Exploring 3Dpol for RNA sequencing in real time

探索 3Dpol 实时 RNA 测序

• 批准号: 9974889
• 负责人: Ya-Ming Hou
• 金额: $ 23.4万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-18 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9974889
• 关键词: Address; Arizona; Base Sequence; Binding; Biological Sciences; Clinic; Complement; Complementary DNA; Complementary RNA; Complex; Crystallization; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Decision Making; Development; Devices; Double-Stranded RNA; Dyes; Electrodes; Electronics; Engineering; Feedback; Gene Expression; Generations; Genetic Transcription; Genome; Genomics; High-Throughput Nucleotide Sequencing; Human Genome; Human poliovirus; Individual; Ions; Label; Masks; Measurement; Measures; Methods; Modification; Monitor; Nucleic Acids; Nucleotides; Polymerase; Process; Property; Proteins; RNA; RNA Sequences; RNA chemical synthesis; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Reader; Reading; Reporting; Reproducibility; Research; Resolution; Scanning Probe Microscopes; Signal Transduction; Signaling Protein; Stretching; Structure; Technology; Testing; Therapeutic; Third Generation Sequencing; Time; Variant; Viral Proteins; Virus Replication; Work; base; clinical practice; conformational conversion; electric field; epigenomics; experimental study; improved; nanopore; novel; novel sequencing technology; nucleobase; response; sequencing platform; single molecule; solid state; tool; transcriptome sequencing; viral RNA

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.

项目摘要 实时RNA测序（RNA-Seq），被称为单分子处的第三代测序 水平是一项重要的技术，它将改善我们对人类基因组的理解。发展的发展 然而，由于RNA的复杂性，实时RNA-Seq受到了挑战 这需要具有单核苷酸分辨率的处理器读取器。而太平洋生物科学（PACBIO）可以 生成长阅读的过程涉及cDNA，它失去了RNA的信息含量。唯一的现实 当前领域中不涉及cDNA的时间RNA-seq是牛津纳米孔技术，是 一次限于一次感测5-7个RNA。我们在这里报告3DPol的酶特征，RNA- 脊髓灰质炎病毒的依赖性RNA聚合酶，可用于开发新的RNA-Seq技术。我们 证明3DPOL一次将RNA模板一个碱基复制，并在高度结构化的RNA上具有加工性。 我们还表明，3DPOL更喜欢发夹底漆来启动RNA合成，产生双链（DS） - 发夹RNA允许在纳米孔中对模板链和互补链进行测序。 我们进一步表明，将3DPOL放置在两个电极之间时，将显示敏感的蛋白电导 在NTP结合上过渡到其会议过渡。我们假设这些功能为 探索以单分子水平的单核苷酸分辨率的直接RNA-Seq的3DPOL。在AIM 1中，我们将 确定3DPol作为RNA的酶读取器的能力和质量。我们将测试3DPOL读取困难的RNA 序列，包括包含转录后修改基碱，均聚物和序列的序列 重复的序列基序。我们将使用3DPol使用纳米孔设备确定RNA读数的质量 一个2D（2方向）平台，该平台序列模板链和复制的链，并具有潜力 提高准确性。这些研究还将确定3DPol的误差签名，这些签名可用于识别 RNA中的修饰碱基。在AIM 2中，我们认为，尽管3DPOL的固有错误率很低（10-5），但该质量 由于后者的技术错误率（10-15％），在纳米孔测序中掩盖了。因此，我们将测试 通过测量蛋白质在电子设备中开发3DPOL以实时测序RNA的可能性 通过聚合酶的电导。我们将设计3DPOL具有两个内置触点以稳定绑扎 到两个电子。我们将使用扫描测量3DPol的蛋白电导，以应对NTP结合 隧道显微镜（STM）。如果成功，STM测量数据将支持一项技术 在固态平台上生成RNA-Seq的长阅读，该平台可产生无需的直接电子读数 用于染料或标签。这项工作是一种令人兴奋的新RNA-seq技术发展的最前沿，该技术将 对RNA研究和临床实践的广泛影响。