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.

项目总结/摘要 过去几十年来，基因组技术的进步大大降低了DNA的成本。 测序和阅读长度的大幅增加，后者是通过开发新的单分子 测序技术。这些进展使得能够探测基因组中被认为是 “暗物质”直到最近，以及新的高质量参考基因组的组装。除了 基因组测序，这些单分子方法开辟了新的应用，探测化学 通过使用光波导探测合成测序的动力学，或者 通过使用纳米孔电区分修饰的碱基。目前，正在努力建立一个强大的 直接RNA测序的方法，以便获得有关RNA序列、表观遗传修饰和 量，可以获得。在单个人类细胞中，只有几皮克的RNA和DNA是可用的， 由于这些核酸中的表观遗传修饰不能成倍增加， 测序技术的目的是减少可以在皮克水平上分析的基因组材料的量。 我们最近开发了一种将皮克级DNA和RNA加载到零模波导中的方法 （ZMW），并且已经证明了长DNA片段的DNA测序，通过制造多孔的 ZMW（PZMW），其中多孔材料嵌入ZMW底部。然而，挑战与 多孔材料的化学性质和寿命限制了该系统的吞吐量。在本提案中，我们 将开发一种全新的直接RNA测序方法， 和RNA碱基修饰信息，只需要皮克级的输入RNA。首先，我们制定了一个 新型ZMW包含嵌入其下方的金属盘电极。在ZMW两端施加电压 ZMW产生电场，有助于DNA和RNA捕获。这些新设备可以极大地 与上一代PZMW相比，吞吐量有所增加， 获得的数据。其次，对于测序引擎，我们将采用MarathonRT，一种超过程反向 通过酶复制将RNA分子转化为互补DNA（cDNA）分子的转录酶 稳健和准确，比目前用于RNA测序的酶更好。第三，我们将采用 先进的单细胞RNA提取和金标准RNA定量方法。以广泛的 初步数据，我们将整合MarathonRT作为引擎，PtZMW作为敏感的序列阅读器， 先进的单细胞分选和RNA提取工具，首次开发定量RNA表达 来自真正单细胞材料的分布（即，无放大）。此外，利用我们的能力， 通过MarathonRT的复制动力学，我们将探测这些RNA分子中保留的化学修饰， 如甲基腺嘌呤和假尿苷。这种独特方法的成功将彻底改变转录组 通过以前所未有的灵敏度为epi/转录组学提供工作流程，从单细胞材料进行分析。