Raman studies of nucleic acids VIII estimation of RNA secondary structure from raman scattering by phosphate-group vibrations

Abstract

Analysis of the Raman spectra of a large number of ribonucleotide monomers and polymers indicates that the frequencies and intensities characteristic of the P-O stretching vibrations provide a basis for the quantitative determination of RNA secondary structure. The intensity ratio of the Raman lines at 815 and 1100 cm −1 is shown to have a value of 1.64 ± 0.04 in completely ordered structures and a value of zero in disordered structures. Ordered structures in this context include double-helical configurations containing A · U or G · C pairs and ordered single-stranded configurations (such as occur in poly(rA) and poly(rC)) but exclude other type of base pairing as well as random-chain configurations. The percentages of RNA nucleotides in such ordered configurations, as computed from Raman intensity measurements, are 16-S RNA ( 95 ± 5), 23-S RNA ( 85 ± 5), tRNA fMet ( 84 ± 3), tRNA Val ( 84 ± 3), tRNA Phe ( 85 ± 3) and R17 RNA ( 87 ± 3 %). Since the present method makes use of spectral transitions originating in the RNA backbone and not in the bases, no limitations are imposed by lack of knowledge of the RNA base sequence. In combination with data obtained from quantitative infrared spectroscopy the Raman results are also shown to yield information on the percentages of RNA nucleotides in ordered single-stranded configurations.