Chemical probe-based Nanopore Sequencing to Selectively Assess the RNA modifications

Abstract

RNA modifications contribute to RNA and protein diversity in eukaryotes and lead to amino acid substitutions, deletions, and changes in gene expression levels. Several methods have developed to profile RNA modifications, however, a less laborious identification of inosine and pseudouridine modifications in the whole transcriptome is still not available. Herein, we address the first step of the above question by sequencing synthetic RNA constructs with inosine and pseudouridine modification using Oxford Nanopore Technology, which is a direct RNA sequencing platform for rapid detection of RNA modification in a relatively less labor-intensive manner. Our analysis of multiple nanopore parameters reveals mismatch error majorly distinguish unmodified versus modified nucleobase. Moreover, we have shown that acrylonitrile selective reactivity with inosine and pseudouridine generates a differential profile between the modified and treated construct. Our results offer a new methodology to harness selectively reactive chemical probe-based modification along with existing direct RNA sequencing methods to profile multiple RNA modifications on a single RNA.

Nanopore dwell time analysis permits sequencing and conformational assignment of pseudouridine in SARS-CoV-2

“…The Ψ sites were spaced >25 nt apart to study them one at a time as they pass from the helicase to the nanopore sensor, an overall distance that spans ~17 nt from the entry of the helicase to the exit of the kmer sensing zone in the protein nanopore. Pseudouridine is base called by Guppy predominantly as U or C, consistent with other studies, 26 and the present work found the ratio is dependent on the local sequence context (Figure 2). The base-calling error for Ψ was greater than U permitting detection of the modification (Figure 3); however, the base-calling errors were sequence context dependent, similar to the base-calling differences.…”

“…When Ψ was present, the nucleotide called was predominantly a mixture of C and U and a low amount of A or G, consistent with prior results. [26][27][28] The new finding herein is the distribution of C and U called at the 13 singly-modified Ψ sites ranged from 10% to 97% C with the remainder predominantly called as U (Figure 2A). Inspection of the sequencedependent base calling results for Ψ did not lead to an obvious sequence context trend.…”

“…have demonstrated the feasibility of this approach. 26,27 Sequencing of the 16s rRNA from E. coli with the MinION™ successfully called m 7 G and Ψ sites, 28 and many examples of calling m 6 A in cellular RNAs have been reported demonstrating that direct sequencing for modifications can work. 27,29,30 Lastly, long-range interactions observed in the nanopore sensor when the modification passes through the motor protein have been noted.…”

Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2

“…has demonstrated the feasibility of this approach. 28 , 29 Sequencing of the 16s rRNA from E. coli with the MinION successfully called m 7 G and Ψ at known sites; 30 additionally, m 6 A and Ψ have been found by base-calling errors in tRNA at known sites, synthetic mRNA, and mRNA from cellular sources. 29 , 31 − 34 While many Ψ sites were identified in the prior studies, our analysis revealed that many sites would be missed on samples with Ψ at unknown locations.…”

RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence

“…A recent review described in details the RNAseq based approaches [31]. Noteworthy, the very recent development of single molecule direct RNA sequencing method by Oxford Nanopore Technologies (ONT, Oxford, UK), is promising for the analysis of modification landscape on a specific RNA sequence, including Ψs [48][49][50][51].…”