Modular access to nucleobase GFP-surrogates: pH-responsive smart probes for ratiometric nucleic acid diagnostics

Area of Science:

• Supramolecular Chemistry
• Biophysical Chemistry
• Nucleic Acid Chemistry

Background:

• Existing green fluorescent protein (GFP) surrogates for nucleic acid applications suffer from low brightness and weak turn-on responses.
• Mimicking the tyrosine (Tyr66) chromophore 4-hydroxybenzylidene-imidazolinone (HBI) is crucial for developing efficient GFP surrogates.
• Fluorescent molecular rotors (FMRs) offer potential for enhanced photophysical properties through twisted intramolecular charge transfer (TICT).

Purpose of the Study:

• To synthesize chalcone-based FMRs as novel GFP surrogates with improved brightness and responsiveness.
• To incorporate these FMRs into DNA and locked nucleic acid (LNA) containing oligonucleotides (duplexes and G-quadruplexes).
• To investigate the structural and photophysical properties of these FMR-modified nucleic acids in various contexts.

Main Methods:

• Modular on-strand aldol synthesis of chalcone-based FMRs.
• Incorporation of FMRs into NarI12 and TBA15 oligonucleotides with canonical and LNA bases.
• Characterization using fluorescence spectroscopy, molecular dynamics simulations, and quantum mechanical calculations.

Main Results:

• FMRs exhibited tunable photophysical properties, with electron-rich chalcones showing dual emission and electron-deficient ones acting as ratiometric indicators.
• Significant turn-on responses (up to 154-fold) were observed in a G-quadruplex to duplex topology switch using LNAs.
• Optimized FMRs achieved high fluorescence quantum yields (Φ_fl up to 0.58) and molar brightness (∼15,000 cm^-1 M^-1) in duplexes.
• A switchable photoinduced electron transfer mechanism enabled a 134-fold turn-on emission response to pH changes.

Conclusions:

• The developed chalcone-based FMRs represent the first successful optimization of GFP surrogates as internal nucleobase replacements.
• These FMRs demonstrate superior brightness and responsiveness compared to previous GFP surrogates in nucleic acid environments.
• The findings suggest these FMRs are promising tools for nucleic acid diagnostics, offering sensitive detection and pH monitoring capabilities.