Open-Endedness in Genelife.

We explore the open-ended nature of evolution in Genelife, an evolutionary extension of Conway's Game of Life cellular automaton in which "live" cell states are endowed at birth with a genome that affects their local dynamics and can be inherited. Both genetic sequences and locally connected spatial patterns are analyzed for novelty, keeping track of all new structures, and innovation is quantified using activity statistics. The impacts of both spatial symmetry breaking with nontotalistic rules and superimposed density regulation of the live state proliferation on the open-ended nature of the evolution are explored. Conditions are found where both genetic and spatial patterns exhibit open-ended innovation. This innovation appears to fall short of functional biological innovation, however, and potential reasons for this are discussed.

Bio-Inspired Dynamically Morphing Microelectronics toward High-Density Energy Applications and Intelligent Biomedical Implants.

Choreographing the adaptive shapes of patterned surfaces to exhibit designable mechanical interactions with their environment remains an intricate challenge. Here, a novel category of strain-engineered dynamic-shape materials, empowering diverse multi-dimensional shape modulations that are combined to form fine-grained adaptive microarchitectures is introduced. Using micro-origami tessellation technology, heterogeneous materials are provided with strategic creases featuring stimuli-responsive micro-hinges that morph precisely upon chemical and electrical cues. Freestanding multifaceted foldable packages, auxetic mesosurfaces, and morphable cages are three of the forms demonstrated herein of these complex 4-dimensional (4D) metamaterials. These systems are integrated in dual proof-of-concept bioelectronic demonstrations: a soft foldable supercapacitor enhancing its power density (≈108 mW cm-2 ), and a bio-adaptive device with a dynamic shape that may enable novel smart-implant technologies. This work demonstrates that intelligent material systems are now ready to support ultra-flexible 4D microelectronics, which can impart autonomy to devices culminating in the tangible realization of microelectronic morphogenesis.

3D nanofabricated soft microrobots with super-compliant picoforce springs as onboard sensors and actuators.

Microscale organisms and specialized motile cells use protein-based spring-like responsive structures to sense, grasp and move. Rendering this biomechanical transduction functionality in an artificial micromachine for applications in single-cell manipulations is challenging due to the need for a bio-applicable nanoscale spring system with a large and programmable strain response to piconewton-scale forces. Here we present three-dimensional nanofabrication and monolithic integration, based on an acrylic elastomer photoresist, of a magnetic spring system with quantifiable compliance sensitive to 0.5 pN, constructed with customized elasticity and magnetization distributions at the nanoscale. We demonstrate the effective design programmability of these 'picospring' ensembles as energy transduction mechanisms for the integrated construction of customized soft micromachines, with onboard sensing and actuation functions at the single-cell scale for microrobotic grasping and locomotion. The integration of active soft springs into three-dimensional nanofabrication offers an avenue to create biocompatible soft microrobots for non-disruptive interactions with biological entities.

Microelectronic Morphogenesis: Smart Materials with Electronics Assembling into Artificial Organisms.

Microelectronic morphogenesis is the creation and maintenance of complex functional structures by microelectronic information within shape-changing materials. Only recently has in-built information technology begun to be used to reshape materials and their functions in three dimensions to form smart microdevices and microrobots. Electronic information that controls morphology is inheritable like its biological counterpart, genetic information, and is set to open new vistas of technology leading to artificial organisms when coupled with modular design and self-assembly that can make reversible microscopic electrical connections. Three core capabilities of cells in organisms, self-maintenance (homeostatic metabolism utilizing free energy), self-containment (distinguishing self from nonself), and self-reproduction (cell division with inherited properties), once well out of reach for technology, are now within the grasp of information-directed materials. Construction-aware electronics can be used to proof-read and initiate game-changing error correction in microelectronic self-assembly. Furthermore, noncontact communication and electronically supported learning enable one to implement guided self-assembly and enhance functionality. Here, the fundamental breakthroughs that have opened the pathway to this prospective path are reviewed, the extent and way in which the core properties of life can be addressed are analyzed, and the potential and indeed necessity of such technology for sustainable high technology in society is discussed.

