Practically all of life's molecular processes, from chemical synthesis to replication, involve enzymesthat carry out their functions through the catalytic transformation of metastable fuels into waste products.Catalytic control of reaction rates will prove to be as useful and ubiquitous in nucleic-acid-based engineeringas it is in biology. Here we report a metastable DNA "fuel" and a corresponding DNA "catalyst" that improveupon the original hybridization-based catalyst system (Turberfield et al.
Phys. Rev. Lett. 90, 118102-1-118102-4) by more than 2 orders of magnitude. This is achieved by identifying and purifying a fuel with akinetically trapped metastable configuration consisting of a "kissing loop" stabilized by flanking helicaldomains; the catalyst strand acts by opening a helical domain and allowing the complex to relax to itsground state by a multistep pathway. The improved fuel/catalyst system shows a roughly 5000-foldacceleration of the uncatalyzed reaction, with each catalyst molecule capable of turning over in excess of40 substrates. With
kcat/
KM 10
7/M/min, comparable to many protein enzymes and ribozymes, this fuelsystem becomes a viable component enabling future DNA-based synthetic molecular machines and logiccircuits. As an example, we designed and characterized a signal amplifier based on the fuel-catalyst system.The amplifier uses a single strand of DNA as input and releases a second strand with unrelated sequenceas output. A single input strand can catalytically trigger the release of more than 10 output strands.