Revised Manuscript Received August 24, 1994® abstract: Electrostatic effects dominate many aspects of nucleic acid behavior in a sequence independent manner. Sequence dependent electrostatic effects are introduced when a polypyrimidine, which contains one or more protonated cytosines, binds in the major groove (Hoogsteen side) of a complementary Watson—Crick double helix. Depending on the number of cytosines in the third strand (global effect) and on their relative position (local effect), the cytosines either enhance or decrease the binding affinityof the third strand, because adjacent protonated cytosines destabilize the third strand binding comparedto cytosines separated by intervening thymines. This local effect (crowding) can reverse the effect of global composition. To investigatethe extent of the local and global electrostatic effects further, two families of oligonucleotides have been synthesized. They share as a common design feature that they all fold sequentially into isosterical intramolecular triple helices by way of hairpin intermediates. This is confirmed by Pi nuclease probing, CD spectroscopy, and UV spectroscopy. The thermalstability of these conformations depends on the sequences, pH, andthe ionic strength and can be summarized as follows: The energy of third strand binding depends on the protonated cytosine content in the Hoogsteen strand. 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 increases with increasing cytosine content below 400 mM Na+, decreases above 400 mM Na+, and is independent of the global composition at 400 mM Na+.

Triple helical RNA complexes have been known since 1957 (Felsenfeld et al., 1957). Subsequently, it was estab-lished that ribo-as well as deoxyribohomopyrimidine polymers can associate with complementary double helical homopurine—homopyrimidine complexes to form triple helices (Riley et al., 1966; Lipsett, 1964; Thiele & Guschl-bauer, 1968; Morgan & Wells, 1968; Amott & Bond, 1973a; Amott & Seising, 1974; Felsenfeld & Miles, 1967; Michelson et al., 1967). Triple helices which contain homopurine strands as third strands also have been observed (Rich, 1958; Howard & Miles, 1977; Thiele et al., 1978; Marck & Thiele, 1978; Amott & Bond, 1973b; Broitman et al., 1987; Letai et al., 1988; Cheng & Pettitt, 1992). The homopyrimidine third strand binds via Hoogsteen base pairing (Hoogsteen, 1959) to the purine bases in the major groove of the Watson—Crick double helix with thymines and/or uracils recognizing only A* T base pairs and protonated cytosines recognizing only G* C base pairs (Amott & Bond, 1973a; Amott & Seising, 1974; Amott & Bond, 1973b; Letai et al., 1988; Amott et al., 1976). The pyrimidine third strand is oriented in parallel to the Watson—Crick homopurine strand (Amott et al., 1976; Hattori et al., 1976; Moser & Dervan, 1987). Initially it was suggested that a change from a B-DNA to an A-DNA conformation occurs on triple helix formation (Amott et al., 1976), but more recent evidence