Development of Nanoscale Technologies for Applications in Single-Molecule Sequencing

Abstract

Recent advancements in next-generation sequencing (NGS) technologies have completely transformed the way in which quantitative genomics and transcriptomics can be done. However, there are a few limitations associated with NGS that restrict the use of these technologies in a clinical setting. For example, the NGS methods have a complicated workflow, can only read short nucleic acid sequences up to about 150 bases, and because these methods use PCR, errors can be introduced, and important modification information can be lost during the amplification process. Furthermore, most of these methods are time-consuming, costly, and require fluorescence labeling. Therefore, new strategies for nucleic acid sequencing that can provide simpler workflow, longer reads, and amplification-free formats that are rapid, accurate, and low cost are required. We propose a novel method for single-molecule nucleic acid sequencing which can address the limitations in NGS. Our strategy, coined as “exonuclease time-of-flight (XToF),” consists of a covalently immobilized exonuclease/exoribonuclease enzyme in a thermoplastic nanofluidic device. The sequentially released mononucleotides by the processive enzymatic activity will be identified via molecular-dependent time-of-flight (ToF) and current transient amplitudes between two in-plane nanopores embedded in a nanofluidic channel. In this study, we lay the groundwork for single-molecule RNA sequencing using the XToF method. Using a microfluidic device, we demonstrated the capability to covalently attach exoribonuclease 1 (XRN1) enzyme to a thermoplastic surface. The covalent immobilization of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Moreover, the ability of immobilized XRN1 to maintain its catalytic activity to digest canonical and methylated RNA transcripts was demonstrated. The processivity and clipping rate of immobilized XRN1 were secured using single-molecule fluorescence measurements of a single RNA transcript. Moreover, the nanofluidic XToF devices were fabricated in thermoplastics via nano-imprint lithography. Studies conducted using labeled RNA demonstrated that there was no statistically significant difference in the clipping rate of the immobilized enzyme due to nano-confinement. Currently, we are working on identifying unlabeled ribonucleotide monophosphates using ToF and current transient amplitudes. In the future, the sequential digestion of ssRNA by immobilized XRN1 will be integrated with the identification of released rNMPs to demonstrate the RNA sequencing using the XToF method. We also report a simple method for tailoring the size of in-plane nanopores fabricated in thermoplastics for single-molecule sensing. We were able to decrease the depth and width of the in-plane nanopores from ~30 x 30 nm to ~10 x 17 nm during the thermal fusion bonding (TFB) process. The cross-sectional area of the in-plane nanopores reduced with increasing pressures during TFB. In-plane nanopore devices assembled at higher TFB pressures showed a higher current transient amplitude when utilized to detect single λ-DNA molecules via resistive pulse sensing. Furthermore, the in-plane nanopore sizes were tuned using TFB pressures to successfully detect ssRNA and single ribonucleotide adenosine monophosphates (rAMPs). However, co-ion exclusion arising due to the high surface charge of the thermoplastic surface reduced the event frequency. To address this issue, the thermoplastic surface was modified using EDC/NHS and ethanolamine. This simple surface modification significantly increased the event frequency from ~1 s-1 to 136 s-1 for a 100 nM concentrated ssRNA.