Programming Temporal DNA Barcodes for Single-Molecule Fingerprinting

Fluorescence microscopy
enables simultaneous observation of the
dynamics of single molecules in a large region of interest. Most traditional
techniques employ either the geometry or the color of single molecules
to uniquely identify (or barcode) different species of interest. However,
these techniques require complex sample preparation and multicolor
hardware setup. In this work, we introduce a time-based amplification-free
single-molecule barcoding technique using easy-to-design nucleic acid
strands. A dye-labeled complementary reporter strand transiently binds
to the programmed nucleic acid strands to emit temporal intensity
signals. We program the DNA strands to emit uniquely identifiable
temporal signals for molecular-scale fingerprinting. Since the reporters
bind transiently to DNA devices, our method offers relative immunity
to photobleaching. We use a single universal reporter strand for all
DNA devices making our design extremely cost-effective. We show DNA
strands can be programmed for generating a multitude of uniquely identifiable
molecular barcodes. Our technique can be easily incorporated with
the existing orthogonal methods that use wavelength or geometry to
generate a large pool of distinguishable molecular barcodes thereby
enhancing the overall multiplexing capabilities of single-molecule
imaging.