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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：
10161799
负责人：
Meni Wanunu
金额：
$ 62.92万
依托单位：
NORTHEASTERN UNIVERSITY
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

来源：
https://reporter.nih.gov/project-details/10161799
关键词：
Access to Information Affect Area Back Benchmarking Binding Caliber Cell Separation Cells Chemicals Chemistry Complementary DNA Complex DNA DNA sequencing DNA-Directed DNA Polymerase Data Daughter Deposition Detection Development Devices Disease Electrodes Engineering Enzyme Kinetics Enzymes Epigenetic Process Eukaryotic Cell Frequencies Funding Future Generations Genetic Transcription Genome Genomic Segment Genomics Goals Gold Health Hela Cells High-Throughput RNA Sequencing Human Image Kinetics Length Light Longevity Measurement Messenger RNA Metals Methodology Methods Modification Molecular Monitor Noise Nucleic Acids Nucleic acid sequencing Oligonucleotides Onset of illness Optics Performance Phase Process Protein Isoforms Proteins Pseudouridine RNA RNA Sequences RNA amplification RNA-Directed DNA Polymerase RNA-Directed RNA Polymerase Reader Reading Research Role Sampling Signal Transduction Silicon Dioxide Site Structure 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

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.

过去几十年来，基因组技术的进步大大降低了DNA的成本