Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C-8)

The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C. H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the Spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(Cs) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent PKC) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C-8) is shifted significantly toward the physiological range, with pK(C) = 5.8 +/- 0.2. The difference in pK(C) between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered.