Re-Reading RNA to Obtain High Accuracy

Project Summary The ultimate goal in the field of RNA sequencing (transcriptomics and epitranscriptomics) is single-molecule, high-accuracy, de novo RNA sequencing, including the ability to sequence modified bases without the need for additional techniques/methods that add time, cost, and sample requirements. Unfortunately, current transcriptomic sequencing technologies are costly, have low accuracy (specifically relative to DNA sequencing approaches), they can require reverse transcription that removes the identity of modified bases (limiting/prohibiting epitranscriptomic investigations), and/or they can use or require processive enzymes that fail to traverse certain types of sequences. During this Phase I SBIR project, we will develop a sequencing technology/methodology that overcomes these short comings, and is capable of state-of-the-art, RNA sequencing and characterization. More specifically, we will develop and demonstrate an approach for controllably and iteratively translocating single RNA molecules back-and-forth through a nanopore reader such that single molecules can be directly and repeatedly sequenced, and corresponding ultra-high accuracy consensus sequence determinations can be made. Furthermore, during this project, we will demonstrate the ability to multiplex the associated measurement, significantly reducing the risk associated with developing a high- throughput, single-molecule sequencing system following this effort. Nanopore-based technology has the potential to enable complete and comprehensive RNA sequencing, and to be a field-leading technology in the future. During this project, we will take steps toward making that future a reality by developing and optimizing all components of our technology and then demonstrating RNA sequencing, including the ability to sequence N6- methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ). We will then show the functionality and advantages of the developed technology by benchmarking our approach against current nanopore-based sequencing approaches.