One-step enzymatic modification of RNA 3′ termini using polymerase θ
Pomerantz, Richard T.
GroupFels Cancer Institute for Personalized Medicine (Temple University)
SubjectNucleic acid enzymology
Permanent link to this recordhttp://hdl.handle.net/20.500.12613/8765
MetadataShow full item record
AbstractSite-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3′ terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3′ termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30–50 2′-deoxyribonucleotides to the 3′ terminus of RNA molecules of various lengths and sequences, and extends RNA 3′ termini with an assortment of 2′-deoxy and 2′,3′-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3′ terminal modification of RNA and potentially enzymatic synthesis of RNA.
CitationCrystal Thomas, Timur Rusanov, Trung Hoang, Taurai Augustin, Tatiana Kent, Imre Gaspar, Richard T Pomerantz, One-step enzymatic modification of RNA 3′ termini using polymerase θ, Nucleic Acids Research, Volume 47, Issue 7, 23 April 2019, Pages 3272–3283, https://doi.org/10.1093/nar/gkz029
Citation to related workOxford University Press
Has partNucleic Acids Research, Vol. 47, Iss. 7
ADA complianceFor Americans with Disabilities Act (ADA) accommodation, including help with reading this content, please contact firstname.lastname@example.org
Except where otherwise noted, this item's license is described as Attribution-NonCommercial CC BY-NC