DNA Polyfluorophores for Real‐Time Multicolor Tracking of Dynamic Biological Systems

Abstract

Dye‐ing to live: Spectral limitations of common organic dyes make it difficult or impossible to visualize and follow multiple biological components in rapidly moving systems. The development of a multispectral set of improved DNA‐scaffolded fluorophores is described. Their use in multicolor cellular imaging (see scheme) and in tracking of biological motions on the subsecond timescale is demonstrated.

In Vitro Fluorogenic Real‐Time Assay of the Repair of Oxidative DNA Damage

“…5′-DMT 3′-cyanoethyl α-pyrene phosphoramidite [67] , 5′-DMT 3′-cyanoethyl α-perylene phosphoramidite [68] , 5′-DMT 3′-cyanoethyl α-phenylethynylpyrene phosphoramidite [69] , 5′-DMT 3′-cyanoethyl β-N,N-dimethyl-2-(quinolin-2-yl)benzo[d]oxazol-6-amine phosphoramidite (QB) [70] , and 5′-DMT 3′-cyanoethyl β-3-chloro-4-fluoro-indole phosphoramidite [62] were prepared according to published methods.…”

“…Probe concentration was determined using fluorophore absorbance (pyrene ε 342 = 39,000 M −1 cm −1 [67] , perylene ε 440 = 39,200 M −1 cm −1 [68] , 2-aminopurine ε 260 = 1,000 M −1 cm −1 [71] , ethenoadenine ε 260 = 4,800 M −1 cm −1 [72] , pyrrolo-dC ε 345 = 3,700 M −1 cm −1 [73] , Dss ε 380 = 31,000 M −1 cm −1 [74] , tCo ε 360 = 9,000 M −1 cm −1 [59] , phenylethynylpyrene ε 363 = 67,000 M −1 cm −1 [69] , and 3-Cl-4-F-indole ε 260 = 13,700 M −1 cm −1 ) [62] . The QB extinction coefficient was calculated to be 22,400 M −1 cm −1 according the Beer-Lambert Law by measuring the absorbances of known concentrations of the QB diol in methanol.…”

Color‐Change Photoswitching of an Alkynylpyrene Excimer Dye

“…[6] ODFs are DNA-like molecules that incorporate multiple fluorescent chromophores as monomers into a single water-soluble assembly so that they interact closely with one another. This close association of chromophores results in complex and useful sequence-dependent changes to fluorescence behavior, which have been exploited in a variety of applications ranging from analyte sensing to multicolor fluorescence imaging with single-wavelength excitation.…”

“…[6] Following up on this observation, we carried out new studies of the SSVV dye, which revealed that, under irradiation at 365 nm (the PEP absorption maximum) it undergoes rapid photobleaching (t 1/2 = 134 s) in air-saturated water in a spectrometer (Figure 1). Strikingly, spectral measurements (as well as inspection by eye) revealed that rather than simply becoming dark, the dye switched emission maximum from 509 nm (green) to 393–413 nm (blue-violet).…”

“…[6,7] Although more studies are needed to better understand the photoswitching mechanisms under varied conditions, we envision future uses of the VV-induced color change in FRAP studies, for super-resolution imaging, and ratiometric measurements of light exposure. It may also have utility as a control mechanism in optogenetics.…”