Selectivity of RNA chain initiation in vitro. 1. Analysis of RNA initiations by two-dimensional thin-layer chromatography of 5'-triphosphate-labeled oligonucleotides

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Abstract
A method for the rapid and quantitative analysis of 5''-terminal oligonucleotides of RNA made in vitro is described. The method involves synthesis of RNA in the presence of [.gamma.-32P]ATP or GTP, isolation of the RNA, and digestion with T1 or pancreatic RNases to release labeled 5''-triphosphate terminated oligonucleotides. The oligonucleotides are then subjected to chromatography on a polyethyleniminecellulose thin-layer systems using 2 M LiCl, 0.01 M EDTA (pH 6.5) in the 1st dimension and 1.5 M LiCl, 1.8 M formic acid, 0.005 M EDTA (pH 2.0) in the second. RNA made with Escherichia coli RNA polymerase and .lambda.cb2, T7, T4 and adenovirus 2 DNA yield characteristic fingerprint patterns. The utility of this method in studying selectivity of in vitro RNA chain initiation is discussed.