Exploring 3Dpol for RNA sequencing in real time

探索 3Dpol 实时 RNA 测序

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 RNA实时测序（RNA-seq），被称为单分子第三代测序水平，是一项重要的技术，将提高我们对人类基因组的了解。然而，由于 RNA 序列和结构的复杂性，实时 RNA 测序一直具有挑战性这需要具有单核苷酸分辨率的连续读取器，而 Pacific Biosciences (PacBio) 可以。生成长读，该过程涉及 cDNA，从而丢失了 RNA 的信息内容。目前RNA-seq领域不涉及cDNA的时候，就是牛津纳米孔技术，它是仅限于一次感测 5-7 个 RNA 碱基，我们在此报告 3Dpol（RNA-）的酶学特征。脊髓灰质炎病毒依赖的RNA聚合酶，这对于开发新的RNA-seq技术很有吸引力。表明 3Dpol 一次复制一个碱基的 RNA 模板，并在高度结构化的 RNA 中具有持续性。我们还表明 3Dpol 更喜欢发夹引物来启动 RNA 合成，生成双链 (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，当放置在两个电极之间时，3Dpol 会显示出敏感的蛋白质电导我们勇敢地说，这些特征为 NTP 结合时的构象转变提供了基础。在目标 1 中，我们将探索 3Dpol 在单分子水平上实现单核苷酸分辨率的直接 RNA 测序。确定 3Dpol 作为 RNA 酶读取器的能力和质量我们将测试 3Dpol 读取困难的 RNA。序列，包括含有转录后修饰碱基、均聚物和我们将使用 Nanopore 装置确定 3Dpol 的 RNA 读取质量。一个 2D（2 向）平台，可对模板链和复制链进行测序，并有可能这些研究还将确定 3Dpol 的错误特征，这对于识别非常有用。在目标 2 中，我们认为虽然 3Dpol 的内在错误率较低 (10-5)，但这种质量。由于后者的技术错误率（10-15%），因此在 Nanopore 测序中被掩盖。可以在电子设备中开发 3Dpol，通过测量蛋白质对 RNA 进行实时测序我们将设计 3Dpol 以拥有两个内置触点以实现稳定的束缚。我们将使用扫描测量 3Dpol 响应 NTP 结合的蛋白质电导。如果成功，STM 测量数据将支持一项技术。在固态平台中生成 RNA-seq 的长读段，无需直接生成电子读数这项工作处于令人兴奋的新 RNA-seq 技术开发的前沿。对 RNA 研究和临床实践产生广泛影响。