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

We have utilized a modular on-strand aldol approach to synthesize chalcone-based fluorescent molecular rotors (FMRs) bearing phenolic oxygen donors that mimic the natural tyrosine (Tyr66) chromophore 4-hydroxybenzylidene-imidazolinone (HBI) within green fluorescent proteins (GFPs). Leveraging the FMRs' propensity to undergo non-radiative decay via twisted intramolecular charge transfer upon excitation within certain microenvironments, we have addressed the longstanding issues of poor brightness (ε _max × Φ _fl) and weak turn-on responses for GFP-surrogates within nucleic acids. To demonstrate its potential and lay the groundwork for future applications, these FMRs were incorporated into NarI12 and TBA15 oligonucleotides with canonical (A, C, T, G) or locked nucleic acids (LNAs) (T_L, A_L) as flanking bases. The resulting duplexes and G-quadruplexes (GQs) were studied using fluorescence spectroscopy, molecular dynamics simulations, and quantum mechanical calculations, yielding a comprehensive understanding of their structural and photophysical properties in DNA, DNA : RNA, and GQ contexts. Electron-rich chalcones favor neutral phenol excitation (ROH) to afford both phenol (ROH*) and phenolate (RO^-*) emission, with the latter generated through an intermolecular excited-state proton transfer process, while electron-deficient chalcones serve as ratiometric excitation indicators, due to their photoacidity. The surrogates display strong turn-on responses (up to 154-fold) in a GQ → duplex topology switch with flanked LNAs, giving Φ _fl up to 0.58 and molar brightness ∼ 15 000 cm^-1 M^-1 in the duplex. By synergizing the NA sequence and probe, we achieve a switchable ON-to-OFF photoinduced electron transfer, resulting in a 134-fold turn-on emission response to pH. Our findings are the first to optimize the performance of GFP-surrogates as internal nucleobase replacements and suggest multiple ways in which they may be useful tools for NA diagnostics.