Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase.
Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase.
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DOI：
10.1073/pnas.2107900118
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发表时间：
2021-10-19
影响因子：
11.1
通讯作者：
Collins K
中科院分区：
文献类型：
--
作者：
Upton HE;Ferguson L;Temoche-Diaz MM;Liu XM;Pimentel SC;Ingolia NT;Schekman R;Collins K
反转录转座子是一种非感染性、可移动的遗传元件，通过RNA中间体在宿主基因组中增殖，RNA中间体通过逆转录酶(RT)酶复制到DNA中。RT对于涉及从RNA中捕获信息的生物技术应用非常重要，因为RNA首先被转化为互补DNA用于检测或测序。在这里，我们对来自两个逆转录元件的RT进行了生化表征，并揭示了几种活动，这些活动使我们能够设计一种简化、高效的工作流程，以确定已处理的RNA池中的RNA序列清单。非逆转录病毒RT活性的独特性质避免了与目前的RNA序列分析方法相关的许多技术问题，在研究、生物技术和诊断中得到了广泛的应用。自私的非长末端重复序列(Non-LtR)逆转录元件和可移动的第二组内含子编码逆转录酶(RT)，可以启动DNA合成，而不需要大量的碱基配对。这些酶的生化特性一直受到重组表达挑战的限制，阻碍了对其性质的了解，以及对其性质的可能开发用于研究和生物技术。我们用家蚕的一个改良的非LTRRT和真细菌的第二组内含子RT研究了代表性的RTS的活性。只有非LTRRT支持稳健和连续的模板跳跃，从多个模板产生一个互补DNA(CDNA)，每个模板端到端复制。我们还发现了RTS的一个意想不到的末端脱氧核苷酸转移酶活性，它将选择的核苷酸(S)添加到单链和/或双链RNA3‘端。结合这两种类型的活性和对双链cDNA产品的非模板核苷酸添加的额外见解，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适配器融合到两个cDNA端。当使用microRNAs参考池(MiRNAs)进行基准测试时，使用重组非LTR逆转录元件RT的有序双模板继电器(OTTR)的文库生产优于所有商业可获得的试剂盒，并与技术要求苛刻的家庭酿造协议的低偏见相媲美。我们应用OTTR来清点从细胞外小泡中纯化的RNA，识别miRNAs以及无数其他非编码RNAs(NcRNAs)和ncRNA片段。我们的结果为自动化友好、低偏差、端到端的复杂ncRNA样本RNA序列清单建立了OTTR的实用程序。
Retrotransposons are noninfectious, mobile genetic elements that proliferate in host genomes via an RNA intermediate that is copied into DNA by a reverse transcriptase (RT) enzyme. RTs are important for biotechnological applications involving information capture from RNA since RNA is first converted into complementary DNA for detection or sequencing. Here, we biochemically characterized RTs from two retroelements and uncovered several activities that allowed us to design a streamlined, efficient workflow for determining the inventory of RNA sequences in processed RNA pools. The unique properties of nonretroviral RT activities obviate many technical issues associated with current methods of RNA sequence analysis, with wide applications in research, biotechnology, and diagnostics. 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.
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