Determination of the base pairing content of ribonucleic acids by infrared spectroscopy

✍ Scribed by George J. Thomas Jr.

• Publisher: Wiley (John Wiley & Sons)
• Year: 1969
• Tongue: English
• Weight: 544 KB
• Volume: 7
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.1969.360070305

✦ Synopsis

A method is described for determiniig the fractions of adenineuracil and guaninecytosine base pain of partly double-helical ribonucleic acids in aqueous solution. The method is based upon the ability to distinguish between paired and unpaired bases by means of infrared spectroscopy of their deuterium oxide solutions in the 150&1800 cm-' frequency region of the infrared spectrum. An application of the method to yeast ribosomal RNA is described. At 30°C, ribosomal RNA is 6 4 f 6% paired (35% GC, 29% AU). The method can be applied to determine the fractions of Watson-Crick base pairs at a given temperature in any RNA containing the four common bases in known ratios.

INTRODIJCTION

Many types of ribonucleic acid, including t-RNA, r-RNA, and 5S-RNAt from a variety of sources, are believed to adopt a secondary structure in solution which is partly double-helical and partly single-stranded.

Recently much attention has been given to the problem of determining the amount of base pairing which contributes to the secondary structure of aqueous RNA. Methods of ultraviolet (UV) absorption and optical rotatory dispersion (ORD) spectroscopy, first described by Felsenfeld and c~llaborators,~~~ have been extensively used in studies of RNA secondary structure. 612

While these studies have contributed to our understanding of the gross features of RNA secondary structure, more detailed knowledge of the extent and kind of base pairing is required. The UV results, in particular, are subject to rather large uncertain tie^'^.'^ and can provide at present only a rough estimate of the double helicity of RNA. This is due in part to the complexity of the broad UV absorption band of RNA which cannot be clearly resolved into components from the four bases separately and thus cannot yield detailed information about the relative amounts of AU and