Pattern selection in a cooperative biochemical in vitro amplification system: the role of parasites.

Previously, numerical simulations have shown that evolving systems can be stabilized against emerging parasites by pattern formation in spatially extended flow reactors. Hence, it can be argued that pattern formation is a prerequisite for any experimental investigation of the biochemical evolution of cooperative function. Here, we study a model of an experimental biochemical system for the cooperative in vitro amplification of DNA strands and show that emerging parasites can induce a complex pattern formation even when no pattern formation occurs without parasites. In an adiabatic approximation where the cooperative amplification reaction is assumed to adapt fast to slowly emerging parasites, the parasite concentration itself acts as a Steuer parameter for the selection of various complex patterns. Without such an adiabatic approximation only transient patterns emerge. As any species can grow for very low concentrations, the parasite is able to infect the entire reactor and the system is finally diluted out. In the experimental biochemical system, however, the species are individual molecules and the growth of spatially separated, non-infected regions becomes feasible. Hence a cutoff threshold for the minimal concentration is applied. In these simulations the otherwise lethal infection by parasites induces the formation of spatiotemporal spirals, and this spatial structure help the host and parasitoid species to survive together. These theoretical results describe an inherent property of cooperative reactions and have an important impact on experimental investigations on the molecular evolution and complex function in spatially extended reactors. Since the formation of the complex pattern is restricted either to a rather large cutoff value or a special choice of the kinetic parameters, we, however, conclude that the persistence of evolving cooperative amplification is not possible in a simple reaction-diffusion reactor. Experimental set-ups with patchy environments, e.g. biomolecular amplification in coupled microstructured flow chambers or in microemulsion, are eligible candidates for the observation of such a self-organized pattern selection.

Complex patterns predicted in an in vitro experimental model system for the evolution of molecular cooperation.

An isothermal biochemical in vitro amplification system with two trans-cooperatively coupled amplifying DNA molecules was investigated homogeneously using a hierarchy of kinetic models and as a simplified reaction-diffusion system. In our model of this recently developed experimental system, no reaction mechanism higher than second order occurs, yet numerical simulations show a variety of complex spatiotemporal patterns which arise in response to finite amplitude perturbations in a flow reactor. In a certain domain of the kinetic parameters the system shows self-replicating spots. These spots can stabilize the cooperative amplification in such evolving systems against emerging parasites. The results are of high relevance for experimental studies on these functional in vitro ecosystems in spatially resolved microstructured reactors.

Monitoring the amplification of CATCH, a 3SR based cooperatively coupled isothermal amplification system, by fluorimetric methods.

Three different types of fluorescence detection methods were employed to monitor amplification of a previously established isothermal cooperatively coupled amplification system as it can serve as a tool for the investigation of fundamental issues in evolutionary optimization. By using 5'IRD-41 fluorescent labeled primers, the intercalating dye TOPRO-1 and a 5'fluorescin/3'DABCYL 4-(4-dimethylamino-phenylazo)benzoic acid labeled ss 24 nt DNA, evolving molecular cooperation is accessible, sequence specifically as well as non-sequence-specifically without using radioactivity.