tModBase v2.0: an expanded database of tRNA modifications with an AI-powered assistant

Chemical modifications of transfer RNAs (tRNAs) are crucial for tRNA stability, codon decoding, translational fidelity, and tRNA-derived small RNA (tsRNA) biogenesis. Changes in these modifications are associated with stress responses, physiological regulation, and human disease, underscoring the need to integrate modification information with experimental evidence and biological context. We previously established tModBase, a web resource to organize and explore tRNA modification-related data. Its first release integrated modification sites, high-throughput sequencing-derived signals, and condition-dependent changes. Since then, tRNA profiling has expanded through modification-specific and direct RNA sequencing strategies, with rapidly growing datasets across species and conditions. Here, we present tModBase v2.0 (http://www.tmodbase2.com/), containing 528 datasets across 23 species, 40 sequencing and library-preparation methods, and 42 modification types. It includes 68 third-generation and 210 modification-specific sequencing datasets, extending coverage of direct-RNA and modification-specific approaches. tModBase v2.0 further integrates sequencing-derived signals, condition-dependent information, and biological annotations linking modifications with enzymes, genetic variants, tsRNAs, diseases, and expression profiles. The LLM- and retrieval-augmented generation-powered assistant tModBot enables natural-language exploration across database modules while grounding responses in retrieved tModBase evidence. tModBase v2.0 therefore provides an expanded resource for investigating the diversity, dynamics, and biological context of tRNA modifications.