Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.

Heterochiral modifications enhance robustness and function of DNA in living human cells.

Oligonucleotide therapeutics are becoming increasingly important as more are approved by the FDA, both for treatment and vaccination. Similarly, dynamic DNA nanotechnology is a promising technique that can be used to sense exogenous input molecules or endogenous biomarkers and integrate the results of multiple sensing reactions in situ via a programmed cascade of reactions. The combination of these two technologies could be highly impactful in biomedicine by enabling smart oligonucleotide therapeutics that can autonomously sense and respond to a disease state. A particular challenge, however, is the limited lifetime of standard nucleic acid components in living cells and organisms due to degradation by endogenous nucleases. In this work, we address this challenge by incorporating mirror-image, ʟ-DNA nucleotides to produce heterochiral "gapmers". We use dynamic DNA nanotechnology to show that these modifications keep the oligonucleotide intact in living human cells for longer than an unmodified strand. To this end, we used a sequential transfection protocol for delivering multiple nucleic acids into living human cells while providing enhanced confidence that subsequent interactions are actually occurring within the cells. Taken together, this work advances the state of the art of ʟ-nucleic acid protection of oligonucleotides and DNA circuitry for applications in vivo.

A geometric framework for reaction enumeration in computational nucleic acid devices.

Cascades of DNA strand displacement reactions enable the design of potentially large circuits with complex behaviour. Computational modelling of such systems is desirable to enable rapid design and analysis. In previous work, the expressive power of graph theory was used to enumerate reactions implementing strand displacement across a wide range of complex structures. However, coping with the rich variety of possible graph-based structures required enumeration rules with complicated side-conditions. This paper presents an alternative approach to tackle the problem of enumerating reactions at domain level involving complex structures by integrating with a geometric constraint solving algorithm. The rule sets from previous work are simplified by replacing side-conditions with a general check on the geometric plausibility of structures generated by the enumeration algorithm. This produces a highly general geometric framework for reaction enumeration. Here, we instantiate this framework to solve geometric constraints by a structure sampling approach in which we randomly generate sets of coordinates and check whether they satisfy all the constraints. We demonstrate this system by applying it to examples from the literature where molecular geometry plays an important role, including DNA hairpin and remote toehold reactions. This work therefore enables integration of reaction enumeration and structural modelling.

Design and Simulation of a Multilayer Chemical Neural Network That Learns via Backpropagation.

The design and implementation of adaptive chemical reaction networks, capable of adjusting their behavior over time in response to experience, is a key goal for the fields of molecular computing and DNA nanotechnology. Mainstream machine learning research offers powerful tools for implementing learning behavior that could one day be realized in a wet chemistry system. Here we develop an abstract chemical reaction network model that implements the backpropagation learning algorithm for a feedforward neural network whose nodes employ the nonlinear "leaky rectified linear unit" transfer function. Our network directly implements the mathematics behind this well-studied learning algorithm, and we demonstrate its capabilities by training the system to learn a linearly inseparable decision surface, specifically, the XOR logic function. We show that this simulation quantitatively follows the definition of the underlying algorithm. To implement this system, we also report ProBioSim, a simulator that enables arbitrary training protocols for simulated chemical reaction networks to be straightforwardly defined using constructs from the host programming language. This work thus provides new insight into the capabilities of learning chemical reaction networks and also develops new computational tools to simulate their behavior, which could be applied in the design and implementations of adaptive artificial life.

Supervised Learning in a Multilayer, Nonlinear Chemical Neural Network.

The development of programmable or trainable molecular circuits is an important goal in the field of molecular programming. Multilayer, nonlinear, artificial neural networks are a powerful framework for implementing such functionality in a molecular system, as they are provably universal function approximators. Here, we present a design for multilayer chemical neural networks with a nonlinear hyperbolic tangent transfer function. We use a weight perturbation algorithm to train the neural network which uses a simple construction to directly approximate the loss derivatives required for training. We demonstrate the training of this system to learn all 16 two-input binary functions from a common starting point. This work thus introduces new capabilities in the field of adaptive and trainable chemical reaction network (CRN) design. It also opens the door to potential future experimental implementations, including DNA strand displacement reactions.

