ELECTROSTATIC EFFECTS IN DNA TRIPLE HELICES

Electrostatic effects dominate many aspects of nucleic acid behavior i n a sequence independent manner. Sequence dependent electrostatic effe cts are introduced when a polypyrimidine, which contains one or more p rotonated cytosines, binds in the major groove (Hoogsteen side) of a c omplementary Watson-Crick double helix. Depending on the number of cyt osines in the third strand (global effect) and on their relative posit ion (local effect), the cytosines either enhance or decrease the bindi ng affinity of the third strand, because adjacent protonated cytosines destabilize the third strand binding compared to cytosines separated by intervening thymines. This local effect (crowding) can reverse the effect of global composition. To investigate the extent of the local a nd global electrostatic effects further, two families of oligonucleoti des have been synthesized. They share as a common design feature that they all fold sequentially into isosterical intramolecular triple heli ces by way of hairpin intermediates. This is confirmed by P-1 nuclease probing, CD spectroscopy, and UV spectroscopy. The thermal stability of these conformations depends on the sequences, pH, and the ionic str ength and can be summarized as follows: The energy of third strand bin ding depends on the protonated cytosine content in the Hoogsteen stran d. It increases with increasing cytosine content (global composition) below pH 7.1 (150 mM Na+), decreases above pH 7.1, and is independent of the cytosine content at pH 7.1. At pH 6.75 the energy of binding in creases with increasing cytosine content below 400 mM Na+, decreases a bove 400 mM Na+, and is independent of the global composition at 400 m M Na+.