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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 (MeOthaU), 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 MeOthaU 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 MeOthaU) 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. [ABSTRACT FROM AUTHOR]

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Fluorescent nucleobase analogues (FBAs) have emerged as powerful tools for understanding nucleic acid systems at the molecular level. However, their application at the single-molecule level has been limited by low brightness and an incomplete understanding of how local chemical environments affect their properties. In this study, we investigate the bright fluorescent pyrimidine analogue ABN in duplex DNA oligonucleotides and study its single-molecule applications. Time-resolved fluorescence spectroscopy reveals its unique tautomeric behavior, including photo-induced double proton transfer, influenced by base-pairing partners. This tautomerization directly impacts ABN's quantum yield and spectral characteristics. By favoring a high quantum yield thymine-like tautomer through base pairing, surface-immobilized ABN-containing DNA duplexes are readily observed as bright spots using single-molecule fluorescence microscopy, exhibiting well-defined single-exponential bleaching kinetics. The brightness and photostability are enhanced by oxygen depletion. These results demonstrate that ABN is unique among FBAs in enabling single-molecule fluorescence studies of oligonucleotides using a standard microscopy setup., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)