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.
Highlights
With the development of generation sequencing (NGS), the field of transcriptomics has seen tremendous advancements creating opportunities for improved diagnostics, prognostics, and treatment of diseases such as cancers and infectious diseases [1,2]
We investigated the mass spectra of both the ribonucleotide monophosphates (rNMPs) mixture and m6A methylated RNA to determine the composition of the overlapped ultra-high-performance liquid chromatography (UPLC) peaks at 4.2 and 4.4 min, which could have arisen from rGMP and m6-rAMP or 8-oxo-guanosine monophosphate
We demonstrated for the first time the covalent attachment of XRN1 onto a solid-support for potential applications in single-molecule RNA exosequencing
Summary
With the development of generation sequencing (NGS), the field of transcriptomics has seen tremendous advancements creating opportunities for improved diagnostics, prognostics, and treatment of diseases such as cancers and infectious diseases [1,2]. There is an increasing interest in the study of post-transcriptional modifications of RNA and their potential role in modulating processes associated with cancer and other diseases [3,4,5,6]. NGS has been a useful technique for identifying specific post-transcriptional modifications, several technical challenges remain [7,8]. Almost all current NGS techniques require library preparation prior to sequencing. The RNA molecules are fragmented and converted to cDNAs using reverse transcription and amplified using PCR, followed by a purification step [7]. Does PCR introduce biases and other artifacts that would affect the identification and quantification of transcripts, and by using these pre-sequencing steps important RNA modification information can be lost [9,10]
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