{"doi":"10.1101/2023.03.12.532289","title":"Nanopore sequencing for the 17 modification types in 36 locations in <i>E. coli</i> ribosomal RNA enables monitoring of stress-dependent changes","abstract":"Abstract Escherichia coli possess the 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Direct RNA nanopore sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to non-modified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knock-out E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modification’s signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. In the 16S rRNA, there resides an m 4 C m modification at site 1402 that decreased with both stressors. Using helicase dwell time, it was determined that the N 4 methyl group is lost during both stressors, and the 2’-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons ( rrn ), and earlier studies aligned the nanopore reads to a single operon ( rrnA ). Here, the reads were aligned to the seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for &gt;16 different RNA modifications in a strand is achievable.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":398710,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.959,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1007710,"name":"Songjun Xiao","orcid":"0000-0001-5220-3406","position":1,"is_corresponding":false},{"id":765973,"name":"Cynthia J. Burrows","orcid":"0000-0001-7253-8529","position":2,"is_corresponding":false},{"id":765972,"name":"Aaron M. Fleming","orcid":"0000-0002-2000-0310","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T01:19:47.744358Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}