Operant conditioning of stochastic chemical reaction networks.

Adapting one's behavior to environmental conditions and past experience is a key trait of living systems. In the biological world, there is evidence for adaptive behaviors such as learning even in naturally occurring, non-neural, single-celled organisms. In the bioengineered world, advances in synthetic cell engineering and biorobotics have created the possibility of implementing lifelike systems engineered from the bottom up. This will require the development of programmable control circuitry for such biomimetic systems that is capable of realizing such non-trivial and adaptive behavior, including modification of subsequent behavior in response to environmental feedback. To this end, we report the design of novel stochastic chemical reaction networks capable of probabilistic decision-making in response to stimuli. We show that a simple chemical reaction network motif can be tuned to produce arbitrary decision probabilities when choosing between two or more responses to a stimulus signal. We further show that simple feedback mechanisms from the environment can modify these probabilities over time, enabling the system to adapt its behavior dynamically in response to positive or negative reinforcement based on its decisions. This system thus acts as a form of operant conditioning of the chemical circuit, in the sense that feedback provided based on decisions taken by the circuit form the basis of the learning process. Our work thus demonstrates that simple chemical systems can be used to implement lifelike behavior in engineered biomimetic systems.

Protecting Heterochiral DNA Nanostructures against Exonuclease-Mediated Degradation.

Heterochiral DNA nanotechnology employs nucleic acids of both chiralities to construct nanoscale devices for applications in the intracellular environment. Interacting directly with cellular nucleic acids can be done most easily using D-DNA of the naturally occurring right-handed chirality; however, D-DNA is more vulnerable to degradation than enantiometric left-handed L-DNA. Here we report a novel combination of D-DNA and L-DNA nucleotides in triblock heterochiral copolymers, where the L-DNA domains act as protective caps on D-DNA domains. We demonstrate that the D-DNA components of strand displacement-based molecular circuits constructed using this technique resist exonuclease-mediated degradation during extended incubations in serum-supplemented media more readily than similar devices without the L-DNA caps. We show that this protection can be applied to both double-stranded and single-stranded circuit components. Our work enhances the state of the art for robust heterochiral circuit design and could lead to practical applications such as in vivo biomedical diagnostics.

Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.

Robust finite automata in stochastic chemical reaction networks.

Finite-state automata (FSA) are simple computational devices that can nevertheless illustrate interesting behaviours. We propose that FSA can be employed as control circuits for engineered stochastic biological and biomolecular systems. We present an implementation of FSA using counts of chemical species in the range of hundreds to thousands, which is relevant for the counts of many key molecules such as mRNAs in prokaryotic cells. The challenge here is to ensure a robust representation of the current state in the face of stochastic noise. We achieve this by using a multistable approximate majority algorithm to stabilize and store the current state of the system. Arbitrary finite state machines can thus be compiled into robust stochastic chemical automata. We present two variants: one that consumes its input signals to initiate state transitions and one that does not. We characterize the state change dynamics of these systems and demonstrate their application to solve the four-bit binary square root problem. Our work lays the foundation for the use of chemical automata as control circuits in bioengineered systems and biorobotics.

Structure sampling for computational estimation of localized DNA interaction rates.

Molecular circuits implemented using molecular components tethered to a DNA tile nanostructure have certain advantages over solution-phase circuits. Tethering components in close proximity increases the speed of reactions by reducing diffusion and improves scalability by enabling reuse of identical DNA sequences at different locations in the circuit. These systems show great potential for practical applications including delivery of diagnostic and therapeutic molecular circuits to cells. When modeling such systems, molecular geometry plays an important role in determining whether the two species interact and at what rate. In this paper, we present an automated method for estimating reaction rates in tethered molecular circuits that takes the geometry of the tethered species into account. We probabilistically generate samples of structure distributions based on simple biophysical models and use these to estimate important parameters for kinetic models. This work provides a basis for subsequent enhanced modeling and design tools for localized molecular circuits.

Robust Heterochiral Strand Displacement Using Leakless Translators.

