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: 10565946
• 负责人: Meni Wanunu
• 金额: $ 83.32万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-11 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10565946
• 关键词: Access to Information; Affect; Area; Benchmarking; Binding; Cell Separation; Cells; Chemicals; Chemistry; Complementary DNA; Complex; DNA; DNA sequencing; DNA-Directed DNA Polymerase; Data; Daughter; Deposition; Detection; Development; Devices; Diameter; Disease; Electrodes; Engineering; Enzyme Kinetics; Enzymes; Epigenetic Process; Eukaryotic Cell; Frequencies; Funding; Future; Generations; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Health; Hela Cells; High-Throughput RNA Sequencing; Human; Image; Kinetics; Length; Longevity; Measurement; Messenger RNA; Metals; Methodology; Methods; Modification; Molecular; Monitor; Noise; Nucleic Acids; Nucleic acid sequencing; Oligonucleotides; Onset of illness; Optics; Performance; Phase; Porosity; Process; Protein Isoforms; Proteins; Pseudouridine; RNA; RNA Sequences; RNA amplification; RNA chemical synthesis; RNA replication; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Reader; Reading; Research; Reverse Transcription; Reverse engineering; Role; Sampling; Signal Transduction; Silicon Dioxide; Site; System; Technology; Testing; Third Generation Sequencing; Time; Transcript; United States National Institutes of Health; Validation; Variant; base; biomacromolecule; cost; dark matter; design; early onset; electric field; epitranscriptome; experimental study; genome sequencing; improved; insight; metallicity; nanofabrication; nanopore; novel; personalized medicine; preservation; reference genome; single cell analysis; single molecule; success; tool; transcriptome; transcriptome sequencing; transcriptomics; voltage; waveguide

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.

过去几十年基因组技术的进步极大地降低了 DNA 成本 测序和读长的大幅增加，后者是由新的单分子的开发提供的 测序技术。这些进步使得探测基因组区域成为可能 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，这些单分子方法开辟了化学探测的新应用 通过使用光波导探索边合成边测序的动力学，或者 通过使用纳米孔电区分修饰碱基。目前，正在努力打造强 直接 RNA 测序的方法，以便获得有关 RNA 序列、表观遗传修饰和 数量，即可获得。在单个人类细胞中，只有几皮克的 RNA 和 DNA，并且 由于这些核酸中的表观遗传修饰不能倍增，这是未来的公认目标 测序技术的目的是减少可在皮克水平上分析的基因组材料的数量。 我们最近开发了一种将皮克级 DNA 和 RNA 加载到零模式波导中的方法 （ZMW），并展示了通过制造多孔材料实现的长 DNA 片段的 DNA 测序 ZMW（PZMW），其中 ZMW 底部嵌入多孔材料。然而，挑战 多孔材料的化学性质和寿命限制了该系统的吞吐量。在这个提案中，我们 将开发一种全新的直接 RNA 测序方法，可实现定量转录组分析 和RNA碱基修饰信息，只需要皮克级的输入RNA。首先，我们开发了一个 新型 ZMW，其下方嵌入金属盘电极。在两端施加电压 ZMW 产生有助于 DNA 和 RNA 捕获的电场。这些新设备可以极大地 与上一代 PZMW 相比，吞吐量有所提高，质量也得到了显着提高 获得的数据。其次，对于测序引擎，我们将采用 MarathonRT，一种超处理逆向 通过酶促复制将 RNA 分子转化为互补 DNA (cDNA) 分子的转录酶 稳健且准确，比目前用于 RNA 测序的酶更加强大和准确。第三，我们将聘用 先进的单细胞 RNA 提取和金标准 RNA 定量方法。广泛的支持 初步数据，我们将集成 MarathonRT 作为引擎，PtZMWs 作为敏感序列读取器， 先进的单细胞分选和RNA提取工具，首次开发定量RNA表达 来自真正的单细胞材料的概况（即无扩增）。此外，利用我们的能力来遵循 通过 MarathonRT 的复制动力学，我们将探测这些 RNA 分子中保存的化学修饰， 例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提供表观/转录组学工作流程，对单细胞材料进行分析。

项目成果

Rapid Identification of DNA Fragments through Direct Sequencing with Electro-Optical Zero-Mode Waveguides.

通过电光零模波导直接测序快速识别 DNA 片段。

• DOI: 10.1002/adma.202209376
• 发表时间: 2022
• 期刊: Advanced materials (Deerfield Beach, Fla.)
• 作者: Farhangdoust,Fatemeh;Alibakhshi,MohammadAmin;Cheng,Feng;Liang,Wentao;Liu,Yongmin;Wanunu,Meni
• 通讯作者: Wanunu,Meni

Author Correction: Unidirectional single-file transport of full-length proteins through a nanopore.

作者更正：全长蛋白质通过纳米孔的单向单文件传输。

• DOI: 10.1038/s41587-023-01995-2
• 发表时间: 2023
• 期刊: Nature biotechnology
• 影响因子: 46.9
• 作者: Yu,Luning;Kang,Xinqi;Li,Fanjun;Mehrafrooz,Behzad;Makhamreh,Amr;Fallahi,Ali;Foster,JoshuaC;Aksimentiev,Aleksei;Chen,Min;Wanunu,Meni
• 通讯作者: Wanunu,Meni

Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing.

• DOI: 10.1038/s41467-023-35858-w
• 发表时间: 2023-01-19
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Tavakoli, Sepideh;Nabizadeh, Mohammad;Makhamreh, Amr;Gamper, Howard;McCormick, Caroline A.;Rezapour, Neda K.;Hou, Ya-Ming;Wanunu, Meni;Rouhanifard, Sara H.
• 通讯作者: Rouhanifard, Sara H.