Programmable and autonomous computing machine made of biomolecules.

Devices that convert information from one form into another according to a definite procedure are known as automata. One such hypothetical device is the universal Turing machine, which stimulated work leading to the development of modern computers. The Turing machine and its special cases, including finite automata, operate by scanning a data tape, whose striking analogy to information-encoding biopolymers inspired several designs for molecular DNA computers. Laboratory-scale computing using DNA and human-assisted protocols has been demonstrated, but the realization of computing devices operating autonomously on the molecular scale remains rare. Here we describe a programmable finite automaton comprising DNA and DNA-manipulating enzymes that solves computational problems autonomously. The automaton's hardware consists of a restriction nuclease and ligase, the software and input are encoded by double-stranded DNA, and programming amounts to choosing appropriate software molecules. Upon mixing solutions containing these components, the automaton processes the input molecule via a cascade of restriction, hybridization and ligation cycles, producing a detectable output molecule that encodes the automaton's final state, and thus the computational result. In our implementation 1012 automata sharing the same software run independently and in parallel on inputs (which could, in principle, be distinct) in 120 microl solution at room temperature at a combined rate of 109 transitions per second with a transition fidelity greater than 99.8%, consuming less than 10-10 W.

Catalytic mechanism of Kdo8P synthase: transient kinetic studies and evaluation of a putative reaction intermediate.

The mechanistic pathway for the reaction catalyzed by Kdo8P synthase has been investigated, and the cyclic bisphosphate 2 has been examined as a putative reaction intermediate. Two parallel approaches were used: (1) chemical synthesis of 2 and evaluation as an alternate substrate for the enzyme and (2) transient kinetic studies using rapid chemical quench methodology to provide direct observation and characterization of putative intermediate(s) during enzyme catalysis. The putative cyclic bisphosphate intermediate 2, possessing the stereochemistry of the beta-pyranose form, was synthesized and evaluated as a substrate and as an inhibitor of Kdo8P synthase. The substrate activity was examined by monitoring the release of anomeric phosphate over time using proton-decoupled 31P NMR spectroscopy. A very similar time course for the formation of inorganic phosphate was found in each experiment and the corresponding control experiment; i.e., no enzyme-catalyzed acceleration in the anomeric phosphate hydrolysis was detected. It was found however that 2 binds to the enzyme and is a competitive inhibitor with respect to phosphoenolpyruvate binding, having a Ki value of 35 microM. In a parallel study, we have performed single-turnover rapid chemical quench experiments to examine both the forward and reverse directions to identify a putative enzyme intermediate(s). Our results clearly demonstrate that the cyclic bisphosphate intermediate 2 does not accumulate under single-enzyme turnover conditions. This observation, coupled with the results obtained through the evaluation of synthetic 2 as a substrate, strongly suggests that the Kdo8P synthase catalytic pathway does not involve the formation of 2 as a reaction intermediate. Taken together, these combined results support the original hypothesis [Hedstrom, L., and Abeles, R. H. (1988) Biochem. Biophys. Res. Commun. 157, 816-820], which suggests a reaction pathway involving an acyclic bisphosphate intermediate 1.