Despite the potential of single-molecule sensing platforms to directly assay a multitude of different biomolecules, multiplexing these techniques to measure and classify many different molecules from a complex sample remains challenging. The most common way to encode molecular identity in a single-molecule tag is to use color, but detector and signal-to-noise considerations limit the number of available color channels in most single-molecule instruments to at most 4-5 colors. However, additional spectroscopic information is present in fluorescence signals that can enable discrimination of chemically different fluorophores that have similar emission spectra but different brightness, fluorescence lifetime, or anisotropy. Here, we demonstrate that the distinct spectroscopic signatures of a series of DNA-based FRET constructs, called <b><u>FluorFRET tags</u></b>, provide a new option for real-time multiplexing at the single-molecule level. By moving donor-acceptor pairs to different locations on a DNA scaffold, we have generated structural variations that produce many different spectroscopic signatures as fluorescent labels. Therefore, each construct can be uniquely identified, but the constructs share a common set of chemical building blocks that will perform robustly and consistently across different environments. We show here the results of these tags attached to different targets and such spectroscopic labels could readily enable the multiplexing of tens or hundreds of unique molecular targets at the single-molecule level.

Engineering FluorFRET tags for spectroscopic multiplexing at the single-molecule level

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