Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, PA, 19140, USA.
European Molecular Biology Laboratory, Heidelberg, Meyerhofstrasse 1, 69117, Germany.
Nucleic Acids Res. 2019 Apr 23;47(7):3272-3283. doi: 10.1093/nar/gkz029.
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.
在各种基础研究和应用研究中，对合成和细胞 RNA 进行特定核苷酸、荧光团和连接化学的位点特异性修饰非常重要。然而，缺乏简单有效的方法来修饰 RNA，例如在 3'末端用特定核苷酸或与各种化学部分偶联的核苷酸类似物进行修饰。在这里，我们开发并表征了一种使用重组人聚合酶θ（Polθ）一步酶法修饰 RNA 3'末端的方法。我们证明 Polθ 可以有效地将 30-50 个 2'-脱氧核苷酸添加到各种长度和序列的 RNA 分子的 3'末端，并使用各种包含功能化学部分的 2'-脱氧和 2'、3'-二脱氧核苷酸类似物扩展 RNA 3'末端，例如高亲和力连接部分和荧光团。与 Polθ 不同，末端脱氧核苷酸转移酶（TdT）根本无法将 RNA 用作底物。总体而言，Polθ 对向 RNA 添加脱氧核苷酸表现出强烈的偏好，但在特定序列环境下也可以相对高效地添加核糖核苷酸。我们预计 Polθ 的这种独特活性将对需要 RNA 3'末端修饰和潜在酶促 RNA 合成的应用变得非常宝贵。
