Toehold Length Research Articles

Activated single nucleotide mismatch determination through toehold-embedded hairpin-mediated strand displacement reaction alongside CRISPR-Cas12a for detection of TP53 point mutation

The CRISPR-Cas12a-based diagnostic platform is renowned for its high sensitivity and, when coupled with the toehold-mediated strand displacement (TMSD) reaction, enables the detection of single nucleotide mismatch (SNM). However, conventional TMSD with the CRISPR-Cas12a system suffers from signal leakage due to overlapping activator sequences and low displacement efficiency. Here, we propose a toehold-embedded hairpin-mediated strand displacement (THMSD) method and leverage the magnesium attenuation effect of CRISPR-Cas12a for selective single-point mutation detection in the TP53 gene. This approach effectively mitigates signal leakage as the hairpin DNA retains partial crRNA targeting sequences within its loop, preventing sequence overlap with the trigger and direct Cas12a activation. Additionally, the magnesium attenuation effect reduces background signal and enhances selectivity. We investigated toehold length and mismatch position to detect SNM with a high discrimination factor (DF). This high DF allows the THMSD/CRISPR-Cas12a system to differentiate between TP53 R273H mutant and TP53 wildtype genes for cancer screening at low abundances, down to 0.1 % with a concentration of 20 pM. When used for screening between H1975 and WI38 cells, the statistically significant difference was attainable with polymerase chain reaction (PCR) template loading as low as 4 ng. These results pave the way for developing CRISPR-Cas12a-based methods to effectively detect TP53 mutations as lung cancer biomarkers.

ANNaMo: Coarse-grained modeling for folding and assembly of RNA and DNA systems.

The folding of RNA and DNA strands plays crucial roles in biological systems and bionanotechnology. However, studying these processes with high-resolution numerical models is beyond current computational capabilities due to the timescales and system sizes involved. In this article, we present a new coarse-grained model for investigating the folding dynamics of nucleic acids. Our model represents three nucleotides with a patchy particle and is parameterized using well-established nearest-neighbor models. Thanks to the reduction of degrees of freedom and to a bond-swapping mechanism, our model allows for simulations at timescales and length scales that are currently inaccessible to more detailed models. To validate the performance of our model, we conducted extensive simulations of various systems: We examined the thermodynamics of DNA hairpins, capturing their stability and structural transitions, the folding of an MMTV pseudoknot, which is a complex RNA structure involved in viral replication, and also explored the folding of an RNA tile containing a k-type pseudoknot. Finally, we evaluated the performance of the new model in reproducing the melting temperatures of oligomers and the dependence on the toehold length of the displacement rate in toehold-mediated displacement processes, a key reaction used in molecular computing. All in all, the successful reproduction of experimental data and favorable comparisons with existing coarse-grained models validate the effectiveness of the new model.

Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one.

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, abiosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limitand a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.

Environmentally Controlled Oscillator with Triplex Guided Displacement of DNA Duplexes.

The use of DNA triplex association is advantageous for the reconfiguration of dynamic DNA nanostructures through pH alteration and can provide environmental control for both structural changes and molecular signaling. The combination of pH-induced triplex-forming oligonucleotide (TFOs) binding with toehold-mediated strand displacement has recently garnered significant attention in the field of structural DNA nanotechnology. While most previous studies use single-stranded DNA to displace or replace TFOs within the triplex, here we demonstrate that pH alteration allows a DNA duplex, with a toehold assistance, to displace TFOs from the components of another DNA duplex. We examined the dependence of this process on toehold length and show that the pH changes allow for cyclic oscillations between two molecular formations. We implemented the duplex/triplex design onto the surface of 2D DNA origami in the form outlining binary digits 0 or 1 and verified the oscillatory conformational changes between the two formations with atomic force microscopy.

Leveraging Steric Moieties for Kinetic Control of DNA Strand Displacement Reactions.

DNA strand displacement networks are a critical part of dynamic DNA nanotechnology and are proven primitives for implementing chemical reaction networks. Precise kinetic control of these networks is important for their use in a range of applications. Among the better understood and widely leveraged kinetic properties of these networks are toehold sequence, length, composition, and location. While steric hindrance has been recognized as an important factor in such systems, a clear understanding of its impact and role is lacking. Here, a systematic investigation of steric hindrance within a DNA toehold-mediated strand displacement network was performed through tracking kinetic reactions of reporter complexes with incremental concatenation of steric moieties near the toehold. Two subsets of steric moieties were tested with systematic variation of structures and reaction conditions to isolate sterics from electrostatics. Thermodynamic and coarse-grained computational modeling was performed to gain further insight into the impacts of steric hindrance. Steric factors yielded up to 3 orders of magnitude decrease in the reaction rate constant. This pronounced effect demonstrates that steric moieties can be a powerful tool for kinetic control in strand displacement networks while also being more broadly informative of DNA structural assembly in both DNA-based therapeutic and diagnostic applications that possess elements of steric hindrance through DNA functionalization with an assortment of chemistries.

