Improved Optical Multiplexing with Temporal DNA Barcodes

Many biochemical events of importance
are complex and dynamic.
Fluorescence microscopy offers a versatile solution to study the dynamics
of biology at the mesoscale. An important challenge in the field is
the simultaneous study of several objects of interest, referred to
as optical multiplexing. For improved multiplexing,
some prior techniques used repeated reporter washing or the geometry
of nanostructures; however, these techniques require complex nanostructure
assembly, multiple reporters, or advanced multistep drift correction.
Here we propose a time-based approach, for improved optical multiplexing,
that uses readily available inexpensive reporters and requires minimal
preparation efforts. We program short DNA strands, referred hereby
as DNA devices, such that they undergo unique conformation
changes in the presence of the dye-labeled reporters. The universal fluorescent reporter transiently binds with the
devices to report their activity. Since each device is programmed
to exhibit different hybridization kinetics, their fluorescent time
trace, referred to as the temporal barcode, will
be unique. We model our devices using continuous-time Markov chains
and use stochastic simulation algorithm to generate their temporal
patterns. We first ran simulation experiments with a small number
of DNA devices, demonstrating several distinct temporal barcodes,
all of which use a single dye color. Later, using nanostructure-based
devices, we designed a much larger pool of temporal barcodes and used
machine learning for classification of these barcodes. Our simulation
experiments and design principles can aid in the experimental demonstration
of the DNA devices.