Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector.
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 RNAI into a vector containing a T7 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 RNAI 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 RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.
Mellits
K H
KH
Cold Spring Harbor Laboratory, New York, NY 11724.
Pe'ery
T
T
Manche
L
L
Robertson
H D
HD
Mathews
M B
MB
D000260:Adenoviruses, Human / Q000235:genetics*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005814:Genes, Viral; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000302:isolation & purification*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013604:T-Phages / Q000235:genetics*; D014158:Transcription, Genetic
D047428:Protein Kinase Inhibitors; D012330:RNA, Double-Stranded; D012367:RNA, Viral; C052835:adenovirus associated RNA; D011494:Protein Kinases