DNA
nanostructures have been shown viable for the creation of complex
logic-enabled sensing motifs. To date, most of these types of devices
have been limited to the interaction with strictly DNA-type inputs.
Restriction endonuclease represents a class of enzyme with endogenous
specificity to DNA, and we hypothesize that these can be integrated
with a DNA structure for use as inputs to trigger structural transformation
and structural rearrangement. In this work, we reconfigured a three-arm
DNA switch, which utilizes a cyclic Förster resonance energy
transfer interaction between three dyes to produce complex output
for the detection of three separate input regions to respond to restriction
endonucleases, and investigated the efficacy of the enzyme targets.
We demonstrate the ability to use three enzymes in one switch with
no nonspecific interaction between cleavage sites. Further, we show
that the enzymatic digestion can be harnessed to expose an active
toehold into the DNA structure, allowing for single-pot addition of
a small oligo in solution.