An APE1 detection method and system based on overhang structure-mediated non-continuous DNA activated LbuCas13a

Abstract

The application discloses an APE1 detection method and system based on a non-continuous DNA activated LbuCas13a mediated by a overhanging structure, and belongs to the technical field of biological detection. The method constructs a structure-switchable double-stranded DNA substrate APS containing an AP site. In the presence of APE1, the APS is specifically cut and releases an activation chain. The activation chain and a preset DNA chain jointly activate an LbuCas13a / crRNA complex, and then trigger the trans-cleavage activity of the LbuCas13a / crRNA complex and cut an RNA fluorescent reporter molecule, so that a detectable fluorescent signal is generated, and qualitative or quantitative analysis of APE1 is realized. The method can be used for clinical serum sample detection. After dilution, the sample can be directly added to a reaction system for detection, and has the advantages of simple operation, high detection sensitivity, good specificity and suitability for complex sample analysis.

Description

Technical Field

[0001] This invention relates to the field of biodetection and molecular diagnostics, specifically to a method and system for detecting APE1 based on discontinuous DNA activation of LbuCas13a mediated by a pendant structure. More specifically, it relates to a method and system for detecting APE1 using a structurally switchable double-stranded DNA switch containing an AP site, which releases a discontinuous DNA activation element under the action of APE1, and synergistically activates the trans-cleavage activity of LbuCas13a with a pre-placed DNA fragment, thereby achieving the detection of APE1 and its application in clinical sample testing. Background Technology

[0002] CRISPR-Cas13a is a class of CRISPR effector proteins with RNA-guided recognition and trans-cleavage activity, showing promising applications in nucleic acid detection and bioanalysis. Its functional output depends on target-induced activation; only when Cas13a forms a complex with the target that supports activation can the relevant catalytic domains enter a cleavable state and trigger subsequent trans-cleavage reactions. Previous studies have shown that Leptotrichia buccalis Cas13a (LbuCas13a), in addition to responding to RNA targets, can also be directly activated by specific DNA substrates without dependence on PAM or PFS, thus expanding the potential applications of Cas13a in non-RNA target detection.

[0003] Currently, most designs related to Cas13a activation are based on the premise of a continuous target sequence; that is, it is generally believed that as long as the target sequence is sufficiently complementary to the crRNA, Cas13a activation can be induced. However, in real biological and analytical systems, nucleic acid sequences are not always in an ideal continuous and fully exposed form, but often exhibit spatial separation, structural confinement, local masking, or discontinuous distribution. In this case, although the target sequence still contains sufficient complementary information, it may not form a stable activation conformation because its spatial configuration is not suitable for effective coordination with the Cas13a-crRNA complex. Therefore, whether discontinuous DNA can activate LbuCas13a, and the key structural factors affecting its activation behavior, still lack clear understanding and usable design basis.

[0004] On the other hand, apurinic / apyrimidinic endonuclease 1 (APE1) is a key enzyme in the DNA base excision repair pathway, capable of recognizing and cleaving double-stranded DNA substrates containing AP sites. Given the close association of APE1 with research on diseases such as tumors, establishing a sensitive, rapid detection method for APE1 applicable to complex sample analysis is of practical significance. While existing APE1 detection technologies have a certain research foundation, there is still room for improvement in terms of operational procedures, system integration, adaptability to complex samples, and signal output stability.

[0005] Against this backdrop, if a novel APE1 detection system can be developed that utilizes the specific cleavage of AP-containing substrates by APE1 to transform the enzymatic recognition event into effective activation of LbuCas13a, and further amplifies the signal through trans-cleavage of Cas13a, it holds promise. Especially in clinical serum sample analysis, detection methods not only need high sensitivity and specificity but also should minimize sample pretreatment steps and maintain stable output. The applicant's research demonstrates that in serum samples from breast cancer patients and healthy donors, the detection system constructed based on the above mechanism can achieve serum APE1 analysis under uniform dilution conditions and exhibits superior discriminative ability compared to the control ELISA method, indicating that this strategy has potential for clinical sample detection applications.

[0006] Therefore, developing a method and system based on the regulation of LbuCas13a activation by discontinuous DNA structure and applicable to APE1 detection, especially clinical sample detection, has significant technical value. Summary of the Invention

[0007] (a) Purpose of the invention

[0008] The main objective of this invention is to provide an APE1 detection method based on discontinuous DNA activation of LbuCas13a mediated by a pendant structure, thereby addressing the problems of cumbersome APE1 detection procedures, limited signal amplification capabilities, and insufficient sensitivity and discrimination in complex samples in existing technologies. This invention further provides the application of the above detection method in clinical serum sample analysis, thereby enabling qualitative or quantitative detection of APE1 in biological samples.

[0009] (II) Technical Solution

[0010] To achieve the above objectives, this invention proposes a CRISPR-LbuCas13a detection method based on the APE1 response, which includes the following steps:

[0011] First, a switchable double-stranded DNA substrate, APS, containing AP sites, was constructed. The APS is a stable double-stranded structure formed by annealing the target strand and the blocking strand, maintaining a relatively closed state in the absence of APE1.

[0012] Subsequently, the sample to be tested is brought into contact with the APS. When APE1 is present in the sample, APE1 can specifically cleave the AP site in the APS, releasing the activation strand; the activation strand, together with the pre-placed DNA strand, acts on the LbuCas13a / crRNA complex, thereby activating the trans-cleavage activity of LbuCas13a.

