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Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.

Background: The emergence of DNA nanotechnology has enabled the systematic design of diverse bionic dissipative behaviors under the precise control of nucleic acid nanodevices. Nevertheless, when compared to the dissipation observed in robust living systems, it is highly desirable to enhance the anti-interference for artificial DNA dissipation to withstand perturbations and facilitate repairs within the complex biological environments., Results: In this study, we introduce strategically designed "trash cans" to facilitate kinetic control over interferences, transforming the stochastic binding of individual components within a homogeneous solution into a competitive binding process. This approach effectively eliminates incorrect binding and the accumulation of systemic interferences while ensuring a consistent pattern of energy fluctuation from response to silence. Remarkably, even in the presence of numerous interferences differing by only one base, we successfully achieve complete system reset through multiple cycles, effectively restoring the energy level to a minimum., Significance: The system was able to operate stably without any adverse effect under conditions of irregular interference, high-abundance interference, and even multiplex interferences including DNA and RNA crosstalk. This work not only provides an effective paradigm for constructing robust DNA dissipation systems but also greatly broadens the potential of DNA dissipation for applications in high-precision molecular recognition and complex biological reaction networks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nonenzymatic self-assembly circuit utilizing hairpin substrates has been developed to be a powerful tool for information transduction, amplification and computation. However, the sensitivity, stability and application of this circuit are impeded by the presence of leakage which refers to undesired triggering in the absence of input. Herein, we proposed a movable toehold principle to suppress leakage and accelerate the catalytic reaction through removing partial hairpin toehold responsible for the leakage and transferring it to the catalyst. With movable toehold, catalytic hairpin assembly (called mtCHA) exhibited an excellent signal-to-background ratio of over 100, high robustness and improved specificity. In more complex circuit, including proximity recognition, signal amplification of small molecules (such as ATP), logic network, autocatalysis circuit and two-layer cascade circuit, mtCHA also demonstrated satisfactory performance. Our findings suggest that mtCHA holds great potential for broader applications, and the approach of repurposing harmful fragments into beneficial candidates can provide valuable insights for other chemical systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

DNA strand displacement plays an essential role in the field of dynamic DNA nanotechnology. However, flexible regulation of strand displacement remains a significant challenge. Most previous regulatory tools focused on controllable activation of toehold and thus limited the design flexibility. Here, we introduce a regulatory tool termed cooperative branch migration (CBM), through which DNA strand displacement can be controlled by regulating the complementarity of branch migration domains. CBM shows perfect compatibility with the majority of existing regulatory tools, and when combined with forked toehold, it permits continuous fine-tuning of the strand displacement rate spanning 5 orders of magnitude. CBM manifests multifunctional regulation ability, including rate fine-tuning, continuous dynamic regulation, reaction resetting, and selective activation. To exemplify the powerful function, we also constructed a nested if-function signal processing system on the basis of cascading CBM reactions. We believe that the proposed regulatory strategy would effectively enrich the DNA strand displacement toolbox and ultimately promote the construction of DNA machines of higher complexity in nucleic acid research and biomedical applications.

Comprehensive Summary Single‐nucleotide variants (SNVs) are crucial in disease development, but their accurate detection is challenging due to their low abundance and interference from wild‐type targets. Although nucleic acid analogs like peptide nucleic acids (PNAs) have been used for SNV detection, they often lack programmable sensitivity and specificity due to poorly calculated thermodynamics and kinetics. Here, we present a computational method for calculating the stacking energy of PNA and DNA hybrids, leveraging nearest neighbor parameters. Validation against experimental data from 16 sequences under varied hybridization conditions yielded good agreement using Bland‐Altman analysis, with all data points falling within the confidence interval. Our findings indicate that PNA‐DNA hybridization is thermodynamically more stable and exhibits kinetics 140‐fold faster than DNA‐DNA hybridization for identical sequences. Utilizing this computational framework, we designed PNA toehold probes, which were screened via simulations and experiments. This combined approach facilitated the identification of highly sensitive and specific PNA toehold probes for single point mutation detection via strand displacement reaction. Our results demonstrate the successful application of PNA toehold probes for detecting point mutations with high sensitivity and specificity, achieving a selective amplification of approximately 200‐fold for variants with a variant allele frequency (VAF) of 0.5% using quantitative polymerase chain reaction. [ABSTRACT FROM AUTHOR]

