Efficient Inhibition of RNA Self-primed Extension by Addition of Competing 3'-capture DNA-improved RNA Synthesis by T7 RNA Polymerase
Overview
Affiliations
In vitro synthesized RNA is used widely in studies of RNA biology, biotechnology and RNA therapeutics. However, in vitro synthesized RNA often contains impurities, such as RNAs with lengths shorter and longer than the expected runoff RNA. We have recently confirmed that longer RNA products are formed predominantly via cis self-primed extension, in which released runoff RNA folds back on itself to prime its own RNA-templated extension. In the current work, we demonstrate that addition of a DNA oligonucleotide (capture DNA) that is complementary to the 3' end of the expected runoff RNA effectively prevents self-primed extension, even under conditions commonly used for high RNA yields. Moreover, the presence of this competing capture DNA during 'high yield' transcription, leads to an increase in the yield of expected runoff RNA by suppressing the formation of undesired longer RNA byproducts.
Bou-Nader C, Link K, Suddala K, Knutson J, Zhang J Nat Commun. 2025; 16(1):2252.
PMID: 40050622 PMC: 11885821. DOI: 10.1038/s41467-025-57519-w.
Bacteriophage RNA polymerases: catalysts for mRNA vaccines and therapeutics.
Nair A, Kis Z Front Mol Biosci. 2024; 11:1504876.
PMID: 39640848 PMC: 11617373. DOI: 10.3389/fmolb.2024.1504876.
Understanding the impact of transcription byproducts and contaminants.
Lenk R, Kleindienst W, Szabo G, Baiersdorfer M, Boros G, Keller J Front Mol Biosci. 2024; 11:1426129.
PMID: 39050733 PMC: 11266732. DOI: 10.3389/fmolb.2024.1426129.
MalagodaPathiranage K, Banerjee R, Martin C Nucleic Acids Res. 2024; 52(17):10607-10618.
PMID: 39011885 PMC: 11417385. DOI: 10.1093/nar/gkae599.
DNA-terminus-dependent transcription by T7 RNA polymerase and its C-helix mutants.
Yu B, Chen Y, Yan Y, Lu X, Zhu B Nucleic Acids Res. 2024; 52(14):8443-8453.
PMID: 38979568 PMC: 11317132. DOI: 10.1093/nar/gkae593.