Exonuclease Time of Flight Sensor for Single Molecule RNA Sequencing

Transcriptomics is advancing disease diagnosis, prognosis, and treatment by uncovering RNA structure complexities, with single-molecule sequencing (SMS) technologies like Oxford Nanopore Technology (ONT) offering notable advantages over next-generation sequencing (NGS). ONT benefits from direct RNA sequencing without amplification, long sequence reads, label-free electrical detection, and a simplified workflow, although it requires substantial sample input (~1 µg). To address this, we are developing the X-ToF, a novel SMS nanofluidic device made from thermoplastic using nano-injection molding, which utilizes an immobilized enzyme, Exoribonuclease 1 (XRN1), to cleave single-stranded RNA into ribonucleotide monophosphates (rNMPs). The X-ToF detects, identifies individual rNMPs by measuring their specific time-of-flight (ToF) or electrophoretic mobility using Resistive Pulse Sensing (RPS), reducing sequencing costs, simplifying library preparation, and enhancing read accuracy. This device comprises a nanofluidic circuit with a solid-phase bioreactor where XRN1 is covalently immobilized to cleave RNA, followed by a nanochannel with dual in-plane nanopore sensors to determine the ToF of each rNMP accurately. In this presentation, we report on utilizing the X-ToF sensor to collect multiple parameters beyond time-of-flight (ToF) for our measurement scheme, such as pore 1,2 dwell times, and pore 1,2 peak amplitudes of each rNMP to create a library for future RNA sequencing. We demonstrate an 95.11% identification accuracy using Principal Component Analysis (PCA) as an unsupervised machine learning technique, achieving 100% accuracy with supervised approach. Additionally, we show the capability to sequence a single RNA molecule within the X-ToF device's bioreactor by reducing the RNA input requirement to 10 pM. The XRN1 enzyme's clipping rate was confirmed as 25 ± 6 nts/s by introducing a new measurement modality, interpeak pair space.