Exploring 3Dpol for RNA sequencing in real time

项目摘要 RNA实时测序(RNA-seq)，称为第三代单分子测序 水平，是一项重要的技术，将提高我们对人类基因组的理解。的发展。 然而，由于rna在序列和结构上的复杂性，实时rna-seq一直具有挑战性。 这需要一个具有单核苷酸分辨率的过程性阅读器。而太平洋生物科学公司(PacBio)可以 产生长阅读时，这个过程涉及到cDNA，这会丢失RNA的信息含量。唯一真正的- 在当前领域中不涉及cDNA的时间RNA-seq是牛津纳米孔技术，它是 仅限于一次检测5-7个碱基的RNA。我们在这里报道了3Dpol的酶特性，即RNA- 脊髓灰质炎病毒依赖的RNA聚合酶，这对开发一种新的RNA-SEQ技术很有吸引力。我们 显示3Dpol一次复制一个碱基的RNA模板，并在高度结构的RNA上进行处理。 我们还表明，3Dpol1更喜欢发夹引物来启动RNA合成，产生双链(DS)- 发夹RNA，允许对纳米孔中的模板链和互补链进行测序。 我们进一步表明，当3Dpol被放置在两个电极之间时，显示出敏感的蛋白质电导 到其在NTP结合时的构象转变。我们假设这些特征提供了 探索在单分子水平上获得单核苷酸分辨率的直接RNA-SEQ的3Dpol.在目标1中，我们将 确定3Dpol作为RNA的酶阅读器的能力和质量。我们将测试3Dpol以读取困难的RNA 序列，包括含有转录后修饰的碱基、均聚体和 重复序列基序。我们将使用中的纳米孔设备来确定3Dpol读取RNA的质量 2D(双向)平台，其对模板链和复制链进行测序，具有潜在的 提高精确度。这些研究还将确定3Dpol的错误特征，这对识别 RNA中的修饰碱基。在目标2中，我们认为虽然3Dpol1的固有错误率很低(10-5)，但这一性质 在纳米孔测序中被掩盖，因为后者的技术错误率(10%-15%)。因此，我们将测试 在通过测量蛋白质进行RNA实时测序的电子设备中开发3Dpol的可能性 通过聚合酶的电导。我们将设计3Dpol拥有两个内置触点，以稳定系绳 连接到两个电极。我们将使用扫描技术测量3Dpol对NTP结合的响应蛋白电导 隧道显微镜(STM)。如果成功，STM测量数据将支持一项将 在固态平台中生成长时间的RNA-seq读数，无需直接进行电子读数 用于染料或标签。这项工作处于令人兴奋的新rna-seq技术开发的前沿，该技术将 对RNA研究和临床实践产生广泛影响。

Project Summary RNA sequencing (RNA-seq) in real time, known as the third-generation sequencing at the single-molecule level, is an important technology that will improve our understanding of the human genome. The development of real-time RNA-seq, however, has been challenging, due to the complexity of RNA in sequence and structure that requires a processive reader with single-nucleotide resolution. While Pacific Biosciences (PacBio) can generate long-reads, the process involves cDNA, which loses the informational content of RNA. The only real- time RNA-seq in the current field that does not involved cDNA is the Oxford Nanopore Technology, which is limited to sensing of 5-7 bases of RNA at a time. We report here enzymatic features of 3Dpol, the RNA- dependent RNA polymerase of poliovirus, that are attractive for developing a new RNA-seq technology. We show that 3Dpol copies the RNA template one base at a time with processivity across highly structured RNA. We also show that 3Dpol prefers a hairpin primer to initiate RNA synthesis, generating a double-stranded (ds)- hairpin RNA that allows sequencing of both the template strand and the complementary strand in a nanopore. We further show that 3Dpol, when placed between two electrodes, displays protein conductance that is sensitive to its conformational transition upon NTP-binding. We hypothesize that these features provide the basis to explore 3Dpol for direct RNA-seq with single-nucleotide resolution at the single-molecule level. In Aim 1, we will determine the ability and quality of 3Dpol as an enzymatic reader of RNA. We will test 3Dpol to read difficult RNA sequences, including sequences that contain post-transcriptionally modified bases, homopolymers, and repeated sequence motifs. We will determine the quality of RNA reading by 3Dpol using the Nanopore device in a 2D (2-directional) platform that sequences both the template strand and the copied strand with the potential to improve accuracy. These studies will also determine error signatures of 3Dpol that are useful for identification of modified bases in RNA. In Aim 2, we consider that while the intrinsic error rate of 3Dpol is low (10-5), this quality is masked in Nanopore sequencing, due to the latter’s technical error rate (10-15%). We will thus test the possibility to develop 3Dpol in an electronic device for real-time sequencing of RNA by measuring protein conductance through the polymerase. We will engineer 3Dpol to possess two built-in contacts for stable tethering to two electrodes. We will measure protein conductance of 3Dpol in response to NTP binding using a scanning tunneling microscope (STM). If successful, data of STM measurements will support a technology that will generate long-reads of RNA-seq in a solid-state platform that produces direct electronic readout without the need for dyes or labels. This work is at the forefront of exciting development of a new RNA-seq technology that will broadly impact on RNA research and clinical practice.