A G6PD genotyping method for two common mutations (c.1376G>T and c.1388G>A) based on multi-code magnetic beads and conjugated strand displacement reaction. [Display omitted] • UDG mediated PCR was constructed to obtain toehold probe for G6PD genotyping. • Genotype c.1376G > T and c.1388G > A sites of G6PD using multi-code magnetic beads. • Compatibility with flow cytometry analysis, fluorescent microscopic image analysis, and spectrofluorimetry. Small variations, even single-nucleotide variants in nucleic acid sequences may exert significant influence in many diseases. The reliable detection and quantification of DNA can provide a variety of information that has significant clinical value, including disease risk assessment, screening, diagnosis, prognosis, the selection of therapies and monitoring. Due to the fact coupling efficiency of dNTPs and purification methodology can limit the chemical synthesis of long stranded probes. We demonstrated a PCR-based method to construct a long-stranded detection probe with high levels of selectivity via a strand displacement reaction with ssDNA target generates by asymmetric PCR. This detection probe was enzymatically generated from a double-stranded DNA duplex, containing a single-stranded active toehold domain. This approach was successfully implemented to genotype human glucose-6-phosphate dehydrogenase (G6PD) deficiency, focusing upon the c.1376G > T and c.1388G > A variants. [ABSTRACT FROM AUTHOR]

Sensitive and specific miRNA detection is essential for the early cancer diagnosis. In this work, we design a fluorescent microRNA biosensor based on exponential amplification reaction (EXPAR) and nicking endonuclease-powered three-dimensional (3-D) bipedal DNA walkers (BDW). Target microRNA initiates EXPAR with the help of polymerase and nicking endonuclease to generate the large number of BDW in solution. The newly generated BDW can be continuously assembled onto polystyrene microsphere track co-modified with fluorescence-labeled DNA strand. Thus, in the presence of nicking endonuclease, the walking machine is activated to produce enhanced fluorescent signal in the supernatant. Besides, we prove that BDW holds the faster walking speed than single-legged DNA walker (SDW) based on comparative study. Under optimal conditions, the proposed amplification method owns a wide linear range from 10 fM to 5 nM with a detection limit down to 5.2 fM. The reaction time of the assay takes about 70 min. The combination of enzyme-assisted EXPAR in solution and enzyme-powered BDW on particle significantly increases the signal amplification efficiency and improves the detection sensitivity. Therefore, our method has enormous potential for the application of BDW-related biosensors. EXPAR-BDW strategy based on exponential amplification reaction-triggered three-dimensional bipedal DNA walkers. Image 1 • A strategy for detection microRNA was constructed based on exponential amplification reaction and 3-D bipedal DNA walkers. • The proposed assay shows high sensitivity towards target microRNA 21 with a detection limit of 5.2 fM. • This method is simple in nucleic acid design and can be completed in short measurement time. [ABSTRACT FROM AUTHOR]

• We design a quasi-steady model of DNAzyme based nanomachine for identifying single nucleotide variants with high specificity. • A non-covalent DNA catalysis reaction was used to tune and optimize the performance of nanomachine. • All reactions were simulated in silico to guide the design of DNA nanomachine. Single nucleotide variants (SNVs) are essential biomarkers both clinically and biologically, and the specific hybridization of matched sequences is crucial to discriminate SNVs. However, the similar sequence would hybridize to probes incorrectly and cause false positive. Herein, we designed a DNAzyme-based nanomachine guided by thermodynamic and kinetic parameters to identify SNVs. The standard Gibbs free energy of the internal loop in the blocked DNAzyme was rationally taken into consideration and a quasi-steady 'toehold exchange' reaction was designed. These simulations in silico enable an optimal performance to discriminate SNVs for various conditions. Additionally, we introduced a DNA fuel to enable non-covalent DNA catalysis reaction, which can tune the performance of nanomachine by changing the concentration of DNA fuel. In this way, we make a satisfactory trade-off between sensitivity and specificity to discriminate SNVs. Collectively, this nanomachine produced discrimination factors between 5 and 31, the detection limit of 0.1 % variant alleles can also be identified. Our work demonstrated a pathway assist to the design of DNA nanomachine through simulation in silico, which hold great potential for the application of complex DNA nanomachines and biosensors. [ABSTRACT FROM AUTHOR]