Customizable NAND logic gate biosensing system based on engineered methylated CRISPR / Cas12a consensus sequence and application thereof

Abstract

The invention discloses a customizable NAND logic gate biosensing system based on an engineered methylated CRISPR / Cas12a consensus sequence and an application of the customizable NAND logic gate biosensing system. According to the system, an engineered methylated CRISPR / Cas12a consensus sequence (MCCS, 5 '-TTTGATC-3') is combined with a hybridization chain reaction triggered by a primer, so that efficient conversion between a methylation state and Cas12a activation is realized. Dam methyltransferase, S-adenosylmethionine (SAM) and DpnI are used as three input signals, and when the three input signals all exist, methylation catalyzed by the Dam methyltransferase promotes DpnI cutting, destroys the continuity of a PAM-original spacer region and enables Cas12a to be inactivated (in a closed state), and a low-fluorescence signal is generated; under any other input combination, the Cas12a-crRNA keeps the activity and outputs a high-fluorescence'turn-on 'signal, so that the ultra-sensitive detection on the activity of the Dam methyltransferase is realized. The system shows high recovery rate and low variability in simulating a real biological sample, and provides a universal molecular calculation driven sensing platform for enzyme activity analysis and inhibitor screening in a complex biological environment.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology, and more specifically, to a customizable NAND logic gate biosensing system based on engineered methylated-CRISPR / Cas12a consensus sequences and its applications. Background Technology

[0002] DNA methylation is one of the most important epigenetic modifications, playing a crucial role in regulating gene expression, maintaining genome stability, and modulating various biological processes. Abnormal DNA methylation patterns are closely associated with tumorigenesis, neurological disorders, bacterial virulence, and antibiotic resistance. DNA methyltransferases (DNA MTases) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to specific internal bases. Among them, DNA adenine methyltransferase (Dam MTase) specifically recognizes the GATC sequence and methylates adenine to generate N. 6 -Methyladenine (6mA). This methylation process is fundamental to prokaryotic DNA replication and mismatch repair, and has also become a biomarker and regulatory signal in microbial pathogenicity and human disease models. Therefore, accurate detection and regulation of DNA MTase activity are crucial for inhibitor screening in epigenetic research, disease diagnosis, and therapeutic development.

[0003] Over the past few decades, various analytical methods have been developed for detecting DNA MTase activity, including radiolabeling assays, high-performance liquid chromatography (HPLC), mass spectrometry (MS), enzyme-linked immunosorbent assays (ELISA), and electrochemical techniques. While these traditional methods offer high accuracy, they typically require multi-step operations, specialized instrumentation, and time-consuming sample preparation. To improve practicality, several fluorescence- or colorimetric assays have been proposed, utilizing nanomaterials, restriction endonucleases, and molecular probes. However, these methods often suffer from limited sensitivity, high background signals, or poor adaptability to complex biological matrices. Furthermore, most existing strategies follow a single-input, single-output design, limiting their ability to integrate multiple biochemical reactions or make logical decisions. Developing intelligent biosensing systems capable of performing logical computations to process enzyme information and output reliable digital signals remains highly desirable.

[0004] Against this backdrop, CRISPR / Cas systems, especially Cas12a, have become powerful tools for molecular diagnostics due to their customizable target recognition and side-branch trans-cleavage activity against single-stranded DNA reporter genes. By coupling with isothermal amplification strategies such as hybridization chain reaction (HCR), catalytic hairpin assembly (CHA), or rolling circle amplification (RCA), CRISPR-based platforms have achieved ultra-sensitive detection of acids and small molecules in nucleic acids. Simultaneously, the concept of molecular logic gates derived from DNA computing has been successfully introduced into biosensing. By integrating CRISPR / Cas systems with specific recognition modules or aptamers, logical operations such as AND, OR, NOR, and INHIBIT have been implemented, enabling multiplex detection, decision-making, and interference-resistant analysis. Despite these advances, logic gate biosensors for enzyme-catalyzed reactions remain rare. Of particular note is the insufficient development of customizable logic systems that can directly couple methylation recognition, restriction cleavage, and CRISPR activation for DNA methyltransferase analysis. Recent studies on asymmetric nanopore logic sensors or MSRE coupled with CRISPR / CHA detection have demonstrated the conceptual feasibility of methylation event analysis, but these systems still rely on independent identification and transduction modules and lack the ability to customize integrated logic at the architectural level. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a customizable NAND logic gate biosensing system based on engineered methylated-CRISPR / Cas12a consensus sequences and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a customizable NAND logic gate biosensing system based on an engineered methylated-CRISPR / Cas12a consensus sequence, wherein the biosensing system converts the DNA methylation state into a Cas12a activation signal by combining the engineered methylated-CRISPR / Cas12a consensus sequence with a primer-triggered hybridization chain reaction. The engineered methylated-CRISPR / Cas12a consensus sequence contains three recognition elements: a PAM sequence for Cas12a recognition, a Dam methyltransferase action site, and a methylation-dependent cleavage site for the restriction endonuclease DpnI. The biosensing system includes: (1) A hybridization chain reaction module for generating a hybridization chain reaction containing an engineered methylated-CRISPR / Cas12a consensus sequence; (2) A three-input NAND logic gate module with Dam methyltransferase, S-adenosylmethionine and DpnI endonuclease as input signals; (3) A CRISPR / Cas12a signal reporting module for outputting corresponding fluorescence signals based on the integrity of the engineered methylated-CRISPR / Cas12a consensus sequence.

[0007] Furthermore, the aforementioned biosensing system includes hairpin probe HP-1, hairpin probe HP-2, primers, S-adenosylmethionine, restriction endonuclease, Cas12a, crRNA, and a fluorescence-quenched reporter probe. The nucleotide sequence of the hairpin probe HP-1 is 5'-ATTTGATCtgaaagttggtgtgGGTGTGGcacaccaactttcaGGCAAAA-3'; The nucleotide sequence of the hairpin probe HP-2 is 5'-CCACACCcacaccaactttcagaTCAAATTTTTGCCtctgaaagttggtgtg-3'; The nucleotide sequence of the primer is 5'-TTTTGCCTCTGAAAGTTGGTGTG-3'; The nucleotide sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUAUCUGAAAGUUGGUGU-3'; The nucleotide sequence of the fluorescence-quenching reporter probe is 5'-FAM-CTCTCATTTTTAGAGAG-BHQ1-3'.

[0008] Furthermore, the detection logic of the above-mentioned biosensor system is as follows: When three input signals are present simultaneously, Dam methyltransferase uses S-adenosylmethionine as a methyl donor to methylate the engineered methylated-CRISPR / Cas12a consensus sequence. The methylated engineered methylated-CRISPR / Cas12a consensus sequence is specifically cleaved by the restriction endonuclease DpnI, thereby disrupting the adjacency relationship between the PAM sequence and the downstream protospacer sequence, preventing the Cas12a-crRNA complex from recognizing and binding, and thus preventing the activation of the trans-cleavage of the fluorescence-quenched reporter probe. The system outputs a low fluorescence signal, defined as logic "0". When at least one of the three input signals is absent, the engineered methylated-CRISPR / Cas12a consensus sequence cannot complete the methylation-cleavage chain reaction. The Cas12a-crRNA complex remains active and cleaves the fluorescence-quenched reporter probe. The system outputs a high fluorescence signal, defined as logic "1". By combining the outputs of high and low fluorescence signals, the NAND gate detection of Dam methyltransferase activity is achieved.

[0009] Furthermore, the nucleotide sequence of the above-mentioned engineered methylated-CRISPR / Cas12a consensus sequence is 5'-TTTGATC-3'.

