Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

Abstract

Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s−1 and a processivity of >10.5 kb at 25°C.

Label‐Free Identification of Single Mononucleotides by Nanoscale Electrophoresis

“…More importantly, molecule overlapping may be avoided in real exonuclease sequencing process with our dual‐nanopore TOF sensor by using enzymes whose clipping rates are lower than TOFs of the clipped mononucleotides in the flight column. For example, the clipping rates of dNMPs and rNMPs digested from λ‐DNA and RNA by λ‐exonuclease and XRN1 immobilized on polymer substrates are 0.9 ms nt −1 (1100 ± 100 nt s −1 ) [ 21 ] and 38 ms nt −1 (26 ± 5 nt s −1 ) [ 45 ] , respectively. The clipping rate of dNMPs is comparable to the measured TOF of this work and thus to reduce the molecule overlapping it may be necessary to modify electrokinetic conditions to slow down the enzyme function.…”

Tailoring Thermoplastic In-Plane Nanopore Size by Thermal Fusion Bonding for the Analysis of Single Molecules

“…Recently, we reported the use of solid-phase XRN1 reactions to sequentially produce rNMPs (5′ → 3′ direction). 70 Previous work from our group also demonstrated the identification of labeled rNMPs via their molecular-dependent electrophoretic mobility (i.e., TOF) in thermoplastic nanochannels; we were able to achieve TOF identification accuracies of >99%.…”

“…For example, we are currently developing a chip-based single-molecule exo-sequencing method, termed exonuclease time-of-flight (X-TOF). ,− This method involves a solid-phase enzymatic reactor coupled to a nanoflight tube that contains dual in-plane nanopores to measure free nucleotides’ TOFs. Recently, we reported the use of solid-phase XRN1 reactions to sequentially produce rNMPs (5′ → 3′ direction) . Previous work from our group also demonstrated the identification of labeled rNMPs via their molecular-dependent electrophoretic mobility ( i.e.…”

Nano-injection molding with resin mold inserts for prototyping of nanofluidic devices for single molecular detection

“…Attachment of XRN1 to the enzymatic nanobioreactor was achieved using EDC/NHS coupling chemistry. 61 To ensure delivery of reagents to the bioreactor, a waveform generator was used to apply a bias voltage. This was done by immersing electrodes into reservoirs of the microchannels that connect to the bioreactor.…”