Steven Magennis, Alexandra E Bailie, Henry G Sansom, Rachel S S Fisher, Ryo Watabe, Yitzhak Tor, Anita C Jones
{"title":"Ultrasensitive detection of a responsive fluorescent thymidine analogue in DNA via pulse-shaped two-photon excitation","authors":"Steven Magennis, Alexandra E Bailie, Henry G Sansom, Rachel S S Fisher, Ryo Watabe, Yitzhak Tor, Anita C Jones","doi":"10.1039/d4cp03391d","DOIUrl":null,"url":null,"abstract":"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 complement 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.","PeriodicalId":99,"journal":{"name":"Physical Chemistry Chemical Physics","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Physical Chemistry Chemical Physics","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1039/d4cp03391d","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CHEMISTRY, PHYSICAL","Score":null,"Total":0}
引用次数: 0
Abstract
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 complement 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.
期刊介绍:
Physical Chemistry Chemical Physics (PCCP) is an international journal co-owned by 19 physical chemistry and physics societies from around the world. This journal publishes original, cutting-edge research in physical chemistry, chemical physics and biophysical chemistry. To be suitable for publication in PCCP, articles must include significant innovation and/or insight into physical chemistry; this is the most important criterion that reviewers and Editors will judge against when evaluating submissions.
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