[0010] A second aspect of the present invention provides a method for constructing the aforementioned customizable NAND logic gate biosensing system, comprising the following steps: S1: Preparation of probe working solution: Hairpin probes HP-1 and HP-2 were respectively subjected to 1× TAE / Na + Mix the buffer solution, denature at 95℃ for 5 min, then cool naturally to room temperature to obtain the corresponding probe working solution, and store at -20℃ for later use; S2: Preparation of HCR products: Mix 6.5 μL of 1× TE buffer, 0.5 μL of MgCl2·6H2O, 2.5 μL of primer, 2.5 μL of hairpin probe HP-1 working solution and 2.5 μL of hairpin probe HP-2 working solution, and perform HCR reaction at 37 °C to obtain reaction solution 1. S3: Construction of a three-input NAND logic gate: Mix 2.5 μL of 10× Dam buffer, 2.5 μL of 10× rCutSmart buffer, 2.5 μL of S-adenosylmethionine, 2 μL of restriction endonuclease DpnI and 1 μL of Dam methyltransferase, incubate at 37 °C for 60 min, and then heat at 80 °C for 20 min to terminate enzyme activity to obtain reaction solution 2. S4: Activation of the CRISPR / Cas12a system: 14.5 μL of reaction solution 1, 10.5 μL of reaction solution 2, 1 μL of Cas12a, 1 μL of crRNA, 8 μL of NE buffer r2.1, 8 μL of DEPC-treated water, and 2 μL of fluorescence-quenched reporter probe were mixed and incubated at 37 °C for 60 min. Then, the emission spectrum in the range of 500–650 nm was recorded using a fluorescence spectrophotometer under 490 nm excitation light, and the peak fluorescence intensity at 520 nm was recorded.

[0011] Furthermore, in step S2 above, the concentration of MgCl2·6H2O used is 120mM; the concentration of the primer used is 0.1μM; the concentration of the hairpin probe HP-1 working solution used is 1μM; the concentration of the hairpin probe HP-2 working solution used is 1μM; and the HCR reaction time is 30min.

[0012] Furthermore, in step S3 above, the concentration of S-adenosylmethionine used is 160 μM; the concentration of the restriction endonuclease DpnI used is 6 U·μL.-1 .

[0013] The third aspect of the present invention provides the application of the customizable NAND logic gate biosensing system described above, and the customizable NAND logic gate biosensing system obtained by the above construction method in the detection of DNA methyltransferases for non-disease diagnosis and treatment purposes; Furthermore, the DNA methyltransferase is a Dam methyltransferase.

[0014] The fourth aspect of the present invention provides the application of the customizable NAND logic gate biosensing system described above, and the customizable NAND logic gate biosensing system obtained by the above construction method in screening DNA methyltransferase inhibitors.

[0015] The beneficial technical effects obtained by this invention are as follows: (1) The present invention constructs an engineered MCCS motif for logic computing: We designed a multifunctional MCCS sequence (5'-TTTGATC-3') that integrates the PAM site of Cas12a, the methylation site of Dam methyltransferase and the DpnI recognition site into a controllable module, thereby realizing seamless signal conversion between methylation and Cas12a activation.

[0016] (2) This invention couples biochemical input signals with CRISPR responses through customizable NAND logic circuits: using Dam methyltransferase, SAM, and DpnI as biochemical input signals, binary fluorescence output is generated based on NAND logic, thereby realizing intelligent judgment for molecular diagnosis. This provides a universal molecular computation-driven biosensing framework for enzyme activity analysis and inhibitor screening in complex biological systems.

[0017] (3) This invention uses primer-triggered HCR for signal amplification: the tandem assembly of dsDNA tandems containing MCCS significantly enhances the trans-cleavage activity of Cas12a, achieving signal amplification over a wide linear range (0.00032~320 U·mL). -1 Ultrasensitive detection within 0.00032 U·mL, with a detection limit as low as 0.00032 U·mL -1 .

[0018] (4) The system of the present invention can be used for inhibitor screening and practical application evaluation of complex samples: it exhibits good anti-interference ability in serum samples and can quantitatively evaluate Dam methyltransferase inhibitors (IC50 of 5-fluorouracil). 50 The IC50 of penicillin G is 1.75 μM. 50 The concentration (11.9 μM) highlights the potential of this system in biomedical and pharmacological applications. Attached Figure Description

[0019] Figure 1 : Schematic diagram of constructing engineered methylated-CRISPR / Cas12a consensus sequences embedded in HCR products.

[0020] Figure 2 : Schematic diagram of the logic operation mechanism of a customizable NAND logic gate biosensing system based on engineered methylated-CRISPR / Cas12a consensus sequences.

[0021] Figure 3 Truth table and logic diagram of a three-input NAND gate.

[0022] Figure 4 Fluorescence feasibility verification diagram of primer-triggered HCR activation of Cas12a.

[0023] Figure 5 : Feasibility characterization diagram of HCR products by gel electrophoresis.

[0024] Figure 6 Compare the fluorescence spectra of the system with and without SAM.

[0025] Figure 7 : Compare the fluorescence spectra of the system with and without Dam methyltransferase.

[0026] Figure 8 Compare the fluorescence spectra of the system with and without DpnI.

[0027] Figure 9 Gel electrophoresis characterization of HCR products under different input conditions.

[0028] Figure 10 A diagram of a DNA double-stranded sequence containing one (dsDNA-1), two (dsDNA-2), three (dsDNA-3), and four (dsDNA-4) MCCS copies.

[0029] Figure 11 Kinetic fluorescence spectra of Cas12a activation containing copy number regulation of one (dsDNA-1), two (dsDNA-2), three (dsDNA-3), and four (dsDNA-4) MCCS.

[0030] Figure 12 Sensitivity measurement plots, linear range and standard curves of a customizable NAND logic gate biosensor system based on engineered methylated-CRISPR / Cas12a consensus sequences.

[0031] Figure 13 : Specificity analysis of a customizable NAND logic gate biosensing system based on engineered methylated-CRISPR / Cas12a consensus sequences.

[0032] Figure 14 Fluorescence and recovery plots of a customizable NAND logic gate biosensor system based on engineered methylated-CRISPR / Cas12a consensus sequences in human serum and whole blood spiked with Dam methyltransferase.

[0033] Figure 15 : Evaluation of fluorescence spectroscopy and IC50 of 5-FU inhibition of Dam methyltransferase using a customizable NAND logic gate biosensor system based on engineered methylated-CRISPR / Cas12a consensus sequences 50 Line graph.

[0034] Figure 16 Evaluation of the fluorescence chromatogram and IC50 of penicillin G inhibiting Dam methyltransferase using a customizable NAND logic gate biosensor system based on engineered methylated-CRISPR / Cas12a consensus sequences. 50 Line graph.

[0035] Figure 17 : in different Mg 2+ Gel electrophoresis images of HCR products obtained at different concentrations and primer concentrations.

[0036] Figure 18 Fluorescence signal and signal-to-noise ratio obtained under different HCR reaction times, different DpnI concentrations, and different SAM concentrations.

[0037] Figure 19 : Fluorescence assays and IC50 assays of 5-FU inhibiting Cas12a activity were performed using a customizable NAND logic gate biosensor system based on engineered methylated-CRISPR / Cas12a consensus sequences. 50 Line graph.

[0038] Figure 20 : Fluorescence assay and IC50 assays to evaluate the penicillin G's inhibitory activity on Cas12a using a customizable NAND logic gate biosensor system based on engineered methylated-CRISPR / Cas12a consensus sequences 50 Line graph.

[0039] Figure 21 Kinetic fluorescence spectra of Cas12a activation regulated by the MCCS functional domain containing PAM site mutant (MT-MCCS-1), methylation / cleavage site mutant (MT-MCCS-2), crRNA complementary DNA domain mutant (MT-Random), and wild type (WT-MCCS, sequence same as dsDNA-1). Detailed Implementation

[0040] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments. However, the following examples are merely examples of this invention and do not represent the scope of protection of this invention. The scope of protection of this invention is determined by the claims.

[0041] The 1× TAE / Na involved in the embodiments of the present invention + The buffer solution is formulated as follows: 10 mM Tris, 1 mM EDTA, 50 mM NaCl; pH=8.0.

