Protonated polynucleotide structures. I. The thermal denaturation of polycytidylic acid in acid solution.

Polycytidylic acid is known to form a double-stranded helical structure that is different from either the A or the B form of DNA. X-ray diffraction studies' indicate the structure to be that of a helix similar to that of poly A,2 with the difference being that only one shared proton is bound between the two cytosine residues, this proton forming a third hydrogen bond (Fig. 1). The two protons in the poly A helix are not involved in R hydrogen bond formation, but stabilize the structure by charge effects. Various physicochemical studies3-6 on poly C have led to the conclusion that the double-helical semiprotonated structure found in the fiber diagrams is also present in solution between pH 3.5 and 5.5. It has R been suggested that during thermal denaturation the lbrtd3' 5 Both the poly A and the FG .Poal y shared proton is libera ed. drogen bonding scheme poly C helix have their bases tilted against each other in a between cytosine residues propeller-like fashion" 2so that the strands have to be iofptohehliCal.(acid) form parallel.7 In reviewing the structures formed by polynucleotides,7 inconsistencies appear between similar data on poly C in the literature. It will be shown that these can be readily resolved by noting that thermal denaturation at pH 4 and pH 5 are two different processes, the first probably involving uptake, the second, loss of proton. Materials and Methods.-Poly C was purchased from Miles Chemical Corporation, Clifton, N.J. It was dissolved in 0.001 M Tris-buffer, pH 7.4, to give a concentration of about 5-10 A260 units per ml. This stock solution was dialyzed against five changes of glass-distilled water. Dilutions were made to the desired pH and ionic strength by diluting with appropriate acetate buffer and/or 2 M NaCl solutionis. pH adjustments were made by adding very small amounts of 1 N acetic acid. pH was measured with a Radiometer 22 pH-meter to 0.02 pH units. The pH-meter was regularly calibrated against standard buffer solutions (Titrisol, E. i\Ierck, Darmstadt, Germany). Thermttal denaturation profiles: Thermal detsaturation profiles were recorded its a modified Bausch atid Lomb Spectroinic 505 spectrophotometer coupled with a Leeds-Northrup-i\IECI 6-chaisisel recorder. The jacketed cuvette holder was thermostated with a Haacke Fe thermostat. One channel of the recorder printed the temperature measured by a glass-embedded Pt-thermo-resistance in a tight Teflotl stopper in the cuvette via a Wheatstotne bridge. The five other channels recorded the absorbance through a potelitiolneter coupled to the pen displacement mechanism of the spectrophotometer. For each sample ois which a thermal denaturation profile was recorded, spectra of the cold and the hot solution were recorded on the spectrophotometer recording drum. In many cases a spectrum of the recooled solution was recorded as well.