Nucleic Acids Research, 1995, Vol. 23, No. 14 2692-2705
© 1995
CHEMISTRY |
UV spectroscopic identification and thermodynamic analysis of protonated third strand deoxycytidine residues at neutrality in the triplex d(C+-T)6:[d(A-G)6d(C–-T)6]; evidence for a proton switch
Department of Molecular Biology, Princeton University Princeton, NJ 08544–1014, USA
*To whom correspondence should be addressed
Received March 28, 1995. Accepted June 6, 1995.
Near-UV difference spectral analysis of the triplex 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 pKa+. Additional support for ion-dlpole 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 triplex and target duplex shows that between pH 5.0 and 8.5 in 0.15 M NaCI/0.005 M MgCI2 the enthalpy of melting (
H°obs) varies from 5.7 to 6.6 kcal.moM for the duplex in a duplex mixture and from 7.3 to 9.7 kcal.moh–11 for third strand dissociation in the triplex mixture. We have extended the condensationscreening 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 
.The number of protons released in the dissociation of the third strand from the target duplex at pH 7.0, n2, is thereby calculated to be 5.5, in good agreement with approximately six third strand dC residues per mole of triplex. This work shows that when third strand binding requires protonated residues that would otherwise be neutral, triplex 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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Guga, M. Janicka, A. Maciaszek, B. Rebowska, and G. Nowak Hoogsteen-Paired Homopurine [RP-PS]-DNA and Homopyrimidine RNA Strands Form a Thermally Stable Parallel Duplex Biophys. J., November 15, 2007; 93(10): 3567 - 3574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. FABREGAT, K. S. KOCH, T. AOKI, A. E. ATKINSON, H. DANG, O. AMOSOVA, J. R. FRESCO, C. L. SCHILDKRAUT, and H. L. LEFFERT Functional pleiotropy of an intramolecular triplex-forming fragment from the 3'-UTR of the rat Pigr gene Physiol Genomics, March 8, 2001; 5(2): 53 - 65. [Abstract] [Full Text] [PDF]
|
|