Polyfluorophores on a DNA Backbone: A Multicolor Set of Labels Excited at One Wavelength

Teo, Yin Nah; Wilson, James N.; Kool, Eric T.

doi:10.1021/ja805502k

Cited by 113 publications

(122 citation statements)

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“…2B) shows a broad range of emission properties upon the same excitation at 360 nm, with emission maxima ranging from 410-670 nm. The spectra closely resemble those previously reported for the ODFs alone (19), which establishes that the protein conjugation does not substantially perturb their emission properties.…”

Section: Resultssupporting

confidence: 84%

“…The DNA-like ODFs chosen contained from one to as many as four fluorescent deoxyriboside monomers, and the set included five fluorophores in different arrangements ( Fig. 1 (15,16,19). A nonfluorescent abasic spacer (S) was also employed at the ends of the ODFs to further increase their aqueous solubility by virtue of the added phosphates.…”

Section: Resultsmentioning

confidence: 99%

“…A nonfluorescent abasic spacer (S) was also employed at the ends of the ODFs to further increase their aqueous solubility by virtue of the added phosphates. In a previous study, we developed ODFs with the sequences 5′-SB, 5′-SSYYYY, 5′-SSEY, 5′-SSEYK, and 5′-SSYKY to generate blue, cyan, green, yellow, and orange emission colors, respectively (19). To better cover the visible spectrum, we synthesized for this study a previously undescribed red-emitting monomer Z ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we described as many as 23 spectrally different ODFs with distinct emission colors upon single wavelength excitation (19). The dyes are water soluble, nontoxic, and stable in vivo (19).…”

Section: Discussionmentioning

confidence: 99%

“…The various forms of energy transfer within these fluorophores, including FRET, exciplex, excimer, H dimer, and other mechanisms (17,18), lead to large Stokes shifts of as much as >250 nm. This property enables them to generate a wide variety of emission colors with a single short-wavelength excitation (19). The emission from delocalized excited states that commonly occurs in ODFs results in their ability to be quenched with high efficiency (20)(21)(22), which has proven useful in the development of turn-on enzyme sensors (23).…”

mentioning

confidence: 99%

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Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Guo

Wang

Dai

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410-670 nm. We developed a new approach to antibody conjugation, using HuisgenSharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340-380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.bioconjugation | immunofluorescence | multiplex T o understand the complexity and dynamics of the molecular interactions in biological systems, the parallel analysis of multiple species, such as different proteins in a cell or cells in a tissue specimen, is often needed (1, 2). The most common mode of imaging for tracking and labeling such species is fluorescence microscopy. For multispecies imaging, this typically requires the use of various fluorophores having distinct excitation and emission wavelengths. Commonly available organic fluorophores are typically used to tag biomolecules for these purposes (3, 4), which allows the visualization of three, or occasionally more, species via the use of separate excitation and emission filters. Using this strategy, one can label multiple cellular antigens, for example, by use of different commercially available dye-labeled secondary antibodies.Although this approach is widely employed, some nonideal factors still exist. One of the major limiting issues of common organic dyes is that they have widely separated absorption spectra. This fact requires the researcher to use specialized filter sets and take a separate image for each dye; the final multicolor image is then constructed by overlaying false-color single-dye images. This approach enforces some restrictions on the researcher and equipment and places limitations on data acquisition. For ex...

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%