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J Am Chem Soc. 2006 Sep 20;128(37):12211-20.

Catalyzed relaxation of a metastable DNA fuel.

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Department of Applied Physics, California Institute of Technology, Pasadena, California 91125, USA.


Practically all of life's molecular processes, from chemical synthesis to replication, involve enzymes that 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 engineering as it is in biology. Here we report a metastable DNA "fuel" and a corresponding DNA "catalyst" that improve upon 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 a kinetically trapped metastable configuration consisting of a "kissing loop" stabilized by flanking helical domains; the catalyst strand acts by opening a helical domain and allowing the complex to relax to its ground state by a multistep pathway. The improved fuel/catalyst system shows a roughly 5000-fold acceleration of the uncatalyzed reaction, with each catalyst molecule capable of turning over in excess of 40 substrates. With k(cat)/K(M) approximately 10(7)/M/min, comparable to many protein enzymes and ribozymes, this fuel system becomes a viable component enabling future DNA-based synthetic molecular machines and logic circuits. 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 sequence as output. A single input strand can catalytically trigger the release of more than 10 output strands.

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