Single-cell direct RNA sequencing using electrical zero-mode waveguides and engineered reverse transcriptases

使用电零模式波导和工程逆转录酶进行单细胞直接 RNA 测序

• 批准号: 10487746
• 负责人: Meni Wanunu
• 金额: $ 12.38万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-11-23 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10487746
• 关键词: Back; Cell Separation; Cells; Chemicals; Chemistry; Complementary DNA; DNA; DNA sequencing; Data; Development; Devices; Electrodes; Enzymes; Epigenetic Process; Equipment; Future; Generations; Genetic Transcription; Genome; Genomics; Goals; Gold; Human; Kinetics; Label; Length; Longevity; Metals; Methods; Modification; Nucleic Acids; Nucleotides; Optics; Pseudouridine; RNA; RNA Sequences; RNA-Directed DNA Polymerase; Reader; Reverse engineering; System; Technology; Time; base; cost; dark matter; electric field; genome sequencing; nanopore; preservation; reference genome; single cell analysis; single molecule; success; tool; transcriptome; transcriptome sequencing; transcriptomics; voltage; waveguide

Project Summary / Abstract Progress in genome technologies over the past few decades has delivered a dramatic cost reduction in DNA sequencing and vast increases in read lengths, the latter afforded by development of new single-molecule sequencing technologies. These advances enabled probing regions of the genome that were considered as “dark matter” up until recently, as well as the assembly of new high-quality reference genomes. In addition to genome sequencing, these single-molecule methods have opened up new applications for probing chemical modifications in DNA, by either probing the kinetics of sequencing-by-synthesis using optical waveguides, or by electrically distinguishing modified bases using nanopores. Currently, efforts are made to create robust methods for direct RNA sequencing, so that information about RNA sequence, epigenetic modifications, and quantity, can be obtained. In a single human cell, only a few picograms of RNA and DNA are available, and since epigenetic modifications in these nucleic acids cannot be multiplied, a recognized goal of future sequencing technologies is to reduce the amount of genomic material that can be analyzed at picogram levels. We have recently developed a method for loading picogram-level DNA and RNA into zero-mode waveguides (ZMWs), and have demonstrated DNA sequencing of a long DNA fragment, achieved by fabricating porous ZMWs (PZMWs) in which a porous material was embedded at the ZMW bottoms. However, challenges with the chemistry and longevity of porous materials have limited the throughput of this system. In this proposal, we will develop an entirely new method for direct RNA sequencing that enables quantitative transcriptome analysis and RNA base modification information, requiring only picogram-level input RNA. First, we have developed a new type of ZMW that contains a metal-disk electrode embedded underneath it. Applying voltage across the ZMWs produces an electric field that assists with DNA and RNA capture. These new devices allow vastly increased throughput over the previous generation PZMWs, as well as substantial quality improvements to the data obtained. Second, for the sequencing engine we will employ MarathonRT, an ultra-processive reverse transcriptase that converts RNA molecules to complementary DNA (cDNA) molecules by enzymatic replication robustly and accurately, more so than currently used enzymes used for RNA sequencing. Third, we will employ advanced single-cell RNA extraction and gold-standard RNA quantification methods. Backed by extensive preliminary data, we will integrate MarathonRT as the engine, PtZMWs as the sensitive sequence readers and advanced single-cell sorting and RNA extraction tools, to develop for the first time quantitative RNA expression profiles from truly single-cell material (i.e., no amplification). Additionally, using our ability to follow the replication kinetics by MarathonRT, we will probe chemical modifications preserved in these RNA molecules, such as methyladenine and pseudouridine. Success in this unique approach will revolutionize transcriptome analysis from single-cell material by providing a workflow for epi/transcriptomics at unprecedented sensitivity.

项目摘要 /摘要 在过去的几十年中，基因组技术的进展已在DNA中进行了巨大的成本修订 测序和读取铅长度的大幅增加，后者通过开发新的单分子提供 测序技术。 直到最近，“暗物质”是新的高质量参考基因组的组装。 基因组测序，这些单分子方法已经开辟了新的应用程序探测化学的应用 通过使用光学波导探测测序的动力学，在DNA中进行修饰，或者 通过使用纳米孔来区分修改的基础，以创建强大 直接RNA测序的方法，因此有关RNA序列，表观遗传修饰的信息以及 可以在单个人类细胞中获得数量，只有少数RNA和DNA的表皮 由于无法乘以脑酸中的表观遗传修饰，这是未来的公认目标 测序技术是减少可以在PICAGRAM级别分析的基因组材料的量。 我们最近开发了一种将二十片片段级DNA和RNA加载到零模式波导中的方法 （ZMWS），并展示了DNA测序 ZMWS（PZMW），其中嵌入了ZMW底部的多孔材料。 多孔材料的化学和寿命限制了该提案中的通行系统 将开发一种直接RNA测序的输入的新方法，以实现定量转录组分析 和RNA基碱的修改信息，仅需要皮克图级输入RNA。 新型的ZMW包含嵌入在其下方的金属盘电极。 ZMW会产生一个有助于DNA和RNA捕获的电场。 Providence Generation Pzmws的虽然提高，以及对您的实质性改进 数据获得的数据，对于测序引擎，我们将使用Marathonrt 通过酶复制将RNA分子转换为编译DNA（cDNA）分子的转录酶 稳健，准确，因此比目前使用的酶用于RNA测序。 高级单细胞RNA提取和金标准RNA定量方法 预启示数据，我们将将Marathonrt作为引擎，PTZMWS作为有风度的序列读取器和 高级单细胞分选和RNA提取工具，首次开发定量RNA表达 训练单细胞材料的剖面（即没有放大）。 Marathonrt的复制动力学，我们将探测这些RNA分子保存的化学修饰， 例如甲基二烯和伪苷。 通过在未经表述的灵敏度下为Epi/转录组学提供工作流程，分析单细胞材料。