Abstract
Modern chemical and biological studies are undergoing a paradigm shift, where understanding the fate of individual cells, in an apparently homogeneous population, is becoming increasingly important. This has inculcated a growing demand for developing strategies that label individual cells with unique fluorescent signatures or barcodes so that their spatiotemporal trajectories can be mapped in real time. Among various approaches, light-regulated methods employing photocaged fluorophores have received particular attention, owing to their fine spatiotemporal control over labelling. However, their multiplexed use to barcode large numbers of cells for interrogating cellular libraries or complex tissues remains inherently challenging, due to the lack of multiple spectrally distinct photoactivated states in the currently available photocaged fluorophores. We report here an alternative multiplexable strategy based on optically controlled host–guest recognition in the cucurbit[7]uril (CB[7]) system that provides spatial control over the positioning of fluorophores to generate distinct barcodes in ‘user-defined’ cells. Using a combination of three spectrally distinct CB[7]-conjugated fluorophores and by sequentially performing cycles of photoactivation and fluorophore encoding, we demonstrate 10-color barcoding in microtubule-targeted fixed cells as well as 7-color barcoding in cell surface glycan targeted live MCF7 cells.
Highlights
A central challenge in biology is to quantitatively understand how each cell processes information and spatiotemporally responds to environmental cues that produce complex and emergent functions or leads to a diseased state
In our molecular design of CADA, we thought to eliminate the impact of synergistic interplay that renders high-affinity binding between CB[7] and ADA by converting the amine residue of ADA to a carbamate functionality in CADA
The formation of a bulky nitrogen center, with the large caging group in the vicinity, hinders the approach of the rigid CB[7] structure towards the hydrophobic ADA core of CADA, which imposes a steric constraint for complex formation (Fig. 1b)
Summary
A central challenge in biology is to quantitatively understand how each cell processes information and spatiotemporally responds to environmental cues that produce complex and emergent functions or leads to a diseased state. When titration was performed with a CADA-Alexa 647 solution that was already irradiated with light, an immediate and near-complete uorescence quenching was observed upon the addition of 1 eq of CB[7]–BHQ3 (Fig. 2c). To probe the feasibility of this CB[7] host–guest system to enable photocontrolled molecular associations in cellular complexities, we rst performed a biomolecular labeling experiment in xed cells under temporal light control.
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