Direct RNA sequencing using electo-optical zero mode waveguides and custom click fluorescent nucleotides - Advances in genome technologies ushered in vast cost reductions in DNA sequencing and increased read lengths, the latter afforded by development of new single-molecule sequencing technologies. As a result, much of the genome’s “dark matter” has been elucidated, and higher-quality reference genomes were made available. In addition to genome sequencing, these single-molecule methods have enabled new applications for probing chemical modifications in DNA, by either probing the kinetics of sequencing-by-synthesis using optical waveguides, or by electrically distinguishing modified bases using nanopores. Despite progress, a critical barrier in genomics is understanding the roles of RNA in biology, which demands methods for quantitative analysis of RNA molecules in a cell. The myriad of types of RNAs in a cell, their dynamic chemical modifications, and their elaborate structural and functional diversity, all hint at a tremendous level of regulation and biological significance. Traditional RNA sequencing methods have primarily relied on conversion to complementary DNA (cDNA) followed by cDNA sequencing using either high-throughput second-generation methods or third generation single-molecule methods, the latter of which offers long reads. Using these methods, some RNA modifications can be read through prior chemical functionalization of the RNA prior to conversion to cDNA (for example, m6A, pseudouridine, A-to-I editing, 1-methyluridine, and dihydrouridine). However, the chemical reactions involved in these methods are not 100% quantitative or specific, and further, detection is often done through incomplete reads due to reverse transcription blocks, which precludes detection of multiple modifications. The only available method for direct RNA sequencing, the Oxford Nanopore Technologies platform, suffers from several drawbacks that include high input requirements, limited ability to probe RNA modifications, and incomplete reads, particularly near the RNA 5’ end. We address these limitations by developing a new single-molecule method that can be scaled to allow long read direct RNA sequencing at high throughputs, all with very low input requirements of several picograms. Building on zero-mode waveguides (ZMWs) originally developed by Pacific Biosciences, we have recently developed electro-optical ZMWs (eZMWs) that allow low-input capture of DNA and RNA molecules. We demonstrated using these devices identification of DNA fragments from low inputs by rapid capture of single molecules and their flash sequencing. Together with the Pyle group, we are developing the integration of MarathonRT, an ultra- processive reverse transcriptase that converts RNA molecules to cDNA molecules with high processivity and accuracy, into our electro-optical eZMWs for direct RNA sequencing. We have already fused MarathonRT to a streptavidin protein and demonstrated its functionality in eZMWs. Here we will build on these developments to develop a direct RNA sequencing method that can detect single base edits and chemical modifications, all with high coverage from single-cell inputs of a few pg per run.
