Research Project
10322603
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.
