Regan Keenan T, Pounder Austin, Johnson Ryan E, Murray Makay T, Glowacki Hannah X, Wetmore Stacey D, Manderville Richard A
Department of Chemistry & Toxicology, University of Guelph Guelph Ontario N1G 2W1 Canada
Department of Chemistry & Biochemistry, University of Lethbridge Lethbridge Alberta T1K 3M4 Canada
Chem Sci. 2025 Mar 13;16(15):6468-6479. doi: 10.1039/d4sc07994a. eCollection 2025 Apr 9.
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 twisted intramolecular charge transfer upon excitation within certain microenvironments, we have addressed the longstanding issues of poor brightness ( × ) 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 12 and TBA15 oligonucleotides with canonical (A, C, T, G) or locked nucleic acids (LNAs) (T, A) 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 up to 0.58 and molar brightness ∼ 15 000 cm M 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.
我们采用了一种模块化的链上羟醛方法来合成基于查尔酮的荧光分子转子（FMR），该转子带有酚氧供体，可模拟绿色荧光蛋白（GFP）中的天然酪氨酸（Tyr66）发色团4-羟基亚苄基咪唑啉酮（HBI）。利用FMR在特定微环境中激发时发生非辐射衰减——扭曲分子内电荷转移的倾向，我们解决了核酸中GFP替代物长期存在的亮度低（×）和开启响应弱的问题。为了证明其潜力并为未来应用奠定基础，将这些FMR纳入以标准（A、C、T、G）或锁定核酸（LNA）（T、A）作为侧翼碱基的12和TBA15寡核苷酸中。使用荧光光谱、分子动力学模拟和量子力学计算对所得的双链体和G-四链体（GQ）进行了研究，从而全面了解了它们在DNA、DNA：RNA和GQ环境中的结构和光物理性质。富电子查尔酮有利于中性酚激发（ROH），以产生酚（ROH*）和酚盐（RO*）发射，后者通过分子间激发态质子转移过程产生，而缺电子查尔酮由于其光酸性而用作比率激发指示剂。这些替代物在具有侧翼LNA的GQ→双链体拓扑结构转换中显示出强烈的开启响应（高达154倍），在双链体中给出高达0.58的量子产率和摩尔亮度~15 000 cm M。通过协同NA序列和探针，我们实现了可切换的开-关光诱导电子转移, 从而产生了对pH值的134倍开启发射响应。我们的研究结果首次优化了作为内部核碱基替代物的GFP替代物的性能，并提出了它们可能成为NA诊断有用工具的多种方式。
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