CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA.
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ABSTRACT:
SUBMITTER: Montagud-Martinez R
PROVIDER: S-EPMC8489798 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA is increasingly being used as an ideal material for the construction of nanoscale structures, circuits, and machines. Toehold-mediated DNA strand displacement reactions play a very important role in these enzyme-free constructions. In this study, the concept of metallo-toehold was utilized to further develop a mechanism for strand displacement driven by Ag+ ions, in which the intercalation of cytosine-cytosine mismatched base pairs on the toeholds provides additional control by varying of the concentration of Ag+ ions. The characteristics of displacement reaction in response to different concentration of Ag+ ions are investigated by fluorescence spectral and non-denaturing polyacrylamide gel electrophoresis. The reaction can successfully occur when the concentration of Ag+ ions is suitabe; excess Ag+ ions block the reaction. Furthermore, the displacement reaction can be tuned and controlled most efficiently under the condition of two C:C mismatched base pairs placed on the six-nt toehold. Based on our research, a mechanism was developed to construct Boolean logic gate AND and OR by employing strand displacement reaction as a tool, Ag+ and Hg2+ as input.
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Project description:We present a simple and effective scheme of a dynamic switch for DNA nanostructures. Under such a framework of toehold-free strand displacement, blocking strands at an excess amount are applied to displace the complementation of specific segments of paired duplexes. The functional mechanism of the scheme is illustrated by modelling the base pairing kinetics of competing strands on a target strand. Simulation reveals the unique properties of toehold-free strand displacement in equilibrium control, which can be leveraged for information processing. Based on the controllable dynamics in the binding of preformed DNA nanostructures, a multi-input-multi-output (MIMO) Boolean function is controlled by the presence of the blockers. In conclusion, we implement two MIMO Boolean functions (one with 4-bit input and 2-bit output, and the other with 16-bit input and 8-bit output) to showcase the controllable dynamics.
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Project description:We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperature and make two experimentally testable predictions: that the displacement is faster if the toehold is placed at the 5' end of the substrate; and that the displacement slows down with increasing temperature for longer toeholds.
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Project description:CRISPR (clustered regularly interspaced short palindromic repeats) technology has achieved great breakthroughs in terms of convenience and sensitivity; it is becoming the most promising molecular tool. However, only two CRISPR activation modes (single and double stranded) are available, and they have specificity and universality bottlenecks that limit the application of CRISPR technology in high-precision molecular recognition. Herein, we proposed a novel CRISPR/Cas12a unrestricted activation mode to greatly improve its performance. The new mode totally eliminates the need for a protospacer adjacent motif and accurately activates Cas12a through toehold-mediated strand displacement and branch migration, which is highly universal and ultra-specific. With this mode, we discriminated all mismatch types and detected the EGFR T790M and L858R mutations in very low abundance. Taken together, our activation mode is deeply incorporated with DNA nanotechnology and extensively broadens the application boundaries of CRISPR technology in biomedical and molecular reaction networks.
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Project description:Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.
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Project description:Cell-free biosensors are powerful platforms for monitoring human and environmental health. Here, we expand their capabilities by interfacing them with toehold-mediated strand displacement circuits, a dynamic DNA nanotechnology that enables molecular computation through programmable interactions between nucleic acid strands. We develop design rules for interfacing a small molecule sensing platform called ROSALIND with toehold-mediated strand displacement to construct hybrid RNA-DNA circuits that allow fine-tuning of reaction kinetics. We use these design rules to build 12 different circuits that implement a range of logic functions (NOT, OR, AND, IMPLY, NOR, NIMPLY, NAND). Finally, we demonstrate a circuit that acts like an analog-to-digital converter to create a series of binary outputs that encode the concentration range of the molecule being detected. We believe this work establishes a pathway to create 'smart' diagnostics that use molecular computations to enhance the speed and utility of biosensors.
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Project description:Dynamic DNA nanotechnology is driving exciting developments in molecular computing, cargo delivery, sensing and detection. Combining this innovative area of research with the progress made in machine learning will aid in the design of sophisticated DNA machinery. Herein, we present a novel framework based on a transformer architecture and a deep learning model which can predict the rate constant of toehold-mediated strand displacement, the underlying process in dynamic DNA nanotechnology. Initially, a dataset of 4450 DNA sequences and corresponding rate constants were generated in-silico using KinDA. Subsequently, a 1D convolution neural network was trained using specific local features and DNA-BERT sequence embedding to produce predicted rate constants. As a result, the newly trained deep learning model predicted toehold-mediated strand displacement rate constants with a root mean square error of 0.76, during testing. These findings demonstrate that DNA-BERT can improve prediction accuracy, negating the need for extensive computational simulations or experimentation. Finally, the impact of various local features during model training is discussed, and a detailed comparison between the One-hot encoder and DNA-BERT sequences representation methods is presented.
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Project description:Molecular circuits crafted from DNA molecules harness the inherent programmability and biocompatibility of DNA to intelligently steer molecular machines in the execution of microscopic tasks. In comparison to combinational circuits, DNA-based temporal circuits boast supplementary capabilities, allowing them to proficiently handle the omnipresent temporal information within biochemical systems and life sciences. However, the lack of temporal mechanisms and components proficient in comprehending and processing temporal information presents challenges in advancing DNA circuits that excel in complex tasks requiring temporal control and time perception. In this study, we engineered temporal logic circuits through the design and implementation of a dual cross-inhibition mechanism, which enables the acceptance and processing of temporal information, serving as a fundamental building block for constructing temporal circuits. By incorporating the dual cross-inhibition mechanism, the temporal logic gates are endowed with cascading capabilities, significantly enhancing the inhibitory effect compared to a cross-inhibitor. Furthermore, we have introduced the annihilation mechanism into the circuit to further augment the inhibition effect. As a result, the circuit demonstrates sensitive time response characteristics, leading to a fundamental improvement in circuit performance. This architecture provides a means to efficiently process temporal signals in DNA strand displacement circuits. We anticipate that our findings will contribute to the design of complex temporal logic circuits and the advancement of molecular programming.
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Project description:The ability to create stimuli-responsive DNA nanostructures has played a prominent role in dynamic DNA nanotechnology. Primary among these is the process of toehold-based strand displacement, where a nucleic acid molecule can act as a trigger to cause conformational changes in custom-designed DNA nanostructures. Here, we add another layer of control to strand displacement reactions through a 'toehold clipping' process. By designing DNA complexes with a photocleavable linker-containing toehold or an RNA toehold, we show that we can use light (UV) or enzyme (ribonuclease) to eliminate the toehold, thus preventing strand displacement reactions. We use molecular dynamics simulations to analyze the structural effects of incorporating a photocleavable linker in DNA complexes. Beyond simple DNA duplexes, we also demonstrate the toehold clipping process in a model DNA nanostructure, by designing a toehold containing double-bundle DNA tetrahedron that disassembles when an invading strand is added, but stays intact after the toehold clipping process even in the presence of the invading strand. This work is an example of combining multiple physical or molecular stimuli to provide additional remote control over DNA nanostructure reconfiguration, advances that hold potential use in biosensing, drug delivery or molecular computation.
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Project description:This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labeled with a fluorophore at its 5'-end, while the other strand is labeled with a quencher at its 3'-end. These two DNA strands are perfectly complementary, but with a 3'-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.
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