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+.