Catalytic-assembly of programmable atom equivalents

Escape from metastable states in self-assembly of colloids is an intractable problem. Unlike the commonly adopted approach of thermal annealing, the recently developed enthalpy-mediated strategy provided a different option to address this dilemma in a dynamically controllable manner at room temperature. However, it required a complex catalytic-assembly DNA strand-displacement circuitry to mediate interaction between multiple components. In this work, we present a simple but effective way to achieve catalytic-assembly of DNA-functionalized colloidal nanoparticles, i.e., programmable atom equivalents, in a far-from-equilibrium system. A removable molecule named "catassembler" that acts as a catalyst was employed to rectify imperfect linkages and help the system escape from metastability without affecting the assembled framework. Notably, catalytic efficiency of the catassembler can be effectively improved by changing the seesaw catassembler in toehold length design or numbers of the repeat units. Leveraging this tractable catalytic-assembly approach, different ordered architectures were easily produced by directly mixing all reactants, as in chemical reactions. By switching bonding identities, solid-solid phase transformations between different colloidal crystals were achieved. This work opens up an avenue for programming colloid assembly in a far-from-equilibrium system.

Deciphering the Design Rules of Toehold-Gated sgRNA for Conditional Activation of Gene Expression and Protein Degradation in Mammalian Cells.

A new class of toehold-gated gRNAs (thgRNAs) has been created to provide conditional gene regulation via RNA-mediated activation. However, the detailed design principles remain elusive. Here, we presented an investigation into the design rules for conditional gRNAs by systematically varying the toehold, stem, and flexible loop regions of thgRNA for optimal gene activation in HeLa cells. We determined that nonspecific interactions between the toehold region and the flexible loop are the main driver for the background leak observed in the OFF state. By trimming the toehold length from 15 to 5 nt, the improved thgNT-F design led to a 38-fold increase in the activated ON state with no observable background leak. The same design rule was successfully adapted to target two different regions on the mCherry mRNA with the same impressive fold change. Using the thgRNA to direct conditional protein degradation, we showed up to 8-fold knockdown of a reporter protein through activating expression of a bifunctional ubiquibody GS2-IpaH9.8. This new strategy may find many new applications for cell culture control or cell therapy by removing unwanted proteins in an RNA-responsive manner.

Kinetics of RNA and RNA:DNA Hybrid Strand Displacement.

In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

Conditional Antisense Oligonucleotides Triggered by miRNA.

Antisense oligonucleotides (ASOs) are single-stranded short nucleic acids that silence the expression of target mRNAs and show increasing therapeutic potential. Since ASOs are internalized by many cell types, both normal and diseased cells, gene silencing in unwanted cells is a significant challenge for their therapeutic use. To address this challenge, we created conditional ASOs that become active only upon detecting transcripts unique to the target cell. As a proof-of-concept, we modified an HIF1α ASO (EZN2968) to generate miRNA-specific conditional ASOs, which can inhibit HIF1α in the presence of a hepatocyte-specific miRNA, miR-122, via a toehold exchange reaction. We characterized a library of nucleic acids, testing how the conformation, thermostability, and chemical composition of the conditional ASO impact the specificity and efficacy in response to miR-122 as a trigger signal. Optimally designed conditional ASOs demonstrated knockdown of HIF1α in cells transfected with exogenous miR-122 and in hepatocytes expressing endogenous miR-122. We confirmed that conditional ASO activity was mediated by toehold exchange between miR-122 and the conditional ASO duplex, and the magnitude of the knockdown depended on the toehold length and miR-122 levels. Using the same concept, we further generated another conditional ASO that can be triggered by miR-21. Our results suggest that conditional ASOs can be custom-designed with any miRNA to control ASO activation in targeted cells while reducing unwanted effects in nontargeted cells.

Kinetics of heterochiral strand displacement from PNA-DNA heteroduplexes.

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

Dynamicallyelongated associative toehold for tuning DNA circuit kinetics and thermodynamics.

