Long-lived Platform Development for Exonuclease-Based Sequencing

基于核酸外切酶的测序的长寿命平台开发

• 批准号: 10322603
• 负责人: Eric Ervin
• 金额: $ 40万
• 依托单位: ELECTRONIC BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322603
• 关键词: Algorithms; Area; Binding; Biological; Biological Markers; Biological Sciences; Biotechnology; Cell Membrane Permeability; Cells; Cleaved cell; Custom; DNA; Detection; Development; Electrodes; Electronics; Ensure; Enzymes; Evaluation; Exonuclease; Foundations; Frequencies; Geometry; Government; Hemolysin; High-Throughput Nucleotide Sequencing; Individual; Ions; Length; Lipid Bilayers; Logic; Longevity; Maintenance; Measurement; Measures; Methodology; Methods; Modeling; Modification; Molecular; Monitor; Noise; Nucleotides; Optics; Performance; Persons; Phase; Preparation; Process; Proteins; Public Health; RNA; RNA Sequences; Reader; Reading; Registries; Regulation; Research; Ribonucleotides; Sampling; Side; Signal Transduction; Site-Directed Mutagenesis; Small Business Technology Transfer Research; Specialist; Surface; System; Systems Development; Systems Integration; Technology; Temperature; Testing; Therapeutic Intervention; Time; Universities; Viscosity; Work; base; improved; innovation; instrument; multimodality; mutant; nanofabrication; nanoparticle; nanopore; novel; novel diagnostics; professor; prognostic; programs; sequencing platform; single molecule; solid state; temporal measurement; transcriptome; transcriptome sequencing; transcriptomics; voltage

Project Summary During this project, Electronic BioSicences (EBS) and Professor Meni Wanunu (at Northeastern University) will develop a true single-molecule, direct, long-lived, RNA sequencer. The developed RNA sequencer will be capable of sequencing RNA with high-accuracy (>99%), as well as iteratively sequencing various samples and quantitatively profiling the RNA content of a given sample, as the first technology of its kind. In order to enable the proposed feat, during this project, we will develop a novel sequencing platform and use it in combination with newly developed custom enzymes, biological nanopore readers, and sequencing methodology. The platform/system will feature vastly increased stability and longevity due to the novel platform developments made during this project. This platform will then be integrated with already developed components, including the NanopatchTM system sold by EBS, which allows for current monitoring of individual biological nanopores with 10- fold lower noise than other commercially available instruments, as well as automated methods for planar lipid bilayer formation and the maintenance of stable protein nanopore insertions, temperature control, and field programable gate array (FPGA) detection logic to automate decisions on the applied voltage bias. Throughout this program, we will further optimize the signal-to-noise ratio and temporal resolution of the measurement, and ultimately the associated based calling. Following the development of the system, we will then demonstrate the associated sequencing methodology through the sequencing of various RNAs, starting with well-behaved control sequences before moving to more complicated, biologically relevant RNA sequences. The different RNA molecules will be sequenced both alone and in mixtures, and the sequence accuracy as well as capture frequency will be analyzed. We will also determine the ability of the system to handle consecutively loaded samples. Upon the completing this project, we will have developed, optimized, and fully demonstrated the world’s first high-accuracy, true single-molecule, direct, long-lived, RNA sequencer. Such technology will transform RNA sequencing and transcriptomics research by enabling direct, highly accurate, fast, and affordable characterization of RNA molecules.

项目摘要 在这个项目中，电子生物科学(EBS)和东北大学的Meni Wanunu教授将 开发一种真正的单分子、直接、长寿命的RNA测序仪。开发的RNA测序仪将是 能够以高精度(&gt；99%)对RNA进行测序，以及迭代地对各种样本和 定量分析给定样本的RNA含量，这是此类技术中的第一项。为了启用 提出的壮举，在这个项目中，我们将开发一个新的测序平台，并将其与 新开发的定制酶、生物纳米孔阅读器和测序方法。这个 由于进行了新的平台开发，平台/系统的稳定性和寿命将大大提高 在这个项目中。然后，该平台将与已经开发的组件集成，包括 EBS出售的NanopatchTM系统，该系统允许使用10-10微米的电流监测单个生物纳米孔 折叠噪音比其他商业仪器更低，以及平面脂类的自动化方法 双层的形成和稳定的蛋白质纳米孔插入、温度控制和电场的维持 可编程门阵列(FPGA)检测逻辑，可自动决定施加的偏压。贯穿始终 本方案中，我们将进一步优化测量的信噪比和时间分辨率，并 最终关联的基于呼叫。随着系统的开发，我们将演示 相关的测序方法，通过对各种RNA进行测序，从行为良好的控制开始 在转移到更复杂的、生物相关的RNA序列之前。不同的RNA 分子将被单独测序和混合测序，测序的准确性以及捕获 将对频率进行分析。我们还将确定系统处理连续加载的能力 样本。在完成这个项目后，我们将开发、优化并充分展示世界上 第一款高精度、真正的单分子、直接、长寿命的RNA测序仪。这样的技术将改变RNA 通过实现直接、高精度、快速和经济实惠的测序和转录组研究 核糖核酸分子的特性。