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

基本信息

  • 批准号:
    10348785
  • 负责人:
  • 金额:
    $ 73.42万
  • 依托单位:
  • 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:
    2020-05-11 至 2024-02-29
  • 项目状态:
    已结题

项目摘要

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.
在过去的几十年里,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,后者是由新的单分子的开发提供的 测序技术。这些进展使人们能够探测基因组中被认为是 “暗物质”直到最近,以及组装新的高质量的参考基因组。除了……之外 基因组测序,这些单分子方法为探测化学物质开辟了新的应用领域 DNA的修饰,通过探索利用光波导合成测序的动力学,或者 通过使用纳米孔对修饰的碱基进行电区分。目前,正在努力创造强大的 直接RNA测序的方法,以便关于RNA序列、表观遗传修饰和 量,可以得到。在单个人类细胞中,只有几个皮克的RNA和DNA可用,并且 由于这些核酸的表观遗传修饰不能倍增,一个公认的未来目标 测序技术是为了减少可以在皮卡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加载到零模波导中的方法 (ZMWs),并展示了通过制造多孔体实现的长DNA片段的DNA测序 ZMWs(PZMWs),在ZMWs底部嵌入了多孔材料。然而,挑战与 多孔材料的化学成分和寿命限制了该系统的处理能力。在这项提案中,我们 将开发一种全新的直接RNA测序方法,使定量转录组分析成为可能 和RNA碱基修饰信息,仅需要皮卡级别的输入RNA。首先,我们开发了一种 一种新型的ZMW,它的下面嵌入了一个金属盘电极。在两端施加电压 ZMWs产生一个电场,帮助捕获DNA和RNA。这些新设备极大地允许 与上一代PZMW相比,提高了吞吐量,并大幅提高了 获得的数据。其次,对于排序引擎,我们将使用马拉松RT,这是一种超过程反向 一种转录酶,通过酶复制将RNA分子转化为互补DNA(CDNA)分子 稳健和准确,比目前用于RNA测序的酶更准确。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。有广泛的支持 初步数据,我们将整合马拉松RT作为引擎,PtZMWs作为敏感的序列阅读器和 先进的单细胞分选和RNA提取工具,首次开发出定量RNA表达 来自真正的单细胞材料的图谱(即没有放大)。此外,使用我们的能力遵循 通过马拉松RT复制动力学,我们将探测保存在这些RNA分子中的化学修饰, 如甲基腺嘌呤和假尿嘧啶。这一独特方法的成功将使转录组发生革命性变化 通过以前所未有的灵敏度提供表观/转录切割工作流程,从单细胞材料进行分析。

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

Meni Wanunu其他文献

Meni Wanunu的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ truncateString('Meni Wanunu', 18)}}的其他基金

Ion Fountain Nanopore Readers for High-Resolution DNA and RNA Sequencing
用于高分辨率 DNA 和 RNA 测序的 Ion Fountain 纳米孔读数器
  • 批准号:
    10204556
  • 财政年份:
    2021
  • 资助金额:
    $ 73.42万
  • 项目类别:
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
  • 财政年份:
    2021
  • 资助金额:
    $ 73.42万
  • 项目类别:
Ion Fountain Nanopore Readers for High-Resolution DNA and RNA Sequencing
用于高分辨率 DNA 和 RNA 测序的 Ion Fountain 纳米孔读数器
  • 批准号:
    10448254
  • 财政年份:
    2021
  • 资助金额:
    $ 73.42万
  • 项目类别:
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:
    10161799
  • 财政年份:
    2020
  • 资助金额:
    $ 73.42万
  • 项目类别:
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
  • 财政年份:
    2020
  • 资助金额:
    $ 73.42万
  • 项目类别:
Direct picogram DNA and RNA sequencing using nanopore Zero-mode waveguides
使用纳米孔零模波导直接进行皮克 DNA 和 RNA 测序
  • 批准号:
    9914480
  • 财政年份:
    2019
  • 资助金额:
    $ 73.42万
  • 项目类别:
Direct picogram DNA and RNA sequencing using nanopore Zero-mode waveguides
使用纳米孔零模波导直接进行皮克 DNA 和 RNA 测序
  • 批准号:
    9356545
  • 财政年份:
    2016
  • 资助金额:
    $ 73.42万
  • 项目类别:

相似海外基金

RII Track-4:NSF: From the Ground Up to the Air Above Coastal Dunes: How Groundwater and Evaporation Affect the Mechanism of Wind Erosion
RII Track-4:NSF:从地面到沿海沙丘上方的空气:地下水和蒸发如何影响风蚀机制
  • 批准号:
    2327346
  • 财政年份:
    2024
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Standard Grant
BRC-BIO: Establishing Astrangia poculata as a study system to understand how multi-partner symbiotic interactions affect pathogen response in cnidarians
BRC-BIO:建立 Astrangia poculata 作为研究系统,以了解多伙伴共生相互作用如何影响刺胞动物的病原体反应
  • 批准号:
    2312555
  • 财政年份:
    2024
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Standard Grant
How Does Particle Material Properties Insoluble and Partially Soluble Affect Sensory Perception Of Fat based Products
不溶性和部分可溶的颗粒材料特性如何影响脂肪基产品的感官知觉
  • 批准号:
    BB/Z514391/1
  • 财政年份:
    2024
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Training Grant
Graduating in Austerity: Do Welfare Cuts Affect the Career Path of University Students?
紧缩毕业:福利削减会影响大学生的职业道路吗?
  • 批准号:
    ES/Z502595/1
  • 财政年份:
    2024
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Fellowship
Insecure lives and the policy disconnect: How multiple insecurities affect Levelling Up and what joined-up policy can do to help
不安全的生活和政策脱节:多种不安全因素如何影响升级以及联合政策可以提供哪些帮助
  • 批准号:
    ES/Z000149/1
  • 财政年份:
    2024
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Research Grant
感性個人差指標 Affect-X の構築とビスポークAIサービスの基盤確立
建立个人敏感度指数 Affect-X 并为定制人工智能服务奠定基础
  • 批准号:
    23K24936
  • 财政年份:
    2024
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
How does metal binding affect the function of proteins targeted by a devastating pathogen of cereal crops?
金属结合如何影响谷类作物毁灭性病原体靶向的蛋白质的功能?
  • 批准号:
    2901648
  • 财政年份:
    2024
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Studentship
ERI: Developing a Trust-supporting Design Framework with Affect for Human-AI Collaboration
ERI:开发一个支持信任的设计框架,影响人类与人工智能的协作
  • 批准号:
    2301846
  • 财政年份:
    2023
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Standard Grant
Investigating how double-negative T cells affect anti-leukemic and GvHD-inducing activities of conventional T cells
研究双阴性 T 细胞如何影响传统 T 细胞的抗白血病和 GvHD 诱导活性
  • 批准号:
    488039
  • 财政年份:
    2023
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Operating Grants
How motor impairments due to neurodegenerative diseases affect masticatory movements
神经退行性疾病引起的运动障碍如何影响咀嚼运动
  • 批准号:
    23K16076
  • 财政年份:
    2023
  • 资助金额:
    $ 73.42万
  • 项目类别:
    Grant-in-Aid for Early-Career Scientists
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了