Stability of triple helices containing RNA and DNA strands: experimental and molecular modeling studies

Abstract

UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.

Prediction of the stability of DNA triplexes.

“…4B). Once again, the model shows no apparent bias in GC content or pH. There are five cases where our prediction is off by -11-13°C (24,(29)(30)(31)(32). In two cases (29,30), our underestimate of tm is likely due to the high spermine concentration used (0.5-1.0 mM).…”

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides

“…These studies suggest that the triple helical structures favor a conformation that is distinct from both canonical A-(C3'-endo sugar puckering) and B-form (C2'-endo puckering) helices. Thermodynamic studies (33,38,39), including our own results on foldback triplexes (manuscript in preparation), show a preference of triple helix structures for a DNA homopurine strand and an RNA Hoogsteen hydrogen bonding third strand consistent with structural evidences. In light of these observations, we propose that the quadruplex destabilization by FTFOs is directly related to their ability to form energetically and structurally stable triplexes.…”

“…We attribute the inability of oligonucleotide 3 to destabilize the RNA quartet structure to its formation of a less stable triple helix with RNA target. Recent studies by others (33)(34)(35)(36)(37)(38)(39) and in our laboratory (manuscript in preparation) on the stabilities of triplexes containing different RNA/DNA compositions revealed that a triplex of DNA-RNA-DNA is not stable (24) because of structural reasons. In solutions containing potassium (100 mM potassium acetate, pH 5.0 buffer), oligonucleotide 3 was less effective in destabilizing DNA quadruplex structure (Fig.…”

Recognition of duplex DNA by RNA polynucleotides

“…Because the pyrimidine motif requires slightly acidic pH to promote protonation of oligonucleotide cytosines (6,19,20), the pH-independent purine motif has been thought to provide a more viable approach for in vivo applications involving DNA oligonucleotides (14,17,18,21). However, whereas RNA can participate as the third strand in triple helices of the pyrimidine motif (22)(23)(24), it is not accommodated in the purine motif (24)(25)(26). Therefore, our initial studies of duplex DNA recognition by oligomeric RNA (24) (Fig.…”

“…If such triple helices exist in natural systems, it would appear essential that they be stabilized at physiological pH by novel structural features or accessory proteins. This limitation is particularly evident because of the inability of the pH-independent purine triple helix motif to accommodate RNA strands (24)(25)(26).…”