Indu Chandrasoma

RNA sequencing is a valuable technique used to identify and analyze the primary sequence of nucleotide bases within an RNA molecule. Its applications span across precision medicine, vaccine development, and Next Generation Sequencing (NGS) technologies. However, existing methods like Illumina sequencing, Ion Torrent (Proton/PGM) sequencing, and SOLiD sequencing have certain limitations, including complex workflows during library preparation, short read lengths (approximately 150 base pairs), amplification errors due to PCR, and the need for fluorescence labeling. To address these limitations, there is a demand for a novel RNA sequencing method that offers improved accuracy, rapidity, cost-effectiveness, operability at low sample concentrations, identification of modified bases, and the ability to read longer sequences.

Considering these requirements, we are currently developing an innovative RNA sequencing method called Exonuclease Time of Flight nanofluidic device (XTOF), which is fabricated using a thermoplastic material. This device comprises five nanochannels that are interconnected to a nano-scale enzymatic reactor situated at the device's core. In our previous work, we have successfully immobilized Exonuclease 1 (XRN1) enzyme within this nanoscale enzymatic reactor. The enzyme functions by progressively clipping RNA molecules into their constituent bases (rAMP, rUMP, rCMP, and rGMP) from the 5' to the 3' end. Once the RNA is cleaved, we achieve detection of individual ribonucleotide monophosphates by electrically shuttling the cleaved bases through a nanochannel containing two in-plane nanopores, each measuring around 10 nm. By analyzing the time each nucleotide takes to traverse these nanopores, known as time of flight (TOF), we can determine the TOF value unique to each ribonucleotide monophosphate, based on its electrophoretic mobility. Ultimately, the TOF values of the ribonucleotides will be utilized to sequence the RNA molecule accurately.

To evaluate the feasibility of our approach, we conducted experiments using a dual in-plane nanopore device that closely mimics the TOF Nano-column within the XTOF device in terms of dimensions. These experiments allowed us to successfully detect all four ribonucleotide monophosphates using the dual in-plane nanopore device.

Currently, we are undertaking preliminary experiments to demonstrate the potential of the XTOF device for RNA sequencing.