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One-step enzymatic modification of RNA 3′ termini using polymerase θ
Thomas, Crystal Hoang, Trung Augustin, Taurai Kent, Tatiana Gaspar, Imre Pomerantz, Richard T.
Thomas, Crystal
Hoang, Trung
Augustin, Taurai
Kent, Tatiana
Gaspar, Imre
Pomerantz, Richard T.
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Journal article
Date
2019-04-23
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Biochemistry
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https://doi.org/10.1093/nar/gkz029
Abstract
Site-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.
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Crystal 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
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Oxford University Press
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Nucleic Acids Research, Vol. 47, Iss. 7
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