The CRISPR effector protein Cas12a has been used for a wide variety of applications such as in vivo gene editing and regulation or in vitro DNA sensing. Here, we add programmability to Cas12a-based ...DNA processing by combining it with strand displacement-based reaction circuits. We first establish a viable strategy for augmenting Cas12a guide RNAs (gRNAs) at their 5' end and then use such 5' extensions to construct strand displacement gRNAs (SD gRNAs) that can be activated by single-stranded RNA trigger molecules. These SD gRNAs are further engineered to exhibit a digital and orthogonal response to different trigger RNA inputs-including full length mRNAs-and to function as multi-input logic gates. We also demonstrate that SD gRNAs can be designed to work inside bacterial cells. Using such in vivo SD gRNAs and a DNase inactive version of Cas12a (dCas12a), we demonstrate logic gated transcriptional control of gene expression in E. coli.
Multicellularity enables the growth of complex life forms as it allows for the specialization of cell types, differentiation and large-scale spatial organization. In a similar way, modular ...construction of synthetic multicellular systems will lead to dynamic biomimetic materials that can respond to their environment in complex ways. To achieve this goal, artificial cellular communication and developmental programs still have to be established. Here, we create geometrically controlled spatial arrangements of emulsion-based artificial cellular compartments containing synthetic in vitro gene circuitry, separated by lipid bilayer membranes. We quantitatively determine the membrane pore-dependent response of the circuits to artificial morphogen gradients, which are established via diffusion from dedicated organizer cells. Utilizing different types of feedforward and feedback in vitro gene circuits, we then implement artificial signalling and differentiation processes, demonstrating the potential for the realization of complex spatiotemporal dynamics in artificial multicellular systems.
Dynamic DNA nanotechnology, a subfield of DNA nanotechnology, is concerned with the study and application of nucleic acid strand-displacement reactions. Strand-displacement reactions generally ...proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination. Through the use of toeholds, which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration, the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude. In addition, the use of toeholds enables the construction of enzyme-free DNA reaction networks exhibiting complex dynamical behavior. A demonstration of this was provided in the year 2000, in which strand displacement reactions were employed to drive a DNA-based nanomachine (Yurke, B.; et al. Nature 2000, 406, 605–608). Since then, toehold-mediated strand displacement reactions have been used with ever increasing sophistication and the field of dynamic DNA nanotechnology has grown exponentially. Besides molecular machines, the field has produced enzyme-free catalytic systems, all DNA chemical oscillators and the most complex molecular computers yet devised. Enzyme-free catalytic systems can function as chemical amplifiers and as such have received considerable attention for sensing and detection applications in chemistry and medical diagnostics. Strand-displacement reactions have been combined with other enzymatically driven processes and have also been employed within living cells (Groves, B.; et al. Nat. Nanotechnol. 2015, 11, 287–294). Strand-displacement principles have also been applied in synthetic biology to enable artificial gene regulation and computation in bacteria. Given the enormous progress of dynamic DNA nanotechnology over the past years, the field now seems poised for practical application.
Nucleic Acid Based Molecular Devices Krishnan, Yamuna; Simmel, Friedrich C.
Angewandte Chemie (International ed.),
March 28, 2011, Letnik:
50, Številka:
14
Journal Article
Recenzirano
In biology, nucleic acids are carriers of molecular information: DNA’s base sequence stores and imparts genetic instructions, while RNA’s sequence plays the role of a messenger and a regulator of ...gene expression. As biopolymers, nucleic acids also have exciting physicochemical properties, which can be rationally influenced by the base sequence in myriad ways. Consequently, in recent years nucleic acids have also become important building blocks for bottom‐up nanotechnology: as molecules for the self‐assembly of molecular nanostructures and also as a material for building machinelike nanodevices. In this Review we will cover the most important developments in this growing field of nucleic acid nanodevices. We also provide an overview of the biochemical and biophysical background of this field and the major “historical” influences that shaped its development. Particular emphasis is laid on DNA molecular motors, molecular robotics, molecular information processing, and applications of nucleic acid nanodevices in biology.
Nucleic acid power: Molecular machines and devices made from DNA or RNA are becoming more complex and more versatile. Nucleic acids have been shown to be an exquisite material for the rational design of simple molecular switches, and researchers now envision the realization of molecular robots for nanotechnology, computers, as well as nanomachines that operate in vivo.
