Recognition of non-standard base pairs by triplex-forming oligonucleotides containing an expanded genetic alphabet

The sequence-specific recognition of double-stranded DNA by biocompatible molecules is fundamental to molecular medicine and synthetic biology. Triplex-forming oligonucleotides (TFOs) enable programmable major groove recognition via Hoogsteen base pairing; however, the limited repertoire of natural nucleobases imposes strict constraints on target sequences and parallel motif triplexes require acidic conditions for stability. Here, we have expanded the triplex recognition space using nucleobases from an artificially expanded genetic information system (AEGIS). Through a systematic evaluation of 120 base triad combinations, we identify at least 12 modular triads that can be combined interchangeably to target duplex DNA containing standard, damaged, or synthetic base pairs with nanomolar affinity at neutral pH. We further demonstrate the versatility of this expanded recognition code by detecting oxidative lesions or AEGIS base pairs in enzymatically assembled duplex constructs using both chemically and enzymatically synthesized TFOs. This generalized framework provides a robust platform for precision gene-targeting, molecular sensing, and nucleic acid nanotechnology.