Nanopore-based technology has the potential to be the future of comprehensive RNA sequencing, i.e., transcriptomics and epitranscriptomics, as it is the only viable path to directly characterizing and profiling long RNA strands without reverse transcription or processive enzymes that fail to traverse certain sequences. The direct characterization enabled by nanopore sequencing will also ultimately lead to detection and characterization of modified bases, a critical requirement for epitranscriptomics. The presently available, state-of-the art RNA sequencing technologies are limited by the method, with the input requiring cDNA intermediates and/or large amounts of costly sample, and the readout featuring a compromise between short read length and low-accuracy. During this program, Electronic BioSciences (EBS) aims to develop a completely new long-read, extremely high-accuracy nanopore-based sequencing technology that will enable de novo epitranscriptomic sequencing, capable of inherent sequence cross-validation and expanded sensitivity for more accurate and versatile RNA sequencing. During this Phase I project, we will build and multiplex an entirely new system, fully assess and optimize the associated workflow/methodology to sequence canonical nucleotides and specific clinically relevant RNA modifications, and demonstrate initial sequencing. At the conclusion of this project, we will have successfully demonstrated and developed an entirely new sequencing system prototype that overcomes the known challenges limiting current RNA sequencing technologies (including other nanopore-based sequencing approaches). The resulting technology development will be a label-free, long-read (>kbases), electronic readout method, capable of providing a new and detailed understanding of RNA and its regulation.
Public Health Relevance Statement: Project Narrative The technology developed during this program will yield an entirely novel and improved method for direct, long-read, high accuracy RNA sequencing, including the ability to identify specific modifications. A high accuracy sequencing technology that does not require cDNA synthesis or utilize processive enzymes/motors (that fail on certain sequences), in addition to accurately distinguishing canonical and modified nucleotides within a RNA strand, will greatly advance transcriptome/epitranscriptome research and propel the associated RNA sequencing-based diagnostics, prognostics, and therapeutic intervention strategies into the future.
Project Terms: Biological Sciences, Biologic Sciences, Bioscience, Life Sciences, Complementary DNA, cDNA, DNA, Deoxyribonucleic Acid, Enzymes, Enzyme Gene, Future, Goals, Gold, instrumentation, Lead, Pb element, heavy metal Pb, heavy metal lead, Libraries, Lipid Bilayers, lipid bilayer membrane, Longevity, Length of Life, life span, lifespan, Methods, Methodology, Motion, Motivation, Nucleotides, Polymers, Research, Reverse Transcription, Non-Polyadenylated RNA, RNA Gene Products, Ribonucleic Acid, RNA, Running, Software, Computer software, Technology, Time, War, Generations, DNA Sequence, base, Label, improved, Phase, Biological, biologic, Ensure, Evaluation, prognostic, Individual, Measurement, Systems Integration, Double-Stranded DNA, dsDNA, ds-DNA, Diagnostic, programs, Investigation, System, Best Practice Analysis, Benchmarking, Church, magnetic, Magnetism, magnetic field, particle, solid state, voltage, tech development, technology development, Stretching, Speed, novel, Position, Positioning Attribute, intervention therapy, Therapeutic Intervention, Regulation, Sampling, single molecule, Intervention Strategies, interventional strategy, Intervention, Genomics, Length, Data, Detection, Motor, Reader, Resolution, Small Business Innovation Research Grant, SBIR, Small Business Innovation Research, Validation, Preparation, Modification, Development, developmental, Image, imaging, epigenomics, cost, nanopore, nano pore, transcriptomics, clinically relevant, clinical relevance, prototype, ChIP-seq, ChIP Sequencing, chromatin immunoprecipitation-sequencing, transcriptome sequencing, RNA Seq, RNA sequencing, RNAseq, transcriptome, global gene expression, global transcription profile, sequencing platform, Third Generation Sequencing, 3rd generation sequencing, epitranscriptomics, epitranscriptome
