UV SPECTROSCOPIC IDENTIFICATION AND THERMODYNAMIC ANALYSIS OF PROTONATED 3RD STRAND DEOXYCYTIDINE RESIDUES AT NEUTRALITY IN THE TRIPLEX D(C+-T)(6)[D(A-G)(6)CENTER-DOT-D(C-T)(6)] - EVIDENCE FOR A PROTON SWITCH

Near-UV difference spectral analysis of the tripler formed from d(C-T)(6) and d(A-G)(6) . d(C-T)(6) in neutral and acidic solution shows that the third strand dC residues are protonated at pH 7.0, far above their intrinsic pK(a). Additional support for ion-dipole interactions between the third strand dC residues and the G . C target base pairs comes from reduced positive dependence of triplet stability on ionic strength below 0.9 M Na+, inverse dependence above 0.9 M Na+ and strong positive dependence on hydrogen ion concentration, Molecular modeling (AMBER) of C:G . C and C+:G . C base triplets with the third strand base bound in the Hoogsteen geometry shows that only the C+:G . C triplet is energetically feasible, van't Hoff analysis of the melting of the tripler and target duplex shows that between pH 5.0 and 8.5 in 0.15 M NaCl/0.005 M MgCl2 the enthalpy of melting (Delta H-obs(o)) varies from 5.7 to 6.6 kcal.mol(-1) for the duplex in a duplex mixture and from 7.3 to 9.7 kcal.mol(-1) for third strand dissociation in the tripler mixture, We have extended the condensation-screening theory of Manning to pH-dependent third strand binding, In this development we explicitly include the H+ contribution to the electrostatic free energy and obtain partial derivative T-m /partial derivative(In[H+]) = 1/2 Delta n(2)/Z(2) R(T-m)(2)/Delta H-2. The number energy of protons released in the dissociation of the third strand from the target duplex at pH 7.0, Delta n(2), is thereby calculated to be 5.5, in good agreement with approximately six third strand dC residues per mole of tripler, This work shows that when third strand binding requires protonated residues that would otherwise be neutral, tripler formation and dissociation are mediated by proton uptake and release, i.e., a proton switch, As a by-product of this study, we have found that at low pH the Watson-Crick duplex d(A-G)(6) . d(C-T)(6) undergoes a transition to a parallel Hoogsteen duplex d(A-G)(6) . d(C+-T)(6).