Long-lived Platform Development for Exonuclease-Based Sequencing

项目摘要 在这个项目中，电子生物科学公司（EBS）和Meni Wanunu教授（东北大学）将 开发一个真正的单分子、直接、长寿命的RNA测序仪。开发的RNA测序仪将是 能够以高准确度（>99%）对RNA进行测序，以及对各种样品进行迭代测序， 定量分析给定样品的RNA含量，这是同类技术中的第一项。为了使 在这个项目中，我们将开发一种新的测序平台，并将其与 新开发的定制酶，生物纳米孔阅读器和测序方法。的 平台/系统的特点将大大增加稳定性和寿命，由于新的平台的发展， 在这个项目中。该平台将与已经开发的组件集成，包括 由EBS出售的NanopatchTM系统，其允许对具有10- 20个纳米孔的单个生物纳米孔进行电流监测。 与其他市售仪器相比，噪声低一倍，以及平面脂质的自动化方法 双层形成和稳定的蛋白质纳米孔插入的维持、温度控制和电场 可编程门阵列（FPGA）检测逻辑，以自动决定所施加的电压偏置。在整个 本方案将进一步优化测量的信噪比和时间分辨率， 最后是相关的基础呼叫。在系统开发之后，我们将演示 通过对各种RNA进行测序的相关测序方法，从行为良好的对照开始 在研究更复杂的生物学相关的RNA序列之前。不同的RNA 分子将被单独和混合物测序，序列准确性以及捕获 频率将被分析。我们还将确定系统处理连续加载的 样品在完成这个项目后，我们将开发，优化，并充分展示世界上最先进的 第一个高精度、真正的单分子、直接、长寿命RNA测序仪。这种技术将改变RNA 测序和转录组学研究，实现直接，高度准确，快速和负担得起的 RNA分子的表征。

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.