From quasispecies to quasispaces: coding and cooperation in chemical and electronic systems.

This contribution addresses the physical roles of spatial structures, either externally imposed or generated through self-assembly, either passive or active, on the physical chemistry of evolution. Starting with simple diffusion in closed capillaries, a one-dimensional space, it covers eight aspects of experimental and theoretical research into the interaction of evolution with spatial structures: in various dimensions, including hitherto unexplored ones, spanning from externally defined physical spaces to actively tailored spaces, assembled by the evolving components themselves. As such, it contains some original research by the author as well as tracing how other insights grew over three decades out of the mentorship of Manfred Eigen in the 1980s. Much of the early interest in spatial structures centres on its role in stabilizing higher order cooperative structures involving the coevolution of different molecules, as the genetic coding system exemplifies. Modern nanotechnology enables the design and construction of genetically encoded variants of smart components that can actively control both the proliferation of molecules and the structuring of space. A key role for this article is to show the continuity in this line of enquiry, beginning with quasispecies and projecting to autonomous microparticles with electronic genomes able to form programmable quasispaces.

Optomagnetic detection of DNA triplex nanoswitches.

We report on optomagnetic dose-dependent detection of DNA triplex-mediated and pH-switchable clusters of functionalised magnetic nanoparticles.

DNA-library assembly programmed by on-demand nano-liter droplets from a custom microfluidic chip.

Nanoscale synthetic biology can benefit from programmable nanoliter-scale processing of DNA in microfluidic chips if they are interfaced effectively to biochemical arrays such as microwell plates. Whereas active microvalve chips require complex fabrication and operation, we show here how a passive and readily fabricated microchip can be employed for customizable nanoliter scale pipetting and reaction control involving DNA. This recently developed passive microfluidic device, supporting nanoliter scale combinatorial droplet generation and mixing, is here used to generate a DNA test library with one member per droplet exported to addressed locations on microwell plates. Standard DNA assembly techniques, such as Gibson assembly, compatible with isothermal on-chip operation, are employed and checked using off-chip PCR and assembly PCR. The control of output droplet sequences and mixing performance was verified using dyes and fluorescently labeled DNA solutions, both on-chip and in external capillary channels. Gel electrophoresis of products and DNA sequencing were employed to further verify controlled combination and functional enzymatic assembly. The scalability of the results to larger DNA libraries is also addressed by combinatorial input expansion using sequential injection plugs from a multiwell plate. Hence, the paper establishes a proof of principle of the production of functional combinatorial mixtures at the nanoliter scale for one sequence per well DNA libraries.

Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems.

Emergence of coding and its specificity as a physico-informatic problem.

We explore the origin-of-life consequences of the view that biological systems are demarcated from inanimate matter by their possession of referential information, which is processed computationally to control choices of specific physico-chemical events. Cells are cybernetic: they use genetic information in processes of communication and control, subjecting physical events to a system of integrated governance. The genetic code is the most obvious example of how cells use information computationally, but the historical origin of the usefulness of molecular information is not well understood. Genetic coding made information useful because it imposed a modular metric on the evolutionary search and thereby offered a general solution to the problem of finding catalysts of any specificity. We use the term "quasispecies symmetry breaking" to describe the iterated process of self-organisation whereby the alphabets of distinguishable codons and amino acids increased, step by step.

On demand nanoliter-scale microfluidic droplet generation, injection, and mixing using a passive microfluidic device.