[0042] The formulation of the 1× TE buffer involved in this embodiment of the invention is: 10mM Tris-HCl, 1mM EDTA; pH=8.0.

[0043] The formulation of the 10× Dam buffer involved in this embodiment of the invention is: 50mM Tris-HCl, 5mM β-mercaptoethanol, 10mM EDTA; pH=7.5.

[0044] The formulation of the 10× rCutSmart buffer involved in this embodiment of the invention is: 50mM potassiumacetate, 20mM Tris-acetate, 10mM magnesium acetate, 100µg / mL recombinant albumin; pH=7.9.

[0045] The formulation of NE buffer r2.1 involved in this embodiment of the invention is as follows: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100µg / mL recombinant albumin; pH=7.9.

[0046] The Cas12a involved in this embodiment of the invention is specifically EnGen® Lba Cas12a (Cpf1).

[0047] The nucleotide sequences involved in the embodiments of this invention are as follows: Hairpin Probe HP-1: 5'-ATTTGATCtgaaagttggtgtgGGTGTGGcacaccaactttcaGGCAAAA-3' Hairpin probe HP-2: 5'-CCACACCcacaccaactttcagaTCAAATTTTTGCCtctgaaagttggtgtg-3' Primer: 5'-TTTTGCCTCTGAAAGTTGGTGTG-3' crRNA: 5'-UAAUUUCUACUAAGUGUAGAUAUCUGAAAGUUGGUGU-3' Fluorescent-quenched reporter probe (F-Q gene reporter probe): 5'-FAM-CTCTCATTTTTAGAGAG-BHQ1-3' ssDNA-1a: 5'-ATTTGATCTGAAAGTTGGTGTG-3' ssDNA-1b: 5'-CCACACCAACTTTCAGATCAAAT-3' ssDNA-2a: 5'-ATTTGATCTGAAAGTTGGTGTGATTTGATCTGAAAGTTGGTGTG-3' ssDNA-2b: 5'-CCACACCAACTTTCAGATCAAATCCACACCAACTTTCAGATCAAAT-3' ssDNA-3a: 5'-ATTTGATCTGAAAGTTGGTGTGATTTGATCTGAAAGTTGGTGTGATTTGATCTGAAAGTTGGTGTG-3' ssDNA-3b: 5'-CCACACCAACTTTCAGATCAAATCCACACCAACTTTCAGATCAAATCCACACCAACTTTCAGATCAAAT-3' ssDNA-4a: 5'-ATTTGATCTGAAAGTTGGTGTGATTTGATCTGAAAGTTGGTGTGATTTGATCTGAAAGTTGGTGTGATTTGATCTGAAAGTTGGTGTG-3' ssDNA-4b: 5'-CCACACCAACTTTCAGATCAAATCCACACCAACTTTCAGATCAAATCCACACCAACTTTCAGATCAAATCCACACCAACTTTCAGATCAAAT-3' MT-ssDNA-1: 5'-ATTGGATCTGAAAGTTGGTGTG-3' MT-ssDNA-2: 5'-CCACACCAACTTTCAGATCCAAT-3' MT-ssDNA-3: 5'-ATTTGCTCTGAAAGTTGGTGTG-3' MT-ssDNA-4: 5'-CCACACCAACTTTCAGACCAAAT-3' MT-Random-1: 5'-ATTTGATCCCTGACTAGGTATG-3' MT-Randon-2: 5'-CCTACCTAGTCAGGGATCAAAT-3' Example 1: Construction of a customizable NAND logic gate biosensing system based on engineered methylated-CRISPR / Cas12a consensus sequences This embodiment constructs a customizable NAND logic gate biosensor system based on an engineered methylated CRISPR / Cas12a consensus sequence for ultrasensitive detection of Dam methyltransferase. This system combines the engineered methylated CRISPR / Cas12a consensus sequence (MCCS, 5'-TTTGATC-3') with a primer-triggered hybridization chain reaction (HCR) to achieve ultrasensitive analysis of Dam methyltransferase.

[0048] To achieve logic-gated recognition of Dam methyltransferase activity, we designed an engineered methylation-CRISPR / Cas12a co-recognition sequence (MCCS, 5'-TTTGATC-3'). This sequence integrates three independent recognition elements: a protospacer adjacent motif (PAM, TTTG) for Cas12a, a Dam methyltransferase action site (GATC), and a methylation-dependent cleavage site for the restriction endonuclease DpnI. To amplify the detection signal, we initiated a hybridization chain reaction (HCR) using primer chains, triggering the continuous hybridization of hairpin probes HP-1 and HP-2, thereby forming a long double-stranded DNA polymer, i.e., the HCR hairpin structure. By embedding the artificially synthesized MCCS into the HCR hairpin structure, a large amount of double-stranded DNA substrate containing methylation sites and PAM regions can be generated, thus providing sufficient input signals for subsequent enzymatic reactions. Figure 1 In this biosensor system, the three molecular inputs—Dam methyltransferase (input 1), its methyl donor S-adenosylmethionine (SAM, input 2), and DpnI (input 3)—collectively determine the final fluorescence output. Figure 2As shown, when all three are present, Dam methyltransferase catalyzes adenine methylation at the GATC site in the MCCS in the presence of SAM, forming GmATC. Subsequently, DpnI specifically recognizes and cleaves this methylation site, inducing precise cleavage at the GmATC site, causing the MCCS to dissociate into two fragments, "TTTGmA" and "TC". This cleavage disrupts the critical adjacency relationship between PAM (TTTG) and its downstream protospacer sequence (crRNA recognition site), preventing Cas12a from binding and activating. Ultimately, the Cas12a-crRNA complex cannot activate the trans-cleavage of the fluorescence-quenched reporter probe, and the system outputs a low fluorescence signal (defined as output "0"). Conversely, when any input is missing, the MCCS remains intact. In this case, the tandem structure of PAM and the protospacer sequence is preserved, allowing it to be effectively recognized and bound by the Cas12a-crRNA complex, thereby triggering a strong trans-cleavage of the fluorescence-quenched reporter probe, resulting in a high fluorescence signal (defined as output "1"). Therefore, this biosensor system can operate as a three-input NAND logic gate: it outputs "0" only when Dam methyltransferase, SAM, and DpnI are all present, and outputs "1" for all other input combinations (see complete logic gate circuit). Figure 3 This logical architecture provides a molecular computing platform with both high sensitivity and high specificity for the analysis of Dam methyltransferase activity.

[0049] Specifically, the construction steps of a customizable NAND logic gate biosensing system based on engineered methylated-CRISPR / Cas12a consensus sequences described in this embodiment are as follows: S1: Preparation of probe working solution: 5 μL of hairpin probes HP-1 and HP-2 with a concentration of 100 μM were respectively mixed with 45 μL of 1× TAE / Na + Mix the buffer solution, denature at 95°C for 5 min, then cool naturally to room temperature to obtain the corresponding probe working solution, and store at -20°C for later use.

[0050] S2: Preparation of HCR products: Mix 6.5 μL of 1× TE buffer, 0.5 μL of 120 mM MgCl2·6H2O, 2.5 μL of 0.1 μM primer, 2.5 μL of 1 μM hairpin probe HP-1 working solution, and 2.5 μL of 1 μM hairpin probe HP-2 working solution, and react at 37 °C for 30 min to obtain the HCR product, i.e., reaction solution 1.

[0051] S3: Construction of a three-input NAND logic gate: Mix 2.5 μL of 10× Dam buffer, 2.5 μL of 10× rCutSmart buffer, 2.5 μL of 160 μM S-adenosylmethionine, 2 μL of 6 U / μL DpnI, and 1 μL of Dam methyltransferase at different concentrations, incubate at 37 °C for 60 min, and then heat at 80 °C for 20 min to terminate enzyme activity, to obtain reaction solution 2.

