Quantitation of ribonucleotides from base-hydrolyzed RNA using capillary zone electrophoresis.

Sir: Ribonucleic acid (RNA) is a biopolymer consisting of four different ribonucleosides connected by phosphate units through 3’-5’ linkages on the ribose rings. Specificity of RNA is derived by the sequence of the four bases, of which two are purines (adenine [A] and guanine [GI) and two are pyrimidines (cytosine [C] and uracil [U]). Unlike in deoxyribonucleic acid (DNA), a double-stranded biopolymer, pairing between purine and pyrimidine bases need not occur in RNA. Consequently, there is much interest in determining the ratio of all four “major” bases in different kinds of RNA, as well as the amount of modified bases (e.g. ribothymidine and pseudouridine) which may constitute up to 15% of tRNA. Additional interest is centered on the determination of the quantity of radiolabel incorporated into RNA and free nucleoside monophosphates (nucleotides) in metabolic studies. Toward these ends, several methods of molecular discrimination have been previously employed to separate nucleotides acquired from complete hydrolysis of RNA. These include paper electrophoresis ( I ) , ion-exchange chromatography (2), high-performance liquid chromatography (HPLC) (3), and isotachophoresis (4) . Recently, capillary zone electrophoresis (CZE) has been shown to be applicable to the problem of nucleotide and oligonucleotide separation (5-7). We report the ability of CZE with UV absorbance detection to distinguish isomers of the four main ribonucleotides, as well as the possible use of this system in quantitation. CZE compares favorably to other separation techniques especially in the areas of limited sample quantity, resolution, and time of analysis. With on-line UV detection (254 nm), CZE provides a 5-min separation of the main ribonucleotides with a detection limit correlating to less than 0.1 ng of RNA.