EaStCHEM School of Chemistry, The University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, UK.
School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow, G12 8QQ, UK.
Phys Chem Chem Phys. 2024 Oct 30;26(42):26823-26833. doi: 10.1039/d4cp03391d.
Fluorescent base analogues (FBAs) are versatile nucleic acid labels that can replace a native nucleobase, while maintaining base pairing and secondary structure. Following the recent demonstration that free FBAs can be detected at the single-molecule level, the next goal is to achieve this level of detection sensitivity in oligonucleotides. Due to the short-wavelength absorption of most FBAs, multiphoton microscopy has emerged as a promising approach to single-molecule detection. We report the multiphoton-induced fluorescence of 5-(5-(4-methoxyphenyl)thiophen-2-yl)-6-aza-uridine (MeOaU), a polarity-sensitive fluorescent thymidine analogue, as a nucleoside, and in two single-stranded deoxyribo-oligonucleotides, with and without their complementary strands. Ensemble steady-state and time-resolved measurements in dioxane, following one-photon and two-photon excitation, reveals both strongly and weakly emissive species, assigned as rotamers, while in Tris buffer there are additional non-emissive states, which are attributed to tautomeric forms populated in aqueous environments. The two-photon (2P) brightness for MeOaU is highest as the free nucleoside in dioxane (10 GM) and lowest as the free nucleoside in Tris buffer (0.05 GM). The species-averaged 2P brightness values in DNA are higher for the single strands (0.66 and 0.82 GM for sequence context AXA and AXT, respectively, where X is MeOaU) than in the duplex (0.31 and 0.25 GM for AXA and AXT, respectively). Using 2P microscopy with pulse-shaped broadband excitation, we were able to detect single- and double-stranded oligos with a molecular brightness of 0.8-0.9 kHz per molecule. This allowed the detection of as few as 7 DNA molecules in the focus, making it the brightest responsive FBA in an oligonucleotide reported to date.
荧光碱基类似物（FBAs）是一种多功能的核酸标记物，可以替代天然碱基，同时保持碱基配对和二级结构。最近的研究表明，游离的 FBAs 可以在单分子水平上被检测到，因此下一个目标是在寡核苷酸中实现这种检测灵敏度。由于大多数 FBAs 的吸收波长较短，多光子显微镜已成为单分子检测的一种很有前途的方法。我们报告了 5-(5-(4-甲氧基苯基)噻吩-2-基)-6-氮杂尿嘧啶（MeOaU）的多光子诱导荧光，MeOaU 是一种极性敏感的荧光胸苷类似物，作为一种核苷，以及在两条单链脱氧核糖寡核苷酸中，无论是否存在其互补链。二氧六环中的单光子和双光子激发后的集总稳态和时间分辨测量揭示了两种强和弱发射物种，分别被分配为旋转异构体，而在 Tris 缓冲液中还有其他非发射状态，这些状态归因于在水相环境中存在的互变异构形式。MeOaU 在二氧六环中的自由核苷（10 GM）中的双光子（2P）亮度最高，而在 Tris 缓冲液中的自由核苷（0.05 GM）中最低。在 DNA 中，单链的物种平均 2P 亮度值（序列 AXA 和 AXT 分别为 0.66 和 0.82 GM，其中 X 为 MeOaU）高于双链（AXA 和 AXT 分别为 0.31 和 0.25 GM）。使用具有脉冲状宽带激发的 2P 显微镜，我们能够检测到分子亮度为 0.8-0.9 kHz/分子的单链和双链寡核苷酸。这使得在焦点中能够检测到多达 7 个 DNA 分子，这是迄今为止在寡核苷酸中报道的最亮的响应性 FBA。