Associative toehold is a powerful concept enabling efficient combinatorial computation in DNA circuit. A longer association length boosts circuit kinetics and equilibrium signal but results in higher leak rate. We reconcile this trade-off by using a hairpin lock design to dynamically elongate the effective associative toehold length in response to the input target. Design guidelines were established to achieve robust elongation without incurring additional leakages. Three hairpin initiators with different combinations of elongated associative toehold (4 → 6 nt, 5 → 8 nt and 6 → 9 nt) were shortlisted from the design framework for further discussion. The circuit performance improved in terms of reaction kinetics, equilibrium signal generated and limit of detection. Overall, the elongated associative toehold served as a built-in function to stabilize and favour the forward, desired reaction when triggered.

Fuel-Driven Transient DNA Strand Displacement Circuitry with Self-Resetting Function.

Toehold-mediated DNA strand displacement (DSD) is a powerful strategy to engineer dynamics in DNA-based devices and for molecular computing. However, facile strategies for autonomously self-resettable DSD cascades with programmable lifetimes are still missing. Here, we concatenate an ATP-powered ligation/restriction network with toehold-mediated DSD reactions realizing ATP-driven transient DSD with self-resetting behavior. The ATP-fueled ligation biases strand displacement reactions by increasing the toehold length and increasing the local concentration by covalent fixation, while the concurrent endonuclease restriction eliminates this bias, allowing to reset the system. The lifetimes and adaptive dynamic steady states for the DSD are engineered by the ATP-fueled uphill-driven nonequilibrium ligation/restriction system. By programming the toeholds for downstream DSD reaction networks, we realize ATP-fueled transient DSD cascades. A higher level of fuel-driven automaton is achieved by combining two subsystems for the transient DSD, where the expelled strands are each other's input strands and the restriction-induced double-stranded DNA melting resets the systems.

Conditional Deoxyribozyme-Nanoparticle Conjugates for miRNA-Triggered Gene Regulation.

DNA-nanoparticle (NP) conjugates have been used to knockdown gene expression transiently and effectively, making them desirable tools for gene regulation therapy. Because DNA-NPs are constitutively active and are rapidly taken up by most cell types, they offer limited control in terms of tissue or cell type specificity. To take a step toward solving this issue, we incorporate toehold-mediated strand exchange, a versatile molecular programming modality, to switch the DNA-NPs from an inactive state to an active state in the presence of a specific RNA input. Because many transcripts are unique to cell subtype or disease state, this approach could one day lead to responsive nucleic acid therapeutics with enhanced specificity. As a proof of concept, we designed conditional deoxyribozyme-nanoparticles (conditional DzNPs) that knockdown tumor necrosis factor α (TNFα) mRNA upon miR-33 triggering. We demonstrate toehold-mediated strand exchange and restoration of TNFα DNAzyme activity in the presence of miR-33 trigger, with optimization of the preparation, configuration, and toehold length of conditional DzNPs. Our results indicate specific and strong ON/OFF response of conditional DzNPs to the miR-33 trigger in buffer. Furthermore, we demonstrate endogenous miR-33-triggered knockdown of TNFα mRNA in mouse macrophages, implying the potential of conditional gene regulation applications using these DzNPs.

Optimizing the Toehold Strategy of On-Chip Nucleic Acid Hybridization Probe for the Discrimination of Single Nucleotide Polymorphism.

The synthetic DNA hybridization probe has proved its importance in biology and biotechnology. In this study, taking advantage of a novel analytical technique called dual polarization interferometry (DPI), the influence of the toehold strategy of on-chip DNA hybridization probe on the discrimination of single nucleotide polymorphism (SNP) was investigated. Through adjusting the toehold length, the toehold strategies of on-chip toehold exchange probe were thoroughly optimized. For the "6/5" probe, an optimal discrimination factor of 78% against the spurious target was achieved. Moreover, the ability of the on-chip probe in SNP discrimination was significantly enhanced compared to its pure solution counterpart. This simple and rapid detection method for SNP discrimination based on the on-chip toehold exchange probe will show great potential in disease diagnosis.

Antibody-controlled actuation of DNA-based molecular circuits

DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody–epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

Engineering chemical reaction modules via programming the assembly of DNA hairpins.

The architect of enzyme-free chemical reaction modules, working as building blocks in implementing complex computing tasks, was achieved by modulating the assembly of DNA hairpins, including non-catalytic and catalytic systems. The performance of heterogeneous outputted sequences, which were programmed on different hairpins for triggering the downstream reaction, was asymmetric in the non-catalytic system, whereas symmetric in the catalytic system. Furthermore, complicated DNA-only chemical modules possessing controllable species of inputs or outputs were constructed based on our strategy. The kinetic studies revealed that the performance of the chemical modules was toehold length correlated; on the basis of which, a DNA concentration monitor was constructed.

