytidines independently of the neighboring bases and position along the strand. However, remarkable sequence-dependent differences in stability were found for (deoxy)thymidines and uridines.
Highlights
It has been shown previously that triplexes form within the pure DNA or RNA backbone context and within mixed RNA and DNA strands, with distinct differences in stability within a given sequence context [8]
Recent analyses by NMR and FT-IR have shown that D:DD and R:DD triple helices have heterogeneous backbone conformations, leading to energetically compromised conformations for certain ribo- and deoxyribonucleotides in the three strands (9 –15)
After affinity chromatography on the immobilized target double helix, utilizing a temperature gradient as the selection criterion, and subsequent deconvolution of the obtained fractions by chemical and analytical means, the molecular features of triplex-forming oligonucleotides (TFOs) leading to enhanced triple helix stability were defined
Summary
It has been shown previously that triplexes form within the pure DNA or RNA backbone context and within mixed RNA and DNA strands, with distinct differences in stability within a given sequence context [8]. The method is based on the synthesis of a combinatorial library of TFOs containing either ribo- or deoxyribonucleosides with the requisite base at each position in the chain Both types of nucleoside units intrinsically prefer different sugar conformations (3Ј-endo for ribonucleosides and 2Ј-endo for deoxyribonucleotides) and give rise to a large variety of backbone conformations in the corresponding TFOs. After affinity chromatography on the immobilized target double helix, utilizing a temperature gradient as the selection criterion, and subsequent deconvolution of the obtained fractions by chemical and analytical means, the molecular features of TFOs leading to enhanced triple helix stability were defined. This method lends itself for use in the assay of oligonucleotides containing new, chemically modified nucleoside analogues, as it does not rely on an enzymatic amplification and in vivo deconvolution step typically used in DNA and RNA selection protocols
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