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Nucleic Acids Research, 1990, Vol. 18, No. 18 5401-5406
© 1990

MOLECULAR BIOLOGY

Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNA1 from a T7 vector

Kenneth H. Mellits, Tsafrira Pe'ery, Lisa Manche, Hugh D. Robertson1 and Michael B. Mathews*

Cold Spring Harbor Laboratory PO Box 100, Cold Spring Harbour, New York, NY 11724 1Department of Biochemistry, Cornell University Medical College New York, NY 10021, USA

*To whom correspondence should be addressed

Received June 27, 1990. Revised August 14, 1990. Accepted August 14, 1990.

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNA1 into a vector containing a 17 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA dependent protein kinase DAI. Exact copies of VA RNA1 were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNA1 and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.


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