[0052] S4: Activation of the CRISPR / Cas12a system: 14.5 μL of reaction solution 1 from step S2, 10.5 μL of reaction solution 2 from step S3, 1 μL of Cas12a (1 μM), 1 μL of crRNA (1 μM), 8 μL of NE buffer r2.1, 8 μL of DEPC-treated water, and 2 μL of FQ gene reporter probe (5 μM) were mixed and incubated at 37 °C for 60 min. The emission spectra in the range of 500–650 nm were then recorded using an F97Pro fluorescence spectrophotometer under 490 nm excitation light, and the performance was evaluated by the peak fluorescence intensity at 520 nm.

[0053] Example 2: Feasibility study of primer-triggered HCR activation of Cas12a To confirm that the hybridization chain reaction can effectively generate the double-stranded DNA substrate required for subsequent logic gate construction, we evaluated the feasibility of primer-triggered HCR activation of the CRISPR / Cas12a system, as follows: S1: Preparation of probe working solution: The same as step S1 in Example 1.

[0054] S2: Preparation of HCR products: Mix 6.5 μL of 1× TE buffer, 0.5 μL of 120 mM MgCl2·6H2O, 2.5 μL of 0.1 μM primer, 2.5 μL of 1 μM hairpin probe HP-1 working solution, and 2.5 μL of 1 μM hairpin probe HP-2 working solution, and react at 37 °C for 30 min to obtain reaction solution 1.

[0055] Mix 6.5 μL of 1× TE buffer, 0.5 μL of 120 mM MgCl2·6H2O, 2.5 μL of 1 μM hairpin probe HP-1 working solution, and 5 μL of ddH2O, and react at 37 °C for 30 min to obtain control reaction solution 1-1.

[0056] Mix 6.5 μL of 1× TE buffer, 0.5 μL of 120 mM MgCl2·6H2O, 2.5 μL of 1 μM hairpin probe HP-2 working solution, and 5 μL of ddH2O, and react at 37 °C for 30 min to obtain control reaction solution 1-2.

[0057] Mix 6.5 μL of 1× TE buffer, 0.5 μL of 120 mM MgCl2·6H2O, 2.5 μL of 0.1 μM primer, 2.5 μL of 1 μM hairpin probe HP-1 working solution, and 2.5 μL of ddH2O, and react at 37 °C for 30 min to obtain control reaction solution 1-3.

[0058] Mix 6.5 μL of 1× TE buffer, 0.5 μL of 120 mM MgCl2·6H2O, 2.5 μL of 0.1 μM primer, 2.5 μL of ddH2O, and 2.5 μL of 1 μM hairpin probe HP-2 working solution, and react at 37 °C for 30 min to obtain control reaction solution 1-4.

[0059] Mix 6.5 μL of 1× TE buffer, 0.5 μL of 120 mM MgCl2·6H2O, 2.5 μL of ddH2O, 2.5 μL of 1 μM hairpin probe HP-1 working solution, and 2.5 μL of 1 μM hairpin probe HP-2 working solution, and react at 37 °C for 30 min to obtain control reaction solutions 1-5.

[0060] S3: Activation of the CRISPR / Cas12a system: 14.5 μL of reaction solution 1, 1 μL of 1 μM Cas12a, 1 μL of 1 μM crRNA, 8 μL of NE buffer r2.1, 8 μL of DEPC-treated water, and 2 μL of 5 μM FQ gene reporter probe were mixed and incubated at 37 °C for 60 min. The emission spectra in the range of 500–650 nm were then recorded using an F97Pro fluorescence spectrophotometer under 490 nm excitation light, and the performance was evaluated using the peak fluorescence intensity at 520 nm. Reaction solution 1 used included reaction solution 1 from step S2 or its series of controls (control reaction solutions 1-1 to 1-5).

[0061] like Figure 4As shown, no significant fluorescence signal was observed in the system whether only a single hairpin structure was present (curve a: HP-1; curve b: HP-2) or when the hairpin structure was paired with primers separately (curve c: HP-1 / primer; curve d: HP-2 / primer). A weak background signal was detected in the control group (curve e) where HP-1 and HP-2 were mixed but no primers were present, which may be due to spontaneous partial hybridization between the two hairpin structures. However, this low-level assembly did not interfere with subsequent logical biosensing, as the target system relies on a pre-formed extended HCR product. Notably, a significant fluorescence enhancement was observed when the primers simultaneously triggered HP-1 and HP-2 (curve f), demonstrating that complete HCR assembly is crucial for efficient activation of Cas12a. Figure 5 The gel electrophoresis results further confirmed that the primers successfully induced HCR. Only simple hairpin structures were observed in lanes a and b (lane a: HP-1; lane b: HP-2), showing clear, rapidly migrating bands at 25–50 bp, without any higher-order structures. After mixing the primers with HP-1 or HP-2 respectively, lane c showed a slower-migrating band near 50 bp, corresponding to the primer / HP-1 hybridization product; while lane d only showed a band consistent with HP-2 alone, indicating that the primers could not hybridize with HP-2. Lane e (HP-1 / HP-2) showed a weak, diffuse band, consistent with the spontaneous dimerization of the hairpin structure. In contrast, lane f (HP-1 / HP-2 / primer) showed clear, step-like bands extending from 150 bp to over 500 bp, with significantly enhanced band signal. This characteristic banding pattern confirms the formation of polymeric HCR products, in which primer-triggered hybridization cycles drive continuous chain extension between HP-1 and HP-2. Fluorescence and electrophoresis results together demonstrate that primer-triggered HCR can stably generate long-chain double-stranded DNA scaffolds carrying MCCS motifs, thus providing a crucial substrate for Cas12a recognition and subsequent NAND logic gate operations.

[0062] Example 3: Feasibility Study of NAND Logic Gate Detection of Dam Methyltransferase To evaluate whether the biosensing system follows the NAND logic rule, we systematically studied the effects of various biochemical inputs on the fluorescence signal, as follows: S1: Preparation of hairpin probe working solution: The same as step S1 in Example 1.

[0063] S2: Preparation of HCR products: The same as step S2 in Example 1.

[0064] S3: Construction of a three-input NAND logic gate: Add 2.5 μL of 10× Dam buffer, 2.5 μL of 10× rCutSmart buffer, 2.5 μL of 160 μM S-adenosylmethionine, and 2 μL of 6 U / μL [unclear - likely a specific compound or solution]. Dpn I was mixed with 1 μL of Dam methyltransferase at a concentration of 800 U / mL and incubated at 37 °C for 60 min, followed by heating at 80 °C for 20 min to terminate enzyme activity, yielding reaction solution 2.

[0065] Add 2.5 μL of 10× Dam buffer, 2.5 μL of 10× rCutSmart buffer, 2.5 μL of ddH2O, and 2 μL of a 6 U / μL solution. Dpn I was mixed with 1 μL of Dam methyltransferase at a concentration of 800 U / mL and incubated at 37 °C for 60 min, followed by heating at 80 °C for 20 min to terminate enzyme activity, yielding control reaction solution 2-1.

[0066] Mix 2.5 μL of 10× Dam buffer, 2.5 μL of 10× rCutSmart buffer, 2.5 μL of 160 μM S-adenosylmethionine, 2 μL of ddH2O, and 1 μL of 800 U / mL Dam methyltransferase. Incubate at 37 °C for 60 min, then heat at 80 °C for 20 min to terminate enzyme activity, to obtain control reaction solution 2-2.

[0067] Add 2.5 μL of 10× Dam buffer, 2.5 μL of 10× rCutSmart buffer, 2.5 μL of 160 μM S-adenosylmethionine, and 2 μL of 6 U / μL [unclear - likely a specific compound or solution]. Dpn Mix I and 1 μL ddH2O, incubate at 37°C for 60 min, then heat at 80°C for 20 min to terminate enzyme activity, to obtain control reaction solution 2-3.

[0068] S4: Activation of the CRISPR / Cas12a system: The reaction solution 2 is basically the same as step S4 in Example 1, except that the reaction solution 2 used includes the reaction solution 2 of step S3 or its series of controls (control reaction solutions 2-1 to 2-3).

