THE CONTRIBUTION OF CYTOSINE PROTONATION TO THE STABILITY OF PARALLELDNA TRIPLE HELICES

The influence of the position of the CG.C+ triplet and the contributio n of protonation at the N3 of the Hoogsteen cytosine residue on the st ability of various sequences of parallel triple helices having the gen eral composition d[(A(5)G)-x-(T5C)-x-(T5C)] and d[(A(4)G(2))-x-(T4C2)- x-(T4C2)], where x is the hexaethylene glycol linker, has been determi ned by NMR, ultraviolet melting and absorbance spectrophotometry. The apparent pK value, i.e. the pH at which the observable has changed by 50% of its range, was typically in the range 6 to 7. However, the NMR spectra unequivocally showed that the pK of the protonated cytosine re sidue must be at least 9.5 for internal positions. This is five units above the pK of the free nucleotide, and represents a free energy of s tabilisation from protonation of >11.5 RT. The pK of terminal cytosine residues is much lower, in the range 6.2 to 7.2, accounting for a fre e energy of stabilisation from protonation of 3.6 to 6 RT. The van't H off enthalpies were determined for the dissociation of the protonated tripler into the duplex + strand, and for the duplex to strand transit ion. The mean value for the duplexes were 23 to 27 kJ mol(-1) base-pai r, and 25 to 30 kJ mol(-1) for the triplexes containing internal CG.C triplets. Good agreement was obtained for the thermodynamic parameter s by the different methods. Free energy differences for the transition between the protonated tripler and the duplex + protonated strand wer e calculated at 298 K. The Delta G of stabilisation of an internal CG. C triplet compared with a terminal CG.C triplet was about 6 kJ mol(-1) ; a similar stabilisation was observed for the triplexes containing tw o CG.C triplets compared with those containing a single CG.C triplet. The very large stabilisation from protonation is too large to be accou nted for by a single hydrogen bond, and is likely to include contribut ions from electrostatic interactions of the positive charge with the p hosphate backbone, and more favourable interactions between neighbouri ng bases owing to the very different electronic properties of the prot onated C. (C) 1998 Academic Press Limited.