Tuning Toehold Length and Temperature to Achieve Rapid, Colorimetric Detection of DNA from the Disassembly of DNA-Gold Nanoparticle Aggregates.

Gold nanoparticles have been widely utilized to achieve colorimetric detection for various diagnostic applications. One of the most frequently used methods for DNA detection involves the aggregation of DNA-modified gold nanoparticles driven by target DNA hybridization. This process, however, is intrinsically slow, limiting its use in rapid diagnostics. Here we take advantage of the reverse process: the disassembly of preformed aggregates triggered by the addition of target DNA via a strand displacement mechanism. A systematic study of the dependence of the disassembly rate on temperature, with and without toeholds, has delivered a system that produces an extremely rapid colorimetric response. Furthermore, using an optimal toehold length of 5 nucleotides, target triggered disassembly is rapid over a wide range of ambient temperatures. Using this overhang system, simple visualization of low picomole amounts of target DNA is possible within 10 min at room temperature.

DNA and DNA computation based on toehold-mediated strand-displacement reactions

biocompatibility. Considering the critical role of DNA less than 150 base pairs (bp) in cellular processes such as regulated gene expression, quantifying the intrinsic bend ability of DNA on a sub-persistence length scale is essential to understanding its molecular functions and the DNA-protein interaction. From the classical point of view, double-stranded DNA is assumed to be stiff and can be treated by semi-flexible chain, but recent studies have yielded contradictory results. A lot of studies tried to prove that the worm-like chain model can be used to fully describe DNA chain. However, recent theoretical and experimental studies indicated that DNA exhibits high flexibility on a short length scale, which cannot be described by the worm-like chain model. Further studies are needed to address the extreme flexibility of DNA on a short length scale. On the basis of the predictability of the double helical structure and the Watson-Crick binding thermodynamics for DNA, a class of DNA reactions can be defined, called toehold-mediated strand-displacement reaction, in which one complementary single-stranded DNA sequence first binds to the dangling toehold domain of the substrate in a pre-hybridized double-stranded DNA, then triggers the strand-displacement reaction, and finally results in the dissociation of the third strand previously bound to the substrate with partial complementarity. In dynamic DNA nanotechnology, isothermal toehold-mediated DNA strand-displacement reaction has been used to design complex nanostructure and nanodevice for molecular computation. The kinetics of the strand-displacement can be modulated using the toehold length. In order to weaken the coupling between the kinetics of strand-displacement and the thermodynamics of the reaction, the concept of toehold exchange was introduced by Winfree et al. to improve the control of strand-displacement kinetics. More importantly, the biomolecular reaction (BM) rate constant of toehold exchange can be analytically derived using the three-step model. Through utilizing strand-displacement reactions and taking advantage of its programmable sequences and precise recognition properties, DNA can be used to build complex circuits which can proceed robustly at constant temperature, achieving specific functions. DNA strand-displacement reaction can be employed to fabricate logic gates, and large and complex circuits for DNA computing, to mimic the naturally occurring occurrence of biological systems. Based on that, DNA circuit can then be used to direct the assembly of nanodevice following the designed pathway, and modulate the chemical reaction networks on the surface of living cell or in cellular systems for biosensing, even program the cellular machinery in the future for genetic diagnostic or gene therapy. In the present paper, we reviewed the proceedings in the fields of DNA structure and conformational changes, and DNA flexibility, discussed the mechanism of DNA strand-displacement reaction at the molecular level, and introduced the recent studies in DNA computation as well as the dynamic DNA nanotechnology, such as self-assembly.

Determination of Adenosine Triphosphate by a Target Inhibited Catalytic Cycle Based on a Strand Displacement Reaction

A strand displacement reaction-based system was developed for the determination of adenosine triphosphate (ATP). It involved an entropy-driven catalytic cycle that directly employed the ATP aptamer as the catalyst. Introduction of ATP into the system induced the catalyst to form the G-quadruplex conformation and inhibited its catalytic activity. All intermediates in the catalytic cycle processes were identified by polyacrylamide gel electrophoresis analysis. When the oligonucleotides were labeled with a carboxyfluorescein fluorophore and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid quencher, this strand displacement reaction-based catalytic system exhibited a “switch-on” response for ATP. Conditions for detecting ATP, such as the toehold length, concentrations of the catalyst and magnesium ion, and incubation temperature, were optimized to obtain a detection limit of 50 nM and a linear response up to 1400 nM of ATP. This target inhibited catalytic cycle provides an enzyme-free biosensing strategy and has potential application in aptamer-based biosensing.

On the biophysics and kinetics of toehold-mediated DNA strand displacement

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.