A possible method for characterizing the secondary structure of ribonucleic acids.

The E(280)/E(260) ratio was found to be suitable for following the ionization of cytosine residues of polynucleotides on the basis of studies with model compounds such as oligoguanylic acid, oligocytidylic acid, a complex formed between polyadenylic acid and polyuridylic acid, and a copolymer of guanylic acid and cytidylic acid, provided that changes in secondary structure were taken into account. The pK of cytosine residues of a polynucleotide in the amorphous form was found to be 4.70 at 25 degrees in 0.1m-sodium phosphate on the basis of titration at 75-85 degrees and on the assumption that the heat of ionization was the same as the value (5.2kcal./mole) found for CMP. In contrast, the pK of cytosine residues in the double-helical form of DNA was found to be about 3.25. These observations were utilized in estimating the fraction of cytosine residues in helical segments of ribosomal RNA, a copolymer of guanylic acid and cytidylic acid, and a copolymer of adenylic acid, guanylic acid, uridylic acid and cytidylic acid. The ionization of guanine and uracil residues was estimated from changes in the E(270)/E(260) ratio and E(230)/E(260) ratio respectively. In the amorphous form of RNA both residues had the same pK, whereas in the double-helical form ionization was suppressed. The fraction of guanine and uracil residues in amorphous segments may be estimated from the titration curves. The difference in the denaturation spectrum of adenine--uracil and guanine--cytosine base pairs at 280mmu was enhanced in acidic solutions whereas E(260) was hardly affected. Hence a comparison of the increments in E(280) and E(260) obtained on increasing the temperature at constant pH may be used to distinguish the melting ranges of helical domains differing in nucleotide composition. In alkaline solutions comparison of the increments in E(260) and E(270) yields similar information. In acidic solutions the fraction of cytosine residues involved in helical secondary structure, the degree of ionization of cytosine residues and the fraction of adenine--uracil base pairs denatured may be estimated from DeltaE(265) and DeltaE(280). In alkaline solutions the fractions of guanine and uracil residues involved in secondary structure and the degrees of ionization of these residues may be estimated from DeltaE(230), DeltaE(245), DeltaE(260) and DeltaE(280).