[0069] like Figure 6 As shown, the system exhibits a strong fluorescence response in the absence of S-adenosylmethionine; however, in the presence of S-adenosylmethionine, it completely inhibits signal output by blocking HCR-mediated substrate formation. Dam methyltransferase ( Figure 7 ) and DpnI restriction endonuclease ( Figure 8The system also exhibits a similar pattern: the addition of any input disrupts the intact MCCS motif, thereby preventing Cas12a activation and ultimately switching the output signal to logic "0". Conversely, when any input is missing, the MCCS site remains intact, activating the trans-cleavage activity of Cas12a and producing a definite logic "1". These results collectively demonstrate that the system conforms to NAND logic behavior—the output signal remains in the "0" state only when the simultaneous presence of the three biochemical inputs (S-adenosylmethionine, Dam methyltransferase, and DpnI) triggers complete inhibition. Figure 9 The electrophoretic analysis shown further validated this mechanism. In lanes a (primer + HP-1 + HP-2 + Dam methyltransferase + DpnI) and b (primer + HP-1 + HP-2 + SAM + DpnI), the lack of S-adenosylmethionine or Dam methyltransferase prevented methylation and subsequent cleavage, resulting in ladder-like bands corresponding to the complete HCR assembly. Although methylation occurred in lane c (primer + HP-1 + HP-2 + SAM + Dam methyltransferase), the lack of DpnI cleavage preserved a significant HCR product. However, in lane d (primer + HP-1 + HP-2 + SAM + Dam methyltransferase + DpnI), the HCR polymer almost completely disappeared, leaving only short cleavage fragments, confirming the effective disruption of the MCCS motif. Fluorescence and electrophoresis data jointly established the NAND logic behavior of the system at both the functional and molecular levels, clarifying that Cas12a activation can only be completely inhibited when SAM, Dam methyltransferase, and DpnI coexist. More importantly, this NAND logic-based mechanism provides a reliable strategy for highly specific differentiation of Dam methyltransferase activity, highlighting the application potential of MCCS integrated logic biosensors in the field of methyltransferase detection.

[0070] Example 4: Kinetic Analysis of MCCS Copy Number Modulation Cas12a Activation To elucidate the effect of the number of units in the engineered methylated-CRISPR / Cas12a consensus sequence (MCCS) on Cas12a-mediated reporter cleavage, we constructed four synthetic double-stranded DNA substrates ( Figure 10The four dsDNA substrates were: dsDNA-1 (formed by annealing ssDNA-1a and ssDNA-1b with only one MCCS unit), dsDNA-2 (formed by annealing ssDNA-2a and ssDNA-3b with two tandem MCCS units), dsDNA-3 (formed by annealing ssDNA-3a and ssDNA-3b with three tandem MCCS units), and dsDNA-4 (formed by annealing ssDNA-4a and ssDNA-4b with four tandem MCCS units. The concentrations of dsDNA-2, dsDNA-3, and dsDNA-4 were adjusted to 0.50 µM, 0.33 µM, and 0.25 µM, respectively, to ensure the total number of MCCS sites was equal to that of 1 µM dsDNA-1. We evaluated the effect of MCCS copy number on Cas12a cleavage kinetics using these four double-stranded DNA substrates instead of the HCR product in Example 1 under conditions with or without Dam methyltransferase, using real-time fluorescence monitoring. Figure 11 As shown, when using dsDNA-1 (single MCCS), the fluorescence signal gradually increases in the reaction without Dam methyltransferase, indicating that the intact MCCS motif can be effectively recognized by the Cas12a-crRNA complex, thereby triggering strong reporter cleavage. However, when Dam methyltransferase, SAM, and... Dpn At step I, the fluorescence rise was significantly suppressed. This suppression stems from adenine methylation catalyzed by Dam methyltransferase—this modification converts GATC to GmATC, making... DpnI cleaves the double strand, thereby disrupting the structural continuity of the PAM-prototype spacer region upon which Cas12a activation depends. Kinetics of dsDNA-2 (containing two tandem MCCSs, half the concentration of dsDNA-1) showed that, under the same total number of MCCSs, the reaction without Dam methyltransferase exhibited a faster and stronger fluorescence increase than dsDNA-1, while the signal of the Dam methyltransferase-treated sample remained suppressed near baseline. Similar results were obtained for dsDNA-3 (containing three tandem MCCSs, one-third the concentration of dsDNA-1). Notably, the reaction without Dam methyltransferase showed the fastest fluorescence rise, while the Dam methyltransferase-treated sample produced almost no signal. Further analysis was extended to dsDNA-4, which contains four tandem MCCS motifs at a concentration only one-quarter that of dsDNA-1. Consistent with the trends of dsDNA-2 and dsDNA-3, the Dam-free MTase reaction of dsDNA-4 produced the strongest and fastest fluorescence kinetics among all constructs, confirming that even with a constant MCCS equivalent, increasing the number of MCCS units correspondingly enhances Cas12a activation. In contrast, Dam-treated dsDNA-4 maintained fluorescence at baseline, indicating that efficient methylation and DpnI digestion still prevented Cas12a activation. Quantitative data acquired at 45, 60, 75, and 90 min clearly showed that the fluorescence intensity of the Dam-free methyltransferase group at each time point was dsDNA-4 > dsDNA-3 > dsDNA-2 > dsDNA-1, while the signal in the Dam-treated group remained consistently at a lower level. For example, at 60 min, the signal of dsDNA-4 reached 30394 au, exceeding that of dsDNA-3 (22692 au), dsDNA-2 (16330 au), and dsDNA-1 (14612 au). Despite the same total MCCS equivalent number, this performance trend may reflect a multi-site synergistic effect: assembling multiple MCCS units on a single double-stranded DNA molecule can increase the local density of the PAM-prototype spacer region, thereby promoting more efficient and even synergistic loading of the Cas12a-crRNA complex and accelerating the trans-cleavage turnover of the reporter molecule. This enhancement effect, driven by structural design, is expected to simultaneously increase the rate and amplitude of fluorescence output, thus achieving faster signal accumulation and providing higher sensitivity for detecting low-abundance methyltransferase targets.