We here present and characterize a programmable nanoliter scale droplet-on-demand device that can be used separately or readily integrated into low cost single layer rapid prototyping microfluidic systems for a wide range of user applications. The passive microfluidic device allows external (off-the-shelf) electronically controlled pinch valves to program the delivery of nanoliter scale aqueous droplets from up to 9 different inputs to a central outlet channel. The inputs can be either continuous aqueous fluid streams or microliter scale aqueous plugs embedded in a carrier fluid, in which case the number of effective input solutions that can be employed in an experiment is no longer strongly constrained (100 s-1000 s). Both nanoliter droplet sequencing output and nanoliter-scale droplet mixing are reported with this device. Optimization of the geometry and pressure relationships in the device was achieved in several hardware iterations with the support of open source microfluidic simulation software and equivalent circuit models. The requisite modular control of pressure relationships within the device is accomplished using hydrodynamic barriers and matched resistance channels with three different channel heights, custom parallel reversible microfluidic I/O connections, low dead-volume pinch valves, and a simply adjustable array of external screw valves. Programmable sequences of droplet mixes or chains of droplets can be achieved with the device at low Hz frequencies, limited by device elasticity, and could be further enhanced by valve integration. The chip has already found use in the characterization of droplet bunching during export and the synthesis of a DNA library.

Addressing, amplifying and switching DNAzyme functions by electrochemically-triggered release of metal ions.

The design of artificial cells, which mimic the functions of native cells, is an ongoing scientific goal. The development of stimuli-responsive chemical systems that stimulate cascaded catalytic transformations, trigger chemical networks, and control vectorial branched transformations and dose-controlled processes, are the minimum requirements for mimicking cell functions. We have studied the electrochemical programmed release of ions from electrodes, which trigger selective DNAzyme-driven chemical reactions, cascaded reactions that self-assemble catalytic DNAzyme polymers, and the ON-OFF switching and dose-controlled operation of catalytic reactions. The addressable and potential-controlled release of Pb2+ or Ag+ ions into an electrolyte that includes a mixture of nucleic acids, results in the metal ion-guided selection of nucleic acids yielding the formation of specific DNAzymes, which stimulate orthogonal reactions or activate DNAzyme cascades.

DNA with 3'-5'-disulfide links--rapid chemical ligation through isosteric replacement.

Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer ligation to reactions of novel oligonucleotides, peptides, and hybrids such as PNA.1 Key requirements for potential diagnostic tools not based on PCR include a fast templated chemical DNA ligation method that exhibits high pairing selectivity, and a sensitive detection method. Here we report on a solid-phase synthesis of oligonucleotides containing 5'- or 3'-mercapto-dideoxynucleotides and their chemical ligations, yielding 3'-5'-disulfide bonds as a replacement for 3'-5'-phosphodiester units. Employing a system designed for fluorescence monitoring, we demonstrate one of the fastest ligation reactions with half-lives on the order of seconds. The nontemplated ligation reaction is efficiently suppressed by the choice of DNA modification and the 3'-5' orientation of the activation site. The influence of temperature on the templated reaction is shown.

Field programmable chemistry: integrated chemical and electronic processing of informational molecules towards electronic chemical cells.

The topic addressed is that of combining self-constructing chemical systems with electronic computation to form unconventional embedded computation systems performing complex nano-scale chemical tasks autonomously. The hybrid route to complex programmable chemistry, and ultimately to artificial cells based on novel chemistry, requires a solution of the two-way massively parallel coupling problem between digital electronics and chemical systems. We present a chemical microprocessor technology and show how it can provide a generic programmable platform for complex molecular processing tasks in Field Programmable Chemistry, including steps towards the grand challenge of constructing the first electronic chemical cells. Field programmable chemistry employs a massively parallel field of electrodes, under the control of latched voltages, which are used to modulate chemical activity. We implement such a field programmable chemistry which links to chemistry in rather generic, two-phase microfluidic channel networks that are separated into weakly coupled domains. Electric fields, produced by the high-density array of electrodes embedded in the channel floors, are used to control the transport of chemicals across the hydrodynamic barriers separating domains. In the absence of electric fields, separate microfluidic domains are essentially independent with only slow diffusional interchange of chemicals. Electronic chemical cells, based on chemical microprocessors, exploit a spatially resolved sandwich structure in which the electronic and chemical systems are locally coupled through homogeneous fine-grained actuation and sensor networks and play symmetric and complementary roles. We describe how these systems are fabricated, experimentally test their basic functionality, simulate their potential (e.g. for feed forward digital electrophoretic (FFDE) separation) and outline the application to building electronic chemical cells.

