Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Zhang, Wei; Liu, Liquan; Liao, Yangwei; Shu, Wan; Tang, Xiaofeng; Dong, Kejun; Ming, Zhihao; Xiao, Xianjin; Wang, Hongbo

doi:10.1016/j.cclet.2021.06.067

Cited by 5 publications

(3 citation statements)

References 25 publications

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“…The former two categories rely on expensive instruments, and real-time PCR-based methods are less sensitive. Post-PCR probe-based assays inherit the advantages of convenience and low cost of normal PCR, and moreover they are compatible with target recognition and signal amplification by various nuclease, such as Flap endonuclease ( 48 ) and endonuclease IV ( 49 ). As we have discussed in the Introduction, Cas12a is unique and distinct compared with those commonly used nucleases due to its ultra-powerful signal amplification efficiency.…”

Section: Resultsmentioning

confidence: 99%

PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA

Zhang

Dong

et al. 2022

Nucleic Acids Research

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Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combining pre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM−SE+ dsDNA). We discovered that CRISPR-Cas12a had special enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005–0.01% for synthesized strands and 0.01–0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.

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Section: Resultsmentioning

confidence: 99%

PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA

Zhang

Dong

et al. 2022

Nucleic Acids Research

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“…This foundational property underpins the malleability and predictability of toehold-mediated strand displacement. This reaction serves as a cornerstone in various applications, including mutation detection [ 1 – 5 ], regulation and surveillance of biological behaviors [ 6 – 11 ], scaffold structuring [ 12 , 13 ], DNA nanomachinery [ 14 – 18 ], coupling with gold nanoparticles [ 19 – 23 ], and implementation within DNA circuits [ 24 – 28 ].…”

Section: Introductionmentioning

confidence: 99%

Controllable and reusable seesaw circuit based on nicking endonucleases

Liao,

Liu,

Liu

et al. 2024

J Nanobiotechnol

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Seesaw circuits are essential for molecular computing and biosensing. However, a notable limitation of seesaw circuits lies in the irreversible depletion of components, precluding the attainment of system recovery and rendering nucleic acid circuits non-reusable. We developed a brand-new method for creating controllable and reusable seesaw circuits. By using the nicking endonucleases Nt.BbvCI and Nt.Alwi, we removed “functional components” while keeping the “skeletal components” for recurrent usage. T-inputs were introduced, increasing the signal-to-noise ratio of AND logic from 2.68 to 11.33 and demonstrating compatibility. We identified the logic switching feature and verified that it does not impair circuit performance. We also built intricate logic circuits, such as OR-AND gate, to demonstrate the versatility of our methodology. This controllable reusability extends the applications of nanotechnology and bioengineering, enhancing the practicality and efficiency of these circuits across various domains.

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“…The selective recognition of short oligonucleotides, such as antisense oligo-DNA and miRNAs, at the single-molecule level is a critical area of research as it enables the development of early cancer diagnosis and drug development. − Nanopore sensing provides an innovative platform for single-molecule characterization of oligonucleotides through monitoring the translocation and analyzing the distinct temporary changes in the ionic current . Solid-state nanopores are considered to be more stable and controllable than biological nanopores, which are often fragile and difficult to manipulate. − This is due to the fact that solid-state nanopores are fabricated using diverse manufacturing techniques, which allows for precise control over their size, shape, and properties, − such as dielectric breakdown (CBD), transmission electron microscopy (TEM) beam, and ion beam irradiation .…”

Section: Introductionmentioning

confidence: 99%

Recognition of Oligonucleotide C by Polydopamine-Coated Solid-State Nanopores

Yang,

Yin,

Chen

et al. 2023

Anal. Chem.

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Selective recognition of short oligonucleotides at the single-molecule level is particularly important for early disease detection and treatment. In this work, polydopamine (PDA)-coated nanopores were prepared via self-polymerization as a solid-state nanopore sensing platform for the recognition of oligonucleotide C (PolyC). The PDA coating possesses abundant active sites, such as indole, amino, carboxyl, catechol, and quinone structures, which had interactions with short oligonucleotides to slow down the translocation rate. PDA-coated nanopores selectively interact with PolyC20 by virtue of differences in hydrogen bonding forces, generating a larger blocking current, while polyA and polyT demonstrated very small blockings. At the same time, PDA-coated nanopores can sensitively distinguish PolyC with different lengths, such as 20, 14, and 10 nt. The functionalization of PDA on the solid-state nanopore provides an opportunity for the rational design of the recognition surface for biomolecules.

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Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Cited by 5 publications

References 25 publications

PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA

PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA

Controllable and reusable seesaw circuit based on nicking endonucleases

Recognition of Oligonucleotide C by Polydopamine-Coated Solid-State Nanopores

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