Molecular computing offers a powerful framework for in situ biosensing and signal processing at the nanoscale. However, for in vivo applications, the use of conventional DNA components can lead to false positive signals being generated due to degradation of circuit components by nuclease enzymes. Here, we use hybrid chiral molecules, consisting of both l- and d-nucleic acid domains, to implement leakless signal translators that enable d-nucleic acid signals to be detected by hybridization and then translated into a robust l-DNA signal for further analysis. We show that our system is robust to false positive signals even if the d-DNA components are degraded by nucleases, thanks to circuit-level robustness. This work thus broadens the scope and applicability of DNA-based molecular computers for practical, in vivo applications.

Domain-Specific Programming Languages for Computational Nucleic Acid Systems.

The construction of models of system behavior is of great importance throughout science and engineering. In bioengineering and bionanotechnology, these often take the form of dynamic models that specify the evolution of different species over time. To ensure that scientific observations and conclusions are consistent and that systems can be reliably engineered on the basis of model predictions, it is important that models of biomolecular systems can be constructed in a reliable, principled, and efficient manner. This review focuses on efforts to address this need by using domain-specific programming languages as the basis for custom design tools for researchers working on computational nucleic acid devices, where a domain-specific language is simply a programming language tailored to a particular application domain. The underlying thesis of our review is that there is a continuum of practical implementation strategies for computational nucleic acid systems, which can all benefit from appropriate domain-specific languages and software design tools. We emphasize the need for specialized yet flexible tools that can be realized using domain-specific languages that compile to more general-purpose representations.

Microcompartments for Protection and Isolation of Nanoscale DNA Computing Elements.

Physical isolation of molecular computing elements holds the potential for increasing system complexity by enabling the reuse of standardized components and by protecting the components from environmental degradation. However, once elements have been compartmentalized, methods for communicating into these compartments are needed. We report the compartmentalization of steroid-responsive DNA aptamers within giant unilamellar vesicles (GUVs) that are permeable to steroid inputs. Monodisperse GUVs are loaded with aptamers using a microfluidic platform. We demonstrate the target-specific activation of individual aptamers within the GUVs and then load two noninterfering aptamers into the same GUV and demonstrate specific responses to all possible combinations of the two input steroids. Crucially, GUVs prevent the degradation of DNA components by nucleases, providing a potential mechanism for deploying nucleic acid components in vivo. Importantly, our compartments also prevent nonspecific cross-talk between complementary strands, thereby providing a method for parallel execution of cross-reacting molecular logic components. Thus, we provide a mechanism for spatially organizing molecular computing elements, which will increase system modularity by allowing standardized components to be reused.

A Logic Programming Language for Computational Nucleic Acid Devices.

Computational nucleic acid devices show great potential for enabling a broad range of biotechnology applications, including smart probes for molecular biology research, in vitro assembly of complex compounds, high-precision in vitro disease diagnosis and, ultimately, computational theranostics inside living cells. This diversity of applications is supported by a range of implementation strategies, including nucleic acid strand displacement, localization to substrates, and the use of enzymes with polymerase, nickase, and exonuclease functionality. However, existing computational design tools are unable to account for these strategies in a unified manner. This paper presents a logic programming language that allows a broad range of computational nucleic acid systems to be designed and analyzed. The language extends standard logic programming with a novel equational theory to express nucleic acid molecular motifs. It automatically identifies matching motifs present in the full system, in order to apply a specified transformation expressed as a logical rule. The language supports the definition of logic predicates, which provide constraints that need to be satisfied in order for a given rule to be applied. The language is sufficiently expressive to encode the semantics of nucleic strand displacement systems with complex topologies, together with computation performed by a broad range of enzymes, and is readily extensible to new implementation strategies. Our approach lays the foundation for a unifying framework for the design of computational nucleic acid devices.

Sequential super-resolution imaging using DNA strand displacement.

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.

A Microsphere-Supported Lipid Bilayer Platform for DNA Reactions on a Fluid Surface.

