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Nanofluidic Device for Single-Molecule RNA Sequencing
Vietz, Chad
Vietz, Chad
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Abstract
Single-molecule sequencing (SMS) technologies, specifically the Oxford Nanopore Technology (ONT), offer a number of advantages over next generation sequencing (NGS) technologies such as the ability to perform direct RNA sequencing negating the need for amplification, long reads, label-free electrical readout, and a simpler workflow. However, the ONT is not without challenges, namely high cost and high mass requirements (~1 µg) even though only a single molecule is sequenced. We are developing a SMS nanofluidic device (X-ToF) that is fabricated in a thermoplastic via nano-injection molding. This device uses an immobilized enzyme, such as XRN1, to processively clip through a single stranded RNA molecule base-by-base and detects and identifies single ribonucleotides using their molecular-dependent time-of-flight (ToF; i.e., electrophoretic mobility). X-ToF has the ability to significantly lower sequencing cost, reduce library preparation time, and significantly improve the call accuracy. The X-ToF chip consists of a network of nanofluidic channels and within this network is placed a solid-phase bioreactor where XRN1 is covalently immobilized for processively cleaving RNA. Following the clipping of RNA, detection of single ribonucleotide monophosphates (rNMPs) is achieved by electrokinetically shuttling cleaved bases through a nanochannel containing two in-plane nanopores. The ToF of each rNMP is determined via the time it takes the rNMPs to pass through the two in-plane nanopores on either end of a nanochannel flight tube. Here, we report work toward the development of this technology focusing on: (1) The fabrication of an immobilized nanoscale enzymatic reactor (INER); and (2) the modification of nanofluidic structures to facilitate the entry of RNA through in-plane nanopores. XRN1 immobilization to PMMA/COC devices was accomplished using EDC/NHS coupling chemistry. Fluorescently-labeled ssRNA molecules were electrokinetically translocated through the X-ToF nanochannels with real-time single-molecule tracking using a fluorescence microscope. We observed electrokinetically translocating RNA in real-time and its association to immobilized XRN1 within the nanoreactor. Digestion of RNA was accomplished by supplying XRN1’s cofactor (Mg+2) and observing the fluorescence decay rate of a single RNA molecule. From this data, the clipping rate was found to be 23 ± 3 nt s-1. Furthermore, by using fluorescently-labeled ssRNA and DNA, the behavior of these biomolecules was studied as they were electrokinetically driven through in-plane nanopores. Observations indicated the failure of these molecules to pass through the in-plane nanopores. A new device was fabricated that contained larger in-plane nanopores and nanostructures that were less abrupt in their depth dimensions. Results from these nanofluidic modifications are presented herein.
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Date
2022-12-31
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University of Kansas
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Keywords
Analytical chemistry, INER, nanofluidics, RNA-seq, sequencing