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，基因组技术的进展促进了DNA的急剧降低 测序和读取长度的大幅度增加，后者通过开发新的单分子提供 测序技术。这些进步使基因组的探测区域被认为是 直到最近，“暗物质”以及新的高质量参考基因组的组装。此外 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，对DNA进行修改，或者 通过以电子方式区分纳米孔。目前，为创造强大的努力而努力 直接RNA测序的方法，因此有关RNA序列，表观遗传修饰和 数量，可以获得。在单个人类细胞中，只有几张RNA和DNA的脱皮图，并且 由于这些核酸中的表观遗传修饰无法倍增，因此公认的未来目标 测序技术是减少可以在皮克图水平上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），并且已经证明了长DNA片段的DNA测序，通过制造多孔来实现 ZMWS（PZMW），其中嵌入了ZMW底部的多孔材料。但是，挑战 多孔材料的化学和寿命限制了该系统的吞吐量。在这个建议中，我们 将开发一种直接RNA测序的全新方法，可以实现定量转录组分析 和RNA碱基修改信息，仅需要皮克图级输入RNA。首先，我们已经开发了 新型的ZMW包含嵌入在其下方的金属盘电极。在整个上施加电压 ZMWS产生一个有助于DNA和RNA捕获的电场。这些新设备允许广泛 上一代PZMW的吞吐量增加，以及对 获得的数据。其次，对于测序引擎，我们将采用超级过程的Marathonrt 通过酶复制将RNA分子转换为完整DNA（cDNA）分子的转录酶 稳健，准确，比目前使用用于RNA测序的酶更重要。第三，我们将雇用 晚期单细胞RNA提取和金标准RNA定量方法。由广泛的支持 初步数据，我们将将Marathonrt作为发动机集成为PTZMWS作为敏感序列读取器，并且 高级单细胞分选和RNA提取工具，首次开发定量RNA表达 来自真正的单细胞材料的曲线（即无扩增）。此外，利用我们遵循的能力 Marathonrt的复制动力学，我们将探测这些RNA分子保存的化学修饰， 例如甲基趋化和伪苷。在这种独特的方法中的成功将彻底改变转录组 通过在前所未有的灵敏度下为情节/转录组学提供工作流程来分析单细胞材料。