[0071] Example 5: Sensitivity Analysis of Biosensor Systems To further verify the performance of the biosensing system constructed in Example 1, we systematically evaluated its analytical performance on Dam methyltransferase. Figure 12The results show different concentrations of Dam methyltransferase (0, 0.00032, 0.0032, 0.032, 0.32, 3.2, 32, 160, 320 U·mL). -1 The fluorescence emission spectrum under [the specified conditions] was observed. As enzyme concentration increased, the fluorescence intensity gradually decreased. This is because more GATC sites in the MCCS motif were methylated, thereby activating [the enzyme / organization]. Dpn I-cleavage disrupts the continuous structure of the PAM-protospacer region, which is essential for Cas12a activation. Normalized fluorescence response heatmaps visually illustrate this concentration-dependent signal quenching trend. Statistical mean fluorescence intensity (±SD) data show highly significant differences between most adjacent concentration groups, indicating excellent discriminative ability of this system over a wide range of enzyme activities. Furthermore, the logarithm of Dam methyltransferase concentration correlates with the fluorescence peak value (F... 520 The curve exhibits a good linear relationship, and the fitted calibration curve is: F 520 =425.4-130.7lg(C_Dam methyltransferase / U·mL) -1 ) (R 2 =0.9968), with a detection range covering 0.00032~320 U·mL. -1 To determine the limit of detection (LOD), we followed the procedures recommended by the International Union of Pure and Applied Chemistry (IUPAC), using the experimentally derived signal-to-noise ratio (S / N) as the basis for evaluation. Specifically, the fluorescence peak intensity at 520 nm was measured 20 times for blank samples (without Dam), from which the average fluorescence value (mean blank) and its standard deviation (SD blank) were calculated. SD blank was taken as the noise (N) of the detection system. Subsequently, 15 measurements were performed on samples with low concentrations of Dam under the same conditions to obtain the corresponding average fluorescence value (mean sample). The S / N ratio was then calculated as: S / N = (mean sample - mean blank) / SD blank. If the calculated S / N exceeded 5, the sample was diluted to half its original concentration, and the S / N measurement was repeated until the S / N value fell within the range of 3–5. -1 ​In summary, these results demonstrate that customizable NAND logic gates, implemented using engineered MCCS sequences and primer-triggered HCR, can effectively convert methylation-triggered cleavage into quantitative and highly sensitive fluorescence readings, thereby supporting the accuracy of DNA methyltransferase analysis and making it suitable for potential biomedical and diagnostic applications. Compared to previously reported methyltransferase assays (Table S2), our customizable NAND logic gate biosensor system achieves a lower LOD compared to several existing methods, including terminal deoxynucleoside transferase-assisted CRISPR activation, multipath SDA-CRISPR / Cas12a tandem systems, cascade reaction colorimetric assays, paper-based G-quadruple fluorescence chips, and autocatalytic HCR circuitry. Although its LOD is similar to that of exponentially amplified reaction-initiated CRISPR / Cas12a and 3D nanopolymer-assisted fluorescence biosensors, our platform offers a shorter detection time (2.5 h), compared to ≥4 h required by most such methods, demonstrating a good balance between sensitivity and efficiency. We also acknowledge that NAND logic operation relies on a multi-enzyme cascade reaction involving HCR amplification, Dam methylation, DpnI digestion, and Cas12a activation. While this multi-step architecture supports programmable logic control, it inevitably increases workflow complexity, reagent costs, and the potential for cumulative variability. These factors can pose challenges when integrating these features into resource-constrained environments or nursing field diagnostic devices.

[0072] Example 5: Specificity Analysis of Biosensor Systems This embodiment presents a rigorous specificity evaluation of the biosensing system proposed in Example 1. Two non-target DNA methyltransferases (...) were selected in the experiment. M.Sss I and EcoR I) As interfering targets, their catalytic recognition sequences are completely different from the GATC sites designed in the MCCS module of Example 1: M.Sss I-specific methylation of cytosine at the "CG" site, while EcoR I methylates the first adenine in the "GAATTC" sequence. For example... Figure 13 As shown, when adding M.Sss I (800 U·mL) -1 ), EcoR I (800 U·mL) -1 In the reaction group of either the enzymes mentioned above or a mixture of both, the fluorescence signal produced was similar to that of the blank control group (approximately 900 au). This indicates that neither enzyme can modify adenine in the MCCS structure. Since adenine is not methylated, DpnThe restriction enzyme I cannot cleave the MCCS site, thus maintaining the continuity of the PAM-protospacer region, thereby effectively activating the Cas12a-crRNA complex and generating a high fluorescence signal. In contrast, the fluorescence signal of the sample containing only Dam methyltransferase was significantly reduced to approximately 220 au, only one-quarter of that of the non-target control group. Even with Dam methyltransferase and M.Sss I and EcoR Even with the coexistence of I, the fluorescence inhibition effect remained stable. These results demonstrate that only Dam methyltransferase-mediated adenine methylation can activate [the inhibition]. Dpn The cleavage function of I interrupts the activation pathway of Cas12a, thereby switching the NAND gate to a "closed" state with low fluorescence output. Notably, even in the presence of excessive non-target methyltransferases, the system exhibits stable performance and strong anti-interference capabilities, ensuring accurate detection of Dam methyltransferases in complex enzymatic environments. To further explore the potential impact of high concentrations of non-target enzymes, we further tested... M.Sss I and EcoR I in the range of 0~4000 U·mL -1 The response was observed within the concentration range. Experimental results showed that even at the highest tested concentration, the fluorescence intensity remained comparable to the control group (approximately 900 au), indicating that nonspecific methyltransferases cannot induce adenine methylation in MCCS and therefore cannot initiate [the process]. Dpn During the I-cutting step, the Cas12a-mediated trans-cutting function remains intact. In summary, this series of experiments demonstrates that this biosensor system possesses excellent target specificity—it can specifically identify the preset Dam methyltransferase activity and convert it into a low-fluorescence output signal, while other common methyltransferases do not cause significant interference even under high concentration conditions. This high specificity further validates the reliability of the MCCS design for accurate DNA methyltransferase analysis in complex biological environments.

[0073] Example 6: Practical Applicability of Biosensing Systems To evaluate the applicability and reliability of the biosensor system proposed in Example 1 in practical bioanalysis, we conducted spiked recovery experiments in human serum and whole blood diluted 50-fold. Three different concentrations (0.032, 0.32, and 3.2 U·mL) were selected for the experiments. -1 The Dam methyltransferase of ) was spiked and measured. Results are as follows: Figure 14As shown, fluorescence intensity was significantly negatively correlated with the concentration of added Dam methyltransferase, indicating that the biosensor system can accurately transduce methylation signals even in complex serum matrices. Further quantitative recovery analysis showed recoveries ranging from 98.16% to 100.03%, with all relative standard deviations (RSDs) below 8.0% and an average RSD of 5.48%, all within acceptable limits. Notably, even in 50-fold diluted whole blood containing significantly higher levels of proteases, nucleases, and other interfering components, the detection method maintained considerable performance, with recoveries ranging from 98.57% to 100.95% and RSDs ranging from 1.98% to 7.11% (average 3.64%). These results confirm that the biosensor system possesses excellent analytical accuracy, good reproducibility, and strong resistance to matrix interference in serum samples, providing a practical basis for its efficient detection of methyltransferases in complex biological environments. The robustness of the fluorescence readings and the high reproducibility on complex sample matrices further support the applicability of this platform in practical detection of methyltransferases and potential clinical applications.

[0074] Example 7: Evaluation of Dam methyltransferase inhibitors Abnormal DNA methyltransferase activity is closely related to the development and progression of various human diseases; therefore, regulating its activity and screening for effective inhibitors is of significant biomedical importance. This study used the biosensor system described in Example 1 to evaluate the inhibitory effects of two representative drugs on Dam methyltransferase: the widely used anticancer agent 5-fluorouracil (5-FU) and the broad-spectrum antibiotic penicillin G. The steps are as follows: S1: Preparation of probe working solution: The same as step S1 in Example 1.

[0075] S2: Preparation of HCR products: The same as step S2 in Example 1.

[0076] S3: Construction of a three-input NAND logic gate: The procedure is basically the same as step S3 in Example 1, except that Dam methyltransferase needs to be co-incubated with the drug to be evaluated at 37°C for 30 minutes before use.

[0077] S4: Activation of the CRISPR / Cas12a system: The same as step S4 in Example 4.

[0078] like Figure 15 and Figure 16As shown, the fluorescence signal gradually increased with increasing concentrations of 5-FU or penicillin G. This phenomenon indicates that both drugs inhibit the activity of Dam methyltransferase, thereby preventing the transfer of methyl groups from SAM to adenine in the MCCS motif. The result is... Dpn I cannot cleave the unmethylated MCCS site, and the PAM-protospacer structure remains intact, thereby reactivating the Cas12a-crRNA complex and inducing potent trans-cleavage of the reporter probe. The bar chart further shows the peak fluorescence intensity (F... 520 The inhibitory efficacy increases in a dose-dependent manner with increasing inhibitor concentration. To quantify the inhibitory efficacy, the relative activity (RA) of Dam methyltransferase is calculated using the formula RA = (Ft - F0) / (Fi - F0): where Ft represents the fluorescence intensity at a specific inhibitor concentration, F0 is the fluorescence value of the blank reaction (without Dam methyltransferase and inhibitor), and Fi is 40 U·mL⁻¹. -1 The fluorescence intensity of Dam methyltransferase without inhibitor was determined. The half-inhibitory concentrations (IC50) of 5-FU and penicillin G were measured by plotting a dose-response curve of RA versus inhibitor concentration. 50 The concentrations were 1.75 μM and 11.9 μM, respectively. These results demonstrate that this biosensor system can serve as a reliable and sensitive tool for screening and quantifying Dam methyltransferase inhibitors, providing new technical support for drug development and clinical applications.

