Project Summary During this program, Electronic BioSciences (EBS) will fully develop and demonstrate a completely new RNA sequencing technology capable of directly sequencing the most common product of RNA editing: inosine (I). Cells diversify the coding potential of their mRNA and regulatory small RNAs by enzymatic conversion of adenosine (A) to I, i.e., A-to-I editing, in which the edited base codes differently in mRNA, guides alternative mRNA splicing, and renders miRNA and tRNA functional in cells. The current inability to directly sequence sites of A-to-I RNA editing with quantitative readout (i.e., profile or determine their ratio within given sequences) seriously limits the ability of researchers to fully understand this epitranscriptomic process in disease. Thus, EBS aims to fill this technology gap by developing a single-molecule sequencing technique capable of directly identifying the four canonical nucleotides along with I, all with high resolution and high accuracy. In turn, such a technology development will improve the understanding of RNA function, including the role and significance of I, to enable better diagnostics, prognostics, and therapeutics. At the end of this Phase II effort, EBS will have developed a beta-prototype I sequencing system and demonstrated its complete functionality and analytical capabilities for a variety of RNA types as well as benchmarked the sequencing results against current state-of-the-art approaches.