# Ratiometric Detection of pH‐Induced i‐Motif Folding Based on a Dual Emissive Cytosine Analog

## TL;DR

A new dual-emissive cytosine analog allows real-time tracking of pH-induced i-motif DNA folding using ratiometric fluorescence.

## Contribution

A novel dual-emissive cytosine analog is introduced for ratiometric detection of i-motif folding states.

## Key findings

- The TCC analog shows distinct emission bands that respond to microenvironmental changes like hydration and protonation.
- The probe distinguishes i-motif folding from duplex DNA through fluorescence enhancement and emission redshift.
- The method enables multiparametric monitoring of DNA structures with high sensitivity.

## Abstract

A dual‐emissive cytosine analog (TCC), based on a 2‐thienyl‐3‐hydroxychromone scaffold, is incorporated into oligodeoxynucleotides to monitor the folding state of DNA i‐motif structures. This modified nucleobase exhibits two distinct emission bands (IN* and IT*), each responding differently to microenvironmental changes, enabling ratiometric detection. The photophysical properties of TCC are systematically characterized in various solvents and DNA contexts, including single‐stranded, double‐stranded, and i‐motif‐forming sequences. The IN*/IT* emission ratio and the wavelength of the IT* band act as robust and orthogonal reporters of hydration, base stacking, and protonation states. In fully paired duplexes, the T* band is quenched and blue‐shifted, while i‐motif folding results in both fluorescence enhancement and a redshift of the T* emission. Additionally, the probe distinguishes mismatched base pairs and abasic sites, offering further insights into local structural defects. Overall, this ratiometric nucleobase analog enables real‐time, multiparametric monitoring of i‐motif folding with high sensitivity, and holds promise for extension to other noncanonical DNA structures. The findings further establish the 3‐hydroxychromone platform as a powerful tool for the rational design of fluorescent sensors targeting dynamic nucleic acid architectures.

A dual‐emissive cytosine analog enables ratiometric fluorescence detection of i‐motif DNA folding. This study reveals a sensitive method to monitor pH‐dependent i‐motif conformational changes, providing a valuable tool for investigating DNA secondary structures in vitro.© 2025 WILEY‐VCH GmbH