RAMAN SIGNATURE OF THE 4-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 f ormed by intercalation of two hemiprotonated and parallel-stranded oli godeoxycytidylate 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 ti tration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that th e Spectral fingerprint associated with N3 protonation of cytosine is d istinct from that of quadruplex formation. The Raman spectrum thus pro vides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 proton ation in DNA. Application to aqueous d(CCCT) and d(Cs) demonstrates th at the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and t he d(CCCT) quadruplex exhibit a midpoint of titration (apparent PKC) o f 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C-8) is shift ed significantly toward the physiological range, with pK(C) = 5.8 +/- 0.2. The difference in pK(C) between the two quadruplexes is equivalen t to a free energy difference of 1.7 kcal/mol at 10 degrees C. The pre sent findings extend the library of Raman conformation markers to deox ycytidylate residues in the novel i quadruplex. The significance of th ese results for probing solution conformations of telomeric DNA sequen ces is also considered.