[0079] Example 8: Condition Optimization In the optimization experiment of this embodiment, we adopt the single variable principle, that is, based on the basic system and steps described in Example 1, we only change the parameter of one factor to be examined each time, while keeping all other conditions consistent with Example 1, so as to accurately evaluate the impact of the factor on the system performance.

[0080] We investigated the Mg content using non-denaturing polyacrylamide gel electrophoresis. 2+ The effects of concentration (i.e., the concentration of MgCl2·6H2O used) and the ratio of primers to hairpin probes on the formation of HCR products. Figure 17 As shown, the yield of high molecular weight HCR products increases with Mg. 2+ The concentration increases with increasing concentration. When the concentration is ≤30mM, only weak low molecular weight bands are observed, indicating insufficient ionic strength, resulting in low hybridization efficiency between the primer and hairpin probe. Mg 2+ When the concentration increased to 60 mM, a ladder-like band corresponding to the HCR product began to appear, indicating that the divalent cation effectively promoted the ordered hybridization process. The product signal reached its peak at 120 mM, indicating that the amplification efficiency was optimal under this condition. However, when the concentration was further increased to 150 mM, the band intensity decreased slightly, which may be due to excess Mg. 2+This weakens the base pairing specificity, thereby inhibiting DNA hybridization. Therefore, 120 mM was selected as the optimal Mg level. 2+ Concentration. Simultaneously, we investigated the effect of primer-to-hairpin probe ratios. With HP-1 and HP-2 concentrations fixed at 1 μM, primer concentrations were adjusted to construct primer: ratio gradients of 0.1:1:1, 0.5:1:1, 1:1:1, 2:1:1, and 3:1:1 (primer:HP-1:HP-2). As the relative primer concentration increased (from 0.1:1:1 to 3:1:1), the band intensity and molecular weight of the HCR product gradually decreased, indicating that polymer elongation was continuously inhibited. At the lowest primer ratio (0.1:1:1), the gel showed continuous high-molecular-weight diffuse bands (>500 bp), typical of efficient and continuous chain elongation. However, as primer concentration increased, short-fragment products gradually became dominant, indicating that excess primers can competitively bind to the trigger region of HP-1, thereby blocking its subsequent hybridization with HP-2 and ultimately inhibiting the elongation process. Therefore, determining a ratio of 0.1:1:1 (primer:HP-1:HP-2) can achieve the most efficient, specific and stable amplification.

[0081] Furthermore, we optimized the HCR reaction time, SAM concentration, and DpnI concentration. Figure 18 As shown, the time dependence of the HCR reaction was assessed by monitoring the fluorescence intensity change at 520 nm: under the condition of Dam methyltransferase deficiency, the fluorescence signal gradually increased with time and reached a plateau at 30 min, indicating that the HCR amplification reaction was basically completed; while the fluorescence signal of the sample containing Dam methyltransferase remained at a weak and stable level, confirming that methylation modification and subsequent cleavage effectively inhibited signal accumulation. The signal-to-noise ratio reached its peak at 30 min and tended to stabilize, thus determining 30 min as the optimal HCR reaction time. The concentration of DpnI endonuclease was further optimized to ensure complete cleavage of the methylated MCCS sequence. As the concentration of DpnI increased, the fluorescence intensity of the group without Dam methyltransferase remained basically unchanged, while the fluorescence of the group containing the enzyme decreased slightly. The results show that when Dam methyltransferase is absent, unmethylated MCCS is not recognized and cleaved by DpnI, and the Cas12a-crRNA system remains fully active, generating a strong signal through trans-cleavage of the reporter molecule; conversely, methylated MCCS can be effectively cleaved by DpnI, thereby blocking Cas12a activation. DpnI was used at a concentration of 6 U·μL -1The signal-to-noise ratio (SNR) was highest at 160 μM SAM, and further increases in enzyme concentration did not improve the SNR, indicating that the methylated substrate was completely digested at this concentration. Finally, the effect of SAM concentration on the catalytic efficiency of Dam methyltransferase was investigated. With increasing SAM concentration, the fluorescence intensity of the control group without Dam methyltransferase remained stable, while the fluorescence signal of the enzyme-containing group gradually weakened, reflecting the enhanced DpnI cleavage due to increased methylation efficiency. The SNR peaked at 160 μM SAM, and higher concentrations did not further improve signal discrimination. These results indicate that 160 μM SAM and 6 U·μL SAM are optimal for signal differentiation. -1 The combined use of DpnI can achieve full methylation and cleavage of the MCCS sequence, providing optimal conditions for high-fidelity activation and signal resolution of the Cas12a system.

[0082] Example 9: Evaluation of the potential nonspecific interference of 5-FU and penicillin G on Cas12a activity To rule out the possibility that 5-FU or penicillin G might interfere with the performance of other enzymes in the biosensor system (especially Cas12a), we conducted additional control experiments, incubating each inhibitor with Cas12a at a fixed Dam methyltransferase concentration (800 U / mL) as described in Example 7, and then evaluating Cas12a-mediated transcleavage activity. Figure 19 As shown, the fluorescence curves of Cas12a were almost identical before and after culture with increasing concentrations of 5-FU (0, 1, 2, 5, 10 μM), with the fluorescence intensity at 520 nm remaining stable around 430 au. To quantitatively assess the effect of 5-FU on Cas12a function, RA was calculated using the equation RA = (Ft - F0) / (Fi - F0), where Ft is the fluorescence intensity in the presence of the inhibitor, F0 is the fluorescence of the blank control group without Cas12a and the inhibitor, and Fi is the fluorescence obtained with 1 μM Cas12a without the inhibitor. The reflectance value of 5-FU remained between 0.95 and 1.00 across all tested concentrations, and the IC50 value was [not specified]. 50 Approaching infinity indicates that 5-FU has no measurable inhibitory effect on Cas12a. These results suggest that Cas12a-crRNA can still bind to unmethylated HCR products and cleave FQ reporter proteins even in the presence of 5-FU. Similar analyses were performed on penicillin G (see...). Figure 20 At concentrations of 0, 5, 10, 20, and 50 μm, the fluorescence intensity was comparable to that of the inhibitor-free control group (approximately 430 au). Dose-response curves further demonstrated the IC50 values. 50Approaching infinity, the calculated RA values ​​remained between 0.96 and 1.00, confirming that penicillin G does not inhibit Cas12a activity. Taken together, these findings indicate that neither 5-FU nor penicillin G directly affects Cas12a-mediated cleavage. Therefore, the fluorescence changes observed in inhibitor screening assays stem from selective inhibition of Dam methyltransferase activity, which alters MCCS methylation and DpnI cleavage efficiency, ultimately regulating Cas12a activation. These results validate the specificity and reliability of MCCS-based NAND logic biosensors in inhibitor evaluation.

[0083] Example 10: Analysis of MCCS domain mutants to evaluate nonspecific CRISPR activation To rule out the possibility of nonspecific CRISPR activation affecting background signals, three mutant controls targeting key functional domains of the MCCS motif were designed: (i) a PAM site mutant, (ii) a methylation / cleavage site mutant, and (iii) a crRNA complementary DNA domain mutant. These mutants, named MT-MCCS-1, MT-MCCS-2, and MT-Random, were obtained by synthesizing dsDNA constructs carrying the corresponding mutations (see [link to documentation]). Figure 21 ).

