Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.
Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.
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DOI：
10.1261/rna.039743.113
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发表时间：
2013-07
期刊：
影响因子：
--
通讯作者：
Lambowitz AM
中科院分区：
文献类型：
--
作者：
Mohr S;Ghanem E;Smith W;Sheeter D;Qin Y;King O;Polioudakis D;Iyer VR;Hunicke-Smith S;Swamy S;Kuersten S;Lambowitz AM
本文描述了 II 组内含子编码的逆转录酶的不寻常特性。它们比逆转录病毒酶具有更高的持续合成能力、保真度和热稳定性。此外，它们具有意想不到的模板切换倾向，这使得它们对于许多克隆应用程序非常有用。移动组 II 内含子编码逆转录酶 (RT)，逆转录酶在内含子移动性（“逆向归巢”）中发挥作用，该过程需要对高度结构化的 2-2.5-kb 内含子 RNA 进行逆转录，具有高持续性和保真度。尽管后者的特性对于 cDNA 合成和下一代 RNA 测序 (RNA-seq) 的应用可能有用，但 II 组内含子 RT 很难纯化而不含内含子 RNA，并且它们作为研究工具的实用性尚未得到系统研究。在这里，我们开发了用于高水平表达和纯化 II 组内含子编码的 RT 作为具有刚性连接、不可切割的溶解性标签的融合蛋白的通用方法，并将它们应用于来自嗜热细菌的 II 组内含子 RT。由此，我们获得了热稳定性 II 族内含子 RT 融合蛋白，其比逆转录病毒 RT 具有更高的持续合成能力、保真度和热稳定性，可在高达 81°C 的温度下合成 cDNA，并且在 qRT-PCR、用于 RNA 结构图谱的毛细管电泳和下一代 RNA 测序方面具有显着优势。此外，我们发现 II 组内含子 RT 在模板转换方面与逆转录病毒酶不同，与新 RNA 模板 3' 末端的碱基配对最少，使得无需 RNA 连接酶步骤即可有效、无缝地将含有 PCR 引物结合位点的接头连接到 cDNA 末端。这种新颖的模板转换活性使得非聚腺苷酸化 RNA（例如 miRNA 或蛋白质结合 RNA 片段）的克隆变得容易且偏差较小。我们的研究结果证明了 II 组内含子 RT 在研究、生物技术和诊断方法方面的新颖生化活性和固有优势，具有潜在的广泛应用。
This paper describes unusual properties of group II intron-encoded reverse transcriptases. They have higher processivity, fidelity, and thermostability than retroviral enzymes. Moreover, they have an unexpected proclivity for template switching that makes them very useful for a number of cloning applications. Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility (“retrohoming”) by a process that requires reverse transcription of a highly structured, 2–2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.
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