Acid multimers of oligodeoxycytidine strands: stoichiometry, base-pair characterization, and proton exchange...

The structure recently proposed for the acid form of the oligonucleotide 5'-d(TC5) is a four-strand "tetrad" in which two parallel-stranded, base-paired duplexes are intimately associated, with their hemiprotonated C-C+ base pairs face-to-face and fully intercalated, in a so-called "i-motif" (Gehring et al., 1993). We use the amino and imino proton spectra to establish the structure and symmetry of the base pairs, properties which are a primary element in the resolution of the acid form describe above. The amino proton spectrum gives the best lower limit (8 x 10(4) s-1) on the rate of the imino proton jumping process which is responsible for the base-pair symmetry. The stoichiometry of the acid form of other deoxycytidine sequences is studied by gel filtration chromatography and in one case by an NMR equilibrium titration. In all cases, i.e., d(C12), d(T2C8T2), d(C4TC4), d(TC5), d(C5), d(C4), d(TC4), d(TC3T), and d(TC3), the acid form elutes as a tetramer. A single-strand component is also present in some cases. But no dimer is observed, except for some samples prepared by quenching from high temperatures. The characteristic H1'-H1' interresidue NOESY cross-peaks of the d(TC5) structure (Gehring et al., 1993) are also found in all the tetramers where they have been searched for, i.e., those of d(T2C8T2), d(C4TC4), d(TC3T), and d(TC3) (not shown), suggesting that these tetramers also are built on the i-motif and that such structures may be formed generally by strands containing a stretch of as little as three deoxycytidines. From the NMR titration of d(TC3), we derive a free energy of -7.6 kJ/mol per cytidine base pair for the formation of the tetramer from single strands. The free energy released by packing a base pair into the i-motif is comparable to that released in forming the base pair itself. Imino proton exchange is limited by base-pair opening, thanks to efficient intrinsic exchange catalysis: this explains the lack of effect of added catalysts. The base-pair lifetime is hundreds of times longer than in any DNA duplex, presumably due to the base-pair intercalation geometry. The variation of the lifetime along the sequence of the d(TC5) tetramer provides support for the recently proposed structure. The internal amino proton exchanges from the open state of the C-C+ pair, at a rate compatible with a pK of 9 appropriate for C+. But the external proton exchanges from the closed state, as with a pK of 17!(ABSTRACT TRUNCATED AT 400 WORDS)