One-step enzymatic modification of RNA 3' termini using polymerase θ.

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 (Pol). We demonstrate that Polefficiently 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 Pol, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polshows 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 Polwill become invaluable for applications requiring 3 terminal modification of RNA and potentially enzymatic synthesis of RNA.