Determination of base pairing in ribonucleic acid by Fourier-transform infrared spectrometry: yeast ribosomal 5S ribonucleic acid

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Abstract
Fourier-transform infrared (FT-IR) spectra of yeast ribosomal 5S RNA were acquired at several temperatures between 30.degree. and 90.degree. C. The difference spectrum between 90.degree. (bases unstacked) and 30.degree. C (bases stacked) provides a measure of base stacking in the RNA. Calibration difference spectra corresponding to stacking of G-C or A-U pairs are obtained from reference FT-IR spectra of poly-(rG).cntdot.poly(rC) minus 5''-GMP and 5''-CMP or poly(rA).cntdot.poly-(rU) minus 5''-AMP and 5''-UMP. The best fit linear combination of the calibration G-C and A-U difference spectra to the 5S RNA (90-30.degree. C) difference spectrum leads to a total of 25 .+-. 3 base pairs (17 G-C pairs + 8 A-U pairs) for the native yeast 5S RNA in the absence of Mg2+. In the presence of Mg2+, an additional 6 base pairs are detected by FT-IR (one G-C and five A-U). FT-IR melting curve midpoints show that A-U and G-C pairs melt together (65 and 63.degree. C) in the presence of Mg2+ but A-U pairs melt before G-C pairs (47 vs.54.degree. C) in the absence of Mg2+.

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