Figure 1: Design and characterization of G6pDH/NAD⁺-assembled DNA tweezers.

(a) Schematic illustration of the mechanics of the DNA tweezer-regulated enzyme nanoreactor: a regulatory oligomer (shown in red) is designed to adopt a ‘GCG’ stem-loop hairpin structure that holds the two arms of the tweezers close together. The addition of a set strand (complementary to the regulatory loop shown in red) to the tweezer structure results in the formation of a DNA double helix between the tweezer arms that separates the G6pDH and NAD⁺ enzyme/cofactor pair (open state). Displacement of the set strands from the regulatory loop by fuel strands leads to the active state (closed) in which G6pDH and the cofactor NAD⁺ are in close proximity. (b) Characterization of the fully assembled tweezers structures: left—ethidium bromide-stained PAGE gel for detecting DNA; right—the same gel visualized by silver stain for detecting proteins. Lane 1: open tweezers with NAD⁺ attached by a poly(T)₂₀ linker; lane 2: open tweezers assembled with G6pDH; lane 3: closed tweezers assembled with G6pDH. All structures were purified using biotin–avidin affinity resins. (c) FRET measurement experiment (Cy3/Cy5 dyes) to characterize the open and closed states of the tweezers. (d) Detection of enzymatic activity in the G6pDH/NAD⁺-assembled tweezers using a phenazine methosulfate (PMS)/resazurin-coupled assay: NAD⁺ is first reduced to NADH by G6pDH. Next, PMS catalyzes electron transfer from NADH to resazurin producing a strongly fluorescent resorufin with an emission of maximum ~590 nm.