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Bright, long-lasting and non-phototoxic organic fluorophores are essential to the continued optimization of a diverse range of imaging applications. While many remarkable advances have been made towards this goal, all currently available technologies remain susceptible to undesirable photophysical phenomena, such as transient (blinking) and irreversible (photobleaching) transitions to dark states as well as related off-target effects including phototoxicities. Dark states arise from triplet state excursions, non-fluorescent electronic configurations from which the rate of return to the excitable ground state is often slow. Such tendencies compromise all fluorescence applications by unpredictably reducing the signal-to-noise ratio (SNR) as well as limiting the total duration of time over which information can be gathered.
The direct conjugation of small-molecule protective agents (PAs) has enabled significant improvements in the photon budget of cyanine-class organic fluorophores spanning the visible spectrum by reducing the lifetime of reactive triplet states through intra-molecular triplet quenching1-5 (Fig. 1). These technologies can now be readily implemented as a general approach to increase the photon yields of a range of chemically and structurally diverse fluorophores by covalently linking PAs in proximity of the fluorogenic center6. Through a partnership with Lumidyne Technologies, Glen Research has created a novel PA-linked phosphoramidite using cyclooctatetraene (COT). This product represents one of many possible iterations demonstrating the utility of a PA in this context, thereby promising investigations that will accelerate the frontiers of nucleic acid imaging.
After the paper, Enhanced photostability of cyanine fluorophores across the visible spectrum, was published,2 Glen Research recognized the impact that these photo-protective agents could have upon the research community by providing a triplet state quencher (TSQ) as a phosphoramidite. It would provide a modular way to improve the photostability (and therefore performance) of virtually any fluorescent dye by coupling a single TSQ vicinal to the fluorophore. This is because all fluorophores have a propensity to undergo intersystem crossing from an excited singlet state to a triplet state. This triplet state is long-lived because relaxation to the ground state is spin-forbidden as the excited state electron has the same spin as the electron in the ground state. In the excited triplet state, the fluorophore can react with dissolved oxygen to produce reactive and cytotoxic singlet oxygen as well as other reactive-oxygen species that lead to photobleaching of the dye. However, even in the absence of molecular oxygen, the fluorophore in the triplet state can undergo redox reactions with solvent and other biomolecules to produce radical cations or anions which again leads to photobleaching of the dye.
While there are a variety of photo-protective species to choose from,1 we decided to use cyclooctatetraene. Cyclooctatetraene (COT) is compatible with DNA synthesis and deprotection conditions and is a well-known triplet state quencher. We chose to use our popular serinol linker to provide sufficient linker length and flexibility for the COT and the dye to interact, leading to efficient relaxation to the ground state from the triplet state. COT Serinol Phosphoramidite (1) is shown in Figure 2.
Figure 2: Structure of Cyclooctatetraene (COT) Serinol Phosphoramidite
(1) COT Serinol Phosphoramidite
To confirm the photo-protective effects of the COT, the sequences 5'-(Cyanine 5)-(COT)-T12-3' and the control 5'-(Cyanine 5)-T12-3' were synthesized. After Glen-Pak™ purification, a fluorescence time course study was performed exciting at 640 nm and observing at 660 nm. The monotonic decrease in fluorescence is indicative of photobleaching of the cyanine 5 fluorophore. From our spectrofluorometric studies, it is clear that the presence of COT limited the amount of photobleaching of the cyanine dye.
A three minute coupling time is recommended for the COT phosphoramidite and the product was found to be compatible with standard DNA deprotection conditions. We have observed an interesting side reaction can occur with COT-labelled oligos. If the oligo, while still bound to the solid support, is thoroughly dried and allowed to sit at room temperature for extended periods, some cleavage of the vicinal phosphodiester linkages can be observed upon deprotection. This side reaction can be completely avoided by deprotecting the oligo immediately after synthesis or storing the column at -20 °C until the deprotection is initiated. This reaction can occur to a lesser extent upon drying the deprotected oligo down.
We thank Roger Altman and Scott Blanchard of Lumidyne for their collaboration in the preparation of this article.
1: Altman RB, Terry DS, Zhou Z, Zheng Q, Geggier P, Kolster RA, Zhao Y, Javitch JA, Warren JD, Blanchard SC. Cyanine fluorophore derivatives with enhanced photostability. Nat Methods. 2011 Nov 13;9(1):68-71.
2: Altman RB, Zheng Q, Zhou Z, Terry DS, Warren JD, Blanchard SC. Enhanced photostability of cyanine fluorophores across the visible spectrum. Nat Methods. 2012 Apr 27;9(5):428-9.
3: Zheng Q, Jockusch S, Zhou Z, Altman RB, Warren JD, Turro NJ, Blanchard SC. On the Mechanisms of Cyanine Fluorophore Photostabilization. J Phys Chem Lett. 2012 Aug 16;3(16):2200-2203
4: Zheng Q, Jockusch S, Zhou Z, Blanchard SC. The contribution of reactive oxygen species to the photobleaching of organic fluorophores. Photochem Photobiol. 2014 Mar-Apr;90(2):448-54.
5: Zheng Q, Juette MF, Jockusch S, Wasserman MR, Zhou Z, Altman RB, Blanchard SC. Ultra-stable organic fluorophores for single-molecule research. Chem Soc Rev.2014 Feb 21;43(4):1044-56.
6: Zheng Q, Jockusch S, Rodríguez-Calero GG, Zhou Z, Zhao H, Altman RB, Abruña HD, Blanchard SC. Intra-molecular Triplet Energy Transfer is a General Approach to Improve Organic Fluorophore Photostability. RSC Photochem & Photobiol Sci 2015 (in press).