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 tha t the third strand dC residues are protonated at pH 7.0, far above the ir intrinsic pK(a). Additional support for ion-dipole interactions bet ween the third strand dC residues and the G . C target base pairs come s from reduced positive dependence of triplet stability on ionic stren gth below 0.9 M Na+, inverse dependence above 0.9 M Na+ and strong pos itive dependence on hydrogen ion concentration, Molecular modeling (AM BER) 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 i s energetically feasible, van't Hoff analysis of the melting of the tr ipler and target duplex shows that between pH 5.0 and 8.5 in 0.15 M Na Cl/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 mix ture, 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 obtai n 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, De lta n(2), is thereby calculated to be 5.5, in good agreement with appr oximately 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 me diated by proton uptake and release, i.e., a proton switch, As a by-pr oduct of this study, we have found that at low pH the Watson-Crick dup lex d(A-G)(6) . d(C-T)(6) undergoes a transition to a parallel Hoogste en duplex d(A-G)(6) . d(C+-T)(6).