We report a versatile microsphere-supported lipid bilayer system that can serve as a general-purpose platform for implementing DNA nanotechnologies on a fluid surface. To demonstrate our platform, we implemented both toehold-mediated strand displacement (TMSD) and DNAzyme reactions, which are typically performed in solution and which are the cornerstone of DNA-based molecular logic and dynamic DNA nanotechnology, on the surface. We functionalized microspheres bearing supported lipid bilayers (µSLBs) with membrane-bound nucleic acid components. Using functionalized µSLBs, we developed TMSD and DNAzyme reactions by optimizing reaction conditions to reduce nonspecific interactions between DNA and phospholipids and to enhance bilayer stability. Additionally, the physical and optical properties of the bilayer were tuned via lipid composition and addition of fluorescently tagged lipids to create stable and multiplexable µSLBs that are easily read out by flow cytometry. Multiplexed TMSD reactions on µSLBs enabled the successful operation of a Dengue serotyping assay that correctly identified all 16 patterns of target sequences to demonstrate detection of DNA strands derived from the sequences of all four Dengue serotypes. The limit of detection for this assay was 3 nM. Furthermore, we demonstrated DNAzyme reactions on a fluid lipid surface, which benefit from free diffusion on the surface. This work provides the basis for expansion of both TMSD and DNAzyme based molecular reactions on supported lipid bilayers for use in molecular logic and DNA nanotechnology. As our system is multiplexable and results in fluid surfaces, it may be of use in compartmentalization and improved kinetics of molecular logic reactions and as a useful building block in a variety of DNA nanotechnology systems.

Modular verification of chemical reaction network encodings via serializability analysis.

Chemical reaction networks are a powerful means of specifying the intended behaviour of synthetic biochemical systems. A high-level formal specification, expressed as a chemical reaction network, may be compiled into a lower-level encoding, which can be directly implemented in wet chemistry and may itself be expressed as a chemical reaction network. Here we present conditions under which a lower-level encoding correctly emulates the sequential dynamics of a high-level chemical reaction network. We require that encodings are transactional, such that their execution is divided by a "commit reaction" that irreversibly separates the reactant-consuming phase of the encoding from the product-generating phase. We also impose restrictions on the sharing of species between reaction encodings, based on a notion of "extra tolerance", which defines species that may be shared between encodings without enabling unwanted reactions. Our notion of correctness is serializability of interleaved reaction encodings, and if all reaction encodings satisfy our correctness properties then we can infer that the global dynamics of the system are correct. This allows us to infer correctness of any system constructed using verified encodings. As an example, we show how this approach may be used to verify two- and four-domain DNA strand displacement encodings of chemical reaction networks, and we generalize our result to the limit where the populations of helper species are unlimited.

A strand graph semantics for DNA-based computation.

DNA nanotechnology is a promising approach for engineering computation at the nanoscale, with potential applications in biofabrication and intelligent nanomedicine. DNA strand displacement is a general strategy for implementing a broad range of nanoscale computations, including any computation that can be expressed as a chemical reaction network. Modelling and analysis of DNA strand displacement systems is an important part of the design process, prior to experimental realisation. As experimental techniques improve, it is important for modelling languages to keep pace with the complexity of structures that can be realised experimentally. In this paper we present a process calculus for modelling DNA strand displacement computations involving rich secondary structures, including DNA branches and loops. We prove that our calculus is also sufficiently expressive to model previous work on non-branching structures, and propose a mapping from our calculus to a canonical strand graph representation, in which vertices represent DNA strands, ordered sites represent domains, and edges between sites represent bonds between domains. We define interactions between strands by means of strand graph rewriting, and prove the correspondence between the process calculus and strand graph behaviours. Finally, we propose a mapping from strand graphs to an efficient implementation, which we use to perform modelling and simulation of DNA strand displacement systems with rich secondary structure.

Logic circuits based on molecular spider systems.

Spatial locality brings the advantages of computation speed-up and sequence reuse to molecular computing. In particular, molecular walkers that undergo localized reactions are of interest for implementing logic computations at the nanoscale. We use molecular spider walkers to implement logic circuits. We develop an extended multi-spider model with a dynamic environment wherein signal transmission is triggered via localized reactions, and use this model to implement three basic gates (AND, OR, NOT) and a cascading mechanism. We develop an algorithm to automatically generate the layout of the circuit. We use a kinetic Monte Carlo algorithm to simulate circuit computations, and we analyze circuit complexity: our design scales linearly with formula size and has a logarithmic time complexity.