# Recognition of Non-standard Base Pairs by Triplex-Forming Oligonucleotides Containing an Expanded Genetic Alphabet

## TL;DR

Scientists expanded the genetic alphabet to create new DNA-targeting molecules that work under normal conditions and can detect DNA damage or synthetic bases.

## Contribution

The study introduces 12 new modular base triplets for triplex DNA recognition at neutral pH using an expanded genetic alphabet.

## Key findings

- New triplets enable nanomolar-affinity DNA targeting at neutral pH.
- The method detects oxidative lesions and synthetic base pairs in DNA.
- Triplex-forming oligonucleotides are synthesized both chemically and enzymatically.

## Abstract

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 requires 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 triplet combinations, we identify at least 12 new modular triplets 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.