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Volume 16 Number 4 1988 Nucleic Acids Research


Accurate in vitro cleavage by RNase III of phosphorothioate-substituted RNA processing signals in
bacteriophage T7 early mRNA


Allen W. Nicholson, Kenneth R. Niebling*, Paul L. McOsker*, and Hugh D. Robertson*


Department of Biological Sciences, Wayne State University, Detroit, MI 48202 and the *Rockefeller University, New York, NY 10021, USA


Received November 3, 1987; Revised and Accepted January 15, 1988

ABSTRACT
To test the ability of an RNA processing enzyme to cleave chemically- modified RNA substrates, RNA transcripts containing RNase III cleavage sites were enzymatically synthesized in vitro to contain specific phosphorothioate diester internucleotide linkages. One transcript (R1.1 RNA) was generated using phage T7 RNA polymerase and a cloned segment of phage T7 DNA containing the R1.1 RNase III processing site. The second transcript was the phage T7 polycistronic early mRNA precursor, which was synthezsized using E. coli RNA polymerase and T7 genomic DNA. The RNA transcripts contained phosphorothioate diester groups at positions including the scissile bonds. The modified RNAs were stable to incubation in Mg2+ containing buffers, and were specifically cleaved by RNase III. RNA oligonucleotide sequence analysis showed that the modified R1.1 RNA processing site was the same as the cononical site and contained a phosphorothioate bond. Furthermore, RNase III cleaved the phosphoro- thioate internucleotide bond with 5' polarity. RNase III cleavage of phosphorothioate substituted T7 polycistronic early mRNA precursor produced the same gel electrophoretic pattern as that obtained with the control transcript. Thus, RNase III cleavage specificity is not altered by phosphorothioate internucleotide linkages.