The use of dynamic, self-assembled DNA nanostructures in the context of nanorobotics requires fast and reliable actuation mechanisms. We therefore created a 55-nanometer-by-55-nanometer DNA-based ...molecular platform with an integrated robotic arm of length 25 nanometers, which can be extended to more than 400 nanometers and actuated with externally applied electrical fields. Precise, computer-controlled switching of the arm between arbitrary positions on the platform can be achieved within milliseconds, as demonstrated with single-pair Förster resonance energy transfer experiments and fluorescence microscopy. The arm can be used for electrically driven transport of molecules or nanoparticles over tens of nanometers, which is useful for the control of photonic and plasmonic processes. Application of piconewton forces by the robot arm is demonstrated in force-induced DNA duplex melting experiments.
Gel‐based artificial organelles have been developed that enable sequence‐specific and programmable localization of cell‐free transcription and translation reactions inside an artificial cellular ...system. To this end, we utilize agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. We show that RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the corresponding DNA addresses. We also demonstrate that mRNA molecules, produced from transcription organelles and controlled by toehold switch riboregulators, are only translated in translation organelles containing their cognate DNA triggers. Spatial confinement of transcription and translation in separate organelles is thus superficially similar to gene expression in eukaryotic cells. Combining communicating gel spheres with specialized functions opens up new possibilities for programming artificial cellular systems at the organelle level.
Chemical processes within biological cells often occur inside organelles. This high degree of intracellular organization has inspired the development of gel‐based artificial organelles that enable sequence‐specific and programmable localization of cell‐free transcription and translation reactions inside an artificial cellular system.
Communication between artificial cells is essential for the realization of complex dynamical behaviors at the multi‐cell level. It is also an important prerequisite for modular systems design, ...because it determines how spatially separated functional modules can coordinate their actions. Among others, molecular communication is required for artificial cell signaling, synchronization of cellular behaviors, computation, group‐level decision‐making processes and pattern formation in artificial tissues. In this review, an overview of various recent approaches to create communicating artificial cellular systems is provided. In this context, important physicochemical boundary conditions that have to be considered for the design of the communicating cells are also described, and a survey of the most striking emergent behaviors that may be achieved in such systems is given.
Molecular communication is an important prerequisite for the realization of complex collective behaviors in a consortia of artificial cell‐scale systems.
Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity ...between components can lead to cross-talk, causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to 1 order of magnitude difference in reaction speed. Finally, we compare two established and one novel technique that speed up TMSD reactions in random sequence pools: a three-letter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.
Nucleic acid strand displacement reactions involve the competition of two or more DNA or RNA strands of similar sequence for binding to a complementary strand, and facilitate the isothermal ...replacement of an incumbent strand by an invader. The process can be biased by augmenting the duplex comprising the incumbent with a single-stranded extension, which can act as a toehold for a complementary invader. The toehold gives the invader a thermodynamic advantage over the incumbent, and can be programmed as a unique label to activate a specific strand displacement process. Toehold-mediated strand displacement processes have been extensively utilized for the operation of DNA-based molecular machines and devices as well as for the design of DNA-based chemical reaction networks. More recently, principles developed initially in the context of DNA nanotechnology have been applied for the de novo design of gene regulatory switches that can operate inside living cells. The article specifically focuses on the design of RNA-based translational regulators termed toehold switches. Toehold switches utilize toehold-mediated strand invasion to either activate or repress translation of an mRNA in response to the binding of a trigger RNA molecule. The basic operation principles of toehold switches will be discussed as well as their applications in sensing and biocomputing. Finally, strategies for their optimization will be described as well as challenges for their operation in vivo.
Triplex nanostructures can be formed in vitro in the promoter region of DNA templates, and it is commonly accepted that these assemblies inhibit the transcription of the downstream genes. Herein, a ...proof of concept highlighting the possibility of the up- or downregulation of RNA transcription is presented. Hybrid DNA-RNA triplex nanostructures were rationally designed to produce bacterial transcription units with switchable promoters. The rate of RNA production was measured using the signal of a transcribed fluorescent RNA aptamer (i.e. Broccoli). Indeed, several designed bacterial promoters showed the ability of induced transcriptional inhibition, while other properly tailored sequences demonstrated switchable enhancement of transcriptional activity, representing an unprecedented feature to date. The use of RNA-regulated transcription units and fluorescent RNA aptamers as readouts will allow the realization of biocomputation circuits characterized by a strongly reduced set of components. Triplex forming RNA oligonucleotides are proposed as smart tools for transcriptional modulation and represent an alternative to current methods for producing logic gates using protein-based components.
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