[0084] like Figure 21 As shown in Figure A, MT-MCCS-1 is constructed by changing the PAM sequence in wild-type MCCS (WT-MCCS) from "TTTG" to "TTGG," thereby disrupting Cas12a recognition. Real-time fluorescence results show that MT-MCCS-1 produces very little background fluorescence regardless of the presence or absence of Dam methyltransferase. This confirms that once the PAM site is damaged, Cas12a cannot bind to or activate its transcleavage function even if other MCCS domains remain intact. Figure 21 B illustrates the behavior of MT-MCCS-2, which retains intact PAM but carries a methylation / DpnI recognition site mutation, changing "GATC" to "GCTC". This mutation simultaneously blocks Dam methyltransferase-mediated methylation and prevents DpnI cleavage. As a result, high fluorescence intensity was observed in both Dam methyltransferase-free and Dam methyltransferase-containing samples, indicating that the uncleaved MCCS allows crRNA-guided recognition and subsequent activation of Cas12a. Notably, both MT-MCCS-1 and MT-MCCS-2 retain the same crRNA complementary DNA region as WT-MCCS; however, in wild-type analysis ( Figure 21 In D), the fluorescence of samples without Dam methyltransferase increased significantly, while the fluorescence of samples containing Dam methyltransferase remained low, indicating that appropriate methylation-related cleavage within the MCCS is crucial for distinguishing between positive and negative results.

[0085] To further validate specificity, MT-Random was designed to retain all functional MCCS domains while replacing the crRNA complementary region (“ACACCAACTTTCAGAT”) with a non-target sequence (“5′-TACCTAGTCAGGGAT-3′”). Figure 21 As shown in Figure C, this mutation results in uniformly low background fluorescence regardless of Dam methyltransferase concentration. The loss of signal activation confirms that Cas12a cannot bind to or cleave the reporter when crRNA-DNA hybridization is canceled. Taken together, these results demonstrate that the integrated MCCS structure and the correct crRNA-complementary DNA domain provide dual assurance for CRISPR specificity. Methylation-dependent structural regulation and crRNA-guided recognition are necessary for successful Cas12a activation, ensuring high sensitivity and strong discrimination in Dam methyltransferase detection.

[0086] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A customizable NAND logic gate biosensing system based on engineered methylated-CRISPR / Cas12a consensus sequences, characterized in that: The biosensing system converts DNA methylation state into Cas12a activation signal by combining engineered methylated-CRISPR / Cas12a consensus sequences with primer-triggered hybridization chain reactions. The engineered methylated-CRISPR / Cas12a consensus sequence contains three recognition elements: a PAM sequence for Cas12a recognition, a Dam methyltransferase action site, and a methylation-dependent cleavage site for the restriction endonuclease DpnI. The biosensing system includes: (1) A hybridization chain reaction module for generating a hybridization chain reaction containing an engineered methylated-CRISPR / Cas12a consensus sequence; (2) A three-input NAND logic gate module with Dam methyltransferase, S-adenosylmethionine and DpnI endonuclease as input signals; (3) A CRISPR / Cas12a signal reporting module for outputting corresponding fluorescence signals based on the integrity of the engineered methylated-CRISPR / Cas12a consensus sequence.

2. The customizable NAND logic gate biosensing system according to claim 1, characterized in that: The biosensing system includes hairpin probe HP-1, hairpin probe HP-2, primers, S-adenosylmethionine, restriction endonuclease, Cas12a, crRNA, and a fluorescence-quenched reporter probe. The nucleotide sequence of the hairpin probe HP-1 is 5'-ATTTGATCtgaaagttggtgtgGGTGTGGcacaccaactttcaGGCAAAA-3'; The nucleotide sequence of the hairpin probe HP-2 is 5'-CCACACCcacaccaactttcagaTCAAATTTTTGCCtctgaaagttggtgtg-3'; The nucleotide sequence of the primer is 5'-TTTTGCCTCTGAAAGTTGGTGTG-3'; The nucleotide sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUAUCUGAAAGUUGGUGU-3'; The nucleotide sequence of the fluorescence-quenching reporter probe is 5'-FAM-CTCTCATTTTTAGAGAG-BHQ1-3'.

3. The customizable NAND logic gate biosensing system according to claim 1, characterized in that: When all three input signals are present, Dam methyltransferase uses S-adenosylmethionine as a methyl donor to methylate the engineered methylated-CRISPR / Cas12a consensus sequence. The methylated engineered methylated-CRISPR / Cas12a consensus sequence is specifically cleaved by the restriction endonuclease DpnI, thereby disrupting the adjacency relationship between the PAM sequence and the downstream protospacer sequence. This prevents the Cas12a-crRNA complex from recognizing and binding to the PAM sequence, thus preventing the activation of the trans-cleavage of the fluorescence-quenched reporter probe. The system outputs a low fluorescence signal, defined as logic "0". When at least one of the three input signals is absent, the engineered methylated-CRISPR / Cas12a consensus sequence cannot complete the methylation-cleavage chain reaction. The Cas12a-crRNA complex remains active and cleaves the fluorescence-quenched reporter probe. The system outputs a high fluorescence signal, defined as logic "1". By combining the high and low fluorescence signals, NAND gate detection of Dam methyltransferase activity is achieved.

4. The customizable NAND logic gate biosensing system according to claim 1, characterized in that: The nucleotide sequence of the engineered methylated-CRISPR / Cas12a consensus sequence is 5'-TTTGATC-3'.

5. A method for constructing a customizable NAND logic gate biosensing system as described in claims 1-4, characterized in that: Includes the following steps: S1: Preparation of probe working solution: The hairpin probe HP-1 and the hairpin probe HP-2 were mixed with 1x TAE / Na + buffer respectively, denatured at 95℃ for 5 min, and then naturally cooled to room temperature to obtain the corresponding probe working solution, which was stored at -20℃ for standby use. S2: Preparation of HCR products: Mix 6.5 μL of 1× TE buffer, 0.5 μL of MgCl2·6H2O, 2.5 μL of primer, 2.5 μL of hairpin probe HP-1 working solution and 2.5 μL of hairpin probe HP-2 working solution, and perform HCR reaction at 37 °C to obtain reaction solution 1. S3: Construction of a three-input NAND logic gate: Mix 2.5 μL of 10× Dam buffer, 2.5 μL of 10× rCutSmart buffer, 2.5 μL of S-adenosylmethionine, 2 μL of restriction endonuclease DpnI and 1 μL of Dam methyltransferase, incubate at 37 °C for 60 min, and then heat at 80 °C for 20 min to terminate enzyme activity to obtain reaction solution 2. S4: Activation of the CRISPR / Cas12a system: 14.5 μL of reaction solution 1, 10.5 μL of reaction solution 2, 1 μL of Cas12a, 1 μL of crRNA, 8 μL of NE buffer r2.1, 8 μL of DEPC-treated water, and 2 μL of fluorescence-quenched reporter probe were mixed and incubated at 37 °C for 60 min. Then, the emission spectrum in the range of 500–650 nm was recorded using a fluorescence spectrophotometer under 490 nm excitation light, and the peak fluorescence intensity at 520 nm was recorded.

6. The construction method according to claim 5, characterized in that: In step S2, the concentration of MgCl2·6H2O used is 120 mM; the concentration of the primer used is 0.1 μM; the concentration of the hairpin probe HP-1 working solution used is 1 μM; the concentration of the hairpin probe HP-2 working solution used is 1 μM; and the HCR reaction time is 30 min.

7. The construction method according to claim 5, characterized in that: In step S3, the concentration of S-adenosyl methionine used is 160 μM; the concentration of restriction enzyme Dpn I used is 6 U·μL -1 .

8. The application of the customizable NAND logic gate biosensor system as described in any one of claims 1 to 4, and the customizable NAND logic gate biosensor system obtained by the construction method described in any one of claims 5 to 7, in the detection of DNA methyltransferases for non-disease diagnosis and treatment purposes.

9. The application according to claim 8, characterized in that: The DNA methyltransferase is Dam methyltransferase.

10. The application of the customizable NAND logic gate biosensing system as described in any one of claims 1 to 4, and the customizable NAND logic gate biosensing system obtained by the construction method described in any one of claims 5 to 7, in screening DNA methyltransferase inhibitors.

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