Salt effects on duplex formation by dGCATGC have been studied with spectroscopic, thermodynamic, and kinetic methods. Circular dichroism spectra indicate different salt conditions have little effect on the structures of the duplex and single strand. NMR chemical shifts indicate the structure of the duplex in 1 M NaCl is similar to that of the B-form determined previously in 0.5 M KCl [Nilges, M., Clore, G. M., Gronenborn, A. M., Brunger, A. T., Karplus, M., & Nilsson, L. (1987) Biochemistry 26, 3718-3733]. Optical melting experiments indicate the effect of Na+ concentration on melting temperature is similar to that expected for a polynucleotide with the same GC content. Laser temperature-jump experiments indicate the effect of Na+ concentration on the rate of duplex formation is much less than is observed for polynucleotides. The observations are consistent with expectations based on a counterion condensation model. This is surprising for a duplex with only 10 phosphates.

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The presence of A n and A n T n tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B‘-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A n tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B‘- to B-DNA. Structural features of A n and A n T n tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)2 (A3T3) and d(CGCATATATGCG)2 [(AT)3]. These studies address the presence of three-centered H-bonds in the B‘ conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A3T3 duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A3T3 duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar−phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH2 and dT C4O vibrational modes suggest that the A3T3 duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)3 duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.

The pentapurine sequence GGAGA, located between 80 and 84 nucleotides downstream of the cleavage site in the self-cleaving antigenomic RNA of hepatitis delta virus, is necessary for highly efficient cleavage and for stability in up to 20 M formamide. Yet much of the cleavage activity lost upon its removal from the 3' end of an 84-nucleotide RNA can be restored by elongation of the 5' end of the RNA with the polypyrimidine sequence found in the virus. We propose that this sequence alteration causes a refolding of the RNA, resulting in a "structural compensation" of the active core of the molecule. Restoration of the self-cleavage activity did not restore the ability to cleave in high concentrations of formamide. Deletion mutagenesis was carried out and supported the alternate RNA folding. The ability to assume more than one active conformation and for one RNA structure to compensate for another in supporting ribozyme activity may be unique to RNA enzymes and could be a useful adaptation in viruses or in prebiotic RNAs.