DNA hybridization catalysts and catalyst circuits

Practically all of life's molecular processes, from chemical synthesis to replication, involve enzymes that carry out their functions through the catalysis of metastable fuels into waste products. Catalytic control of reaction rates will prove to be as useful and ubiquitous in DNA nanotechnology as it is in biology. Here we present experimental results on the control of the decay rates of a metastable DNA "fuel". We show that the fuel complex can be induced to decay with a rate about 1600 times faster than it would decay spontaneously. The original DNA hybridization catalyst [15] achieved a maximal speed-up of roughly 30. The fuel complex discussed here can therefore serve as the basic ingredient for an improved DNA hybridization catalyst. As an example application for DNA hybridization catalysts, we propose a method for implementing arbitrary digital logic circuits

Catalyzed relaxation of a metastable DNA fuel

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, 118102-1118102-4) 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 ≈ 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

Improving the Selectivity and Reducing the Leakage of DNA Strand Displacement Systems

Because of the elegance of Watson-Crick base pairing and the programmability of toehold-mediated strand displacement, DNA is a model material for designing, building, and testing molecular assemblies. DNA assemblies are categorized as structural when they are at thermodynamic equilibrium and dynamic when they are not. Through programmed perturbations, metastable assemblies perform physical, chemical, and computational work. When integrated into a diagnostic package, disease-specific nucleic acid sequences can be identified, amplified, and analyzed via standard DNA nanotechnology rules. In order for these rules to make an impact, two critical challenges in the field have been undertaken in this dissertation. First, the selectivity to distinguish an on-target sequence from off-target sequences, with a resolution of a single-nucleotide mutation, has been explored by site-specifically integrating locked nucleic acids into DNA sequences. Locked nucleic acids are RNA analogues that have higher thermal and hence mechanical stability than RNA and DNA. Second, the initiation of metastable chemical reaction networks, in the absence of on-target sequences, has been explored to suppress network leakage; which is the single greatest problem in dynamic DNA nanotechnology. To address this challenge, original catalytic substrates were designed, built, and tested to increase the energy barrier of the leakage reactions without sacrificing the performance of the favorable catalytic reactions. The experimental results showed that site-specific integration of LNA into DNA sequences improved the sequence selectivity by over 2 orders of magnitude. They also showed that network leakage could be suppressed by 2 orders of magnitude by decoupling the leakage pathway from the catalytic pathway in the original catalytic substrates. When combined, these results constitute a substantial contribution to the field of dynamic DNA nanotechnology and represent important steps towards the creation of low-cost, early-stage diagnostic tools for difficult to detect diseases such as lung, breast, and pancreatic cancers