PARTIAL PURIFICATION OF SOLUBLE RNA

Abstract

A method of fractionation of S-RNA molecules has been described which is based on the ability of periodate to oxidize to aldehydes the unesterified 2[image] and 3[image] hydroxyl groups of the terminal ribosyl group of S-RNA. S-RNA previously stripped of amino acids is relabeled with a single amino acid, L-valine, which becomes esterified to the 3[image] or 2[image] hydroxyl group of the terminal ribose, thus blocking periodate oxidation of this amino acid-bearing RNA molecule. The remaining free 3[image] and 2[image] hydroxyl groups of the terminal ribosyl groups present in the remainder of the molecules in the S-RNA are oxidized by periodate. 2-Hydroxy-3-naphthoic acid hydrazide is now added, and forms a hydrazone with the aldehyde groups. Upon addition of o-dianisidine to this solution of hydrazono-RNA, a blue dye is formed by coupling of the diazonium compound with the hydroxy-naphthoic acid hydrazono-RNA. This dye-bound RNA is sufficiently different in solubility from the original RNA in concentrated phosphate buffer at pH 7.5 to be separable by stepwise addition of n-propanol. A partial purification of valine-RNA has been achieved, with thus far a 12-fold enhancement of specific activity. This procedure appears to have a general applicability to the separation of other aminoacyl-RNA molecules from the family of closely related RNA molecules generally believed to play a role in the coding of amino acids for the sequence-aligning step in protein synthesis.