Living technology: exploiting life's principles in technology.

The concept of living technology-that is, technology that is based on the powerful core features of life-is explained and illustrated with examples from artificial life software, reconfigurable and evolvable hardware, autonomously self-reproducing robots, chemical protocells, and hybrid electronic-chemical systems. We define primary (secondary) living technology according as key material components and core systems are not (are) derived from living organisms. Primary living technology is currently emerging, distinctive, and potentially powerful, motivating this review. We trace living technology's connections with artificial life (soft, hard, and wet), synthetic biology (top-down and bottom-up), and the convergence of nano-, bio-, information, and cognitive (NBIC) technologies. We end with a brief look at the social and ethical questions generated by the prospect of living technology.

Evolutionary self-organization in complex fluids.

This paper explores the ability of molecular evolution to take control of collective physical phases, making the first decisive step from independent replicators towards cell-like collective structures. We develop a physical model of replicating combinatorial molecules in a ternary fluid of hydrocarbons, amphiphiles and water. Such systems are being studied experimentally in various laboratories to approach the synthesis of artificial cells, and are also relevant to the origin of cellular life. The model represents amphiphiles by spins on a lattice (with Ising coupling in the simplest case), coupled to replicating molecules that may diffuse on the lattice and react with each other. The presence of the replicating molecules locally modulates the phases of the complex fluid, and the physical replication process and/or mobility of the replicating molecules is influenced by the local amphiphilic configuration through an energetic coupling. Consequently, the replicators can potentially modify their environment to enhance their own replication. Through this coupling, the system can associate hereditary properties, and the potential for autonomous evolution, to self-assembling mesoscale structures in the complex fluid. This opens a route to analyse the evolution of artificial cells. The models are studied using Monte Carlo simulation, and demonstrate the evolution of phase control. We achieve a unified combinatorial framework for the description of isotropic families of spin-lattice models of complex phases, opening up the physical study of their evolution.

Optimization and design of oligonucleotide setup for strand displacement amplification.

Several advantages of strand displacement amplification (SDA) as an all-purpose DNA amplification reaction are due to it isothermal mechanism. The major problem of isothermal amplification mechanism is the accumulation of non-predictable byproduct especially for longer incubation time and low concentrations of initial template DNA. New theoretical strategies to tackle the difficulties regarding the specificity of the reaction are experimentally verified. Besides improving the reaction conditions, the stringency of primer hybridization can be distinctly improved by computer based sequence prediction algorithms based on the thermodynamic stability of DNA hybrid a described by the partition function of the hybridization reaction. An alternative SDA mechanism, with sequences developed by this means is also investigated.

Folding stabilizes the evolution of catalysts.

Sequence folding is known to determine the spatial structure and catalytic function of proteins and nucleic acids. We show here that folding also plays a key role in enhancing the evolutionary stability of the intermolecular recognition necessary for the prevalent mode of catalytic action in replication, namely, in trans, one molecule catalyzing the replication of another copy, rather than itself. This points to a novel aspect of why molecular life is structured as it is, in the context of life as it could be: folding allows limited, structurally localized recognition to be strongly sensitive to global sequence changes, facilitating the evolution of cooperative interactions. RNA secondary structure folding, for example is shown to be able to stabilize the evolution of prolonged functional sequences, using only a part of this length extension for intermolecular recognition, beyond the limits of the (cooperative) error threshold. Such folding could facilitate the evolution of polymerases in spatially heterogeneous systems. This facilitation is, in fact, vital because physical limitations prevent complete sequence-dependent discrimination for any significant-size biopolymer substrate. The influence of partial sequence recognition between biopolymer catalysts and complex substrates is investigated within a stochastic, spatially resolved evolutionary model of trans catalysis. We use an analytically tractable nonlinear master equation formulation called PRESS (McCaskill et al., Biol. Chem. 382: 1343-1363), which makes use of an extrapolation of the spatial dynamics down from infinite dimensional space, and compare the results with Monte Carlo simulations.