Jm. Benevides et al., RAMAN SIGNATURE OF THE 4-STRANDED INTERCALATED CYTOSINE MOTIF IN CRYSTAL AND SOLUTION STRUCTURES OF DNA DEOXYCYTIDYLATES D(CCCT) AND D(C-8), Biochemistry, 35(18), 1996, pp. 5747-5755
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.