Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale. Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3′ ends of single- and/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.

自私的、非长末端重复 (non-LTR) 逆转录元件和移动组 II 内含子编码逆转录酶 (RT)，无需引物和模板的大量碱基配对即可启动 DNA 合成。这些酶的生化表征受到重组表达挑战的限制，阻碍了对其特性的理解以及将其特性用于研究和生物技术的可能性。我们使用来自家蚕的改良非 LTR RT和来自直肠真杆菌的II 组内含子 RT研究了代表性 RT 的活性. 只有非 LTR RT 支持稳健和连续的模板跳跃，从几个模板中产生一个互补 DNA (cDNA)，每个模板都端到端复制。我们还发现了 RT 的意外末端脱氧核苷酸转移酶活性，可将选择的核苷酸添加到单链和/或双链 RNA 或 DNA 的 3' 末端。将这两种类型的活动与关于向双链 cDNA 产品添加非模板核苷酸的额外见解相结合，我们开发了一种简化的协议，用于在单个 RT 反应中将下一代测序接头融合到两个 cDNA 末端。当使用 microRNA (miRNA) 参考池进行基准测试时，使用重组非 LTR 逆转录元件 RT 由有序双模板中继 (OTTR) 生产的文库优于所有市售试剂盒，并与技术要求高的自制方案的低偏差相媲美。我们将 OTTR 应用于从细胞外囊泡中纯化的 RNA 清单，识别 miRNA 以及无数其他非编码 RNA (ncRNA) 和 ncRNA 片段。我们的结果确立了 OTTR 在复杂 ncRNA 样本的自动化友好、低偏差、端到端 RNA 序列库存方面的效用。