[0013] Furthermore, the cleavage product is added to a detection system containing LbuCas13a protein, crRNA, pre-placed DNA strand, RNA reporter molecule, and reaction buffer. The activated LbuCas13a further cleaves the RNA reporter molecule and generates a detectable signal, thereby enabling qualitative or quantitative analysis of APE1 in the sample.

[0014] Preferably, the APS is formed by mixing the target strand containing AP sites and the blocking strand in a buffer solution, followed by heating to denature and slow cooling to anneal, thus forming a stable double-stranded structure.

[0015] Preferably, the APE1 cleavage reaction is carried out in a buffer system, and the cleavage is terminated by heating after the reaction is completed.

[0016] Preferably, the CRISPR–LbuCas13a detection system includes LbuCas13a protein, crRNA, pre-placed DNA strand, RNA reporter molecule, and reaction buffer; the RNA reporter molecule is an RNA molecule labeled with a fluorescent group at one end and a quencher group at the other end.

[0017] Preferably, the detectable signal is a fluorescence signal, and APE1 in the test sample is analyzed by detecting fluorescence intensity, fluorescence growth rate, or endpoint fluorescence value.

[0018] The present invention also proposes the application of the above detection method in the detection of APE1 in serum samples.

[0019] Preferably, the sample to be tested is a serum sample. Before detection, the serum sample is diluted with buffer and then added to the CRISPR–LbuCas13a detection system for reaction to reduce the interference of the serum matrix on the detection results.

[0020] Preferably, the serum sample is a serum sample from a breast cancer patient and / or a serum sample from a healthy donor.

[0021] In some embodiments, the present invention may also employ the commercial APE1 ELISA method to perform parallel testing on the same batch of samples, so as to compare and analyze the detection effect of the method of the present invention.

[0022] This invention constructs an analytical method for APE1 detection based on a structurally switchable substrate and CRISPR-LbuCas13a signal amplification. Its core design involves introducing a double-stranded DNA switch (APS) containing an AP site. In the absence of APE1, the APS remains in a relatively stable, closed state, making it difficult for the system to generate a significant signal. However, when APE1 is present in the sample, the AP site in the APS is specifically cleaved, releasing the activation strand. This activation strand, along with a pre-placed b14-o8, acts on the LbuCas13a / crRNA complex, activating its trans-cleavage activity, ultimately cleaving the fluorescent reporter RNA and generating a detection signal. The entire detection principle is as follows: Figure 1 As shown in the figure. Subsequently, the substrate assembly and APE1 cleavage process were verified by PAGE. The results showed that APS could form the expected structure and cleave in the presence of APE1, thus effectively converting the APE1 cleavage event into the subsequent Cas13a activation process. The corresponding results are shown in the figure. Figure 2 Further fluorescence kinetics results showed that the system maintained only a low background in the absence of APE1, while the fluorescence signal increased rapidly over time after the addition of APE1, indicating that the system can effectively distinguish the presence or absence of the target. These results are detailed in [link to related document]. Figure 3 To balance the stability of APS in the uncut state and the release efficiency of the activating chain after cleavage, the complementary length of the blocking chain was further screened. Results showed that an appropriate complementary length could achieve a better signal-to-noise ratio and more stable detection output. The relevant results are shown in [link to relevant results]. Figure 4 .

[0023] Under optimized conditions, different concentrations of APE1 were detected. The results showed that as the APE1 concentration increased, the fluorescence signal output by the system gradually increased, exhibiting a clear concentration-dependent relationship. This indicates that the method of this invention can not only achieve qualitative identification of APE1 but also be further used for its quantitative analysis. This part of the results corresponds to... Figure 5 , Figure 6 Meanwhile, to evaluate the specificity of this method, APE1 was compared with enzymes such as UDG, FEN1, Lambda exonuclease, and Nt. BbvCI. The results showed that only APE1 could elicit a significant fluorescence response, while other enzymes produced low background noise, indicating that the method of this invention has good detection specificity. The relevant results are shown in [link to relevant results]. Figure 7 .

[0024] After establishing the in vitro analysis system, this invention was further applied to clinical serum sample testing. A total of 77 serum samples were collected, including 47 serum samples from pathologically confirmed breast cancer patients and 30 serum samples from healthy individuals, all collected from the First Affiliated Hospital of Chongqing Medical University. All samples were uniformly diluted to 10% serum working solution before testing to reduce serum matrix interference. The overall sample processing and testing procedure is described below. Figure 8 Under the same pretreatment conditions, all clinical samples were tested using ACROSS and a commercially available APE1 ELISA, respectively. The heatmap results showed that ACROSS exhibited a clearer separation trend between the breast cancer group and the healthy control group, while the ELISA results still showed some overlap between groups. Figure 9 .

[0025] Further distribution analysis of the detection signals from the two methods revealed that the normalized fluorescence signal obtained by ACROSS was significantly higher in the breast cancer group than in the healthy group, with less overall overlap in distribution. In contrast, while the normalized OD value detected by ELISA was also elevated in the breast cancer group, its dynamic range was narrower, and inter-group overlap was more pronounced. Therefore, ACROSS demonstrated better sample discrimination ability in terms of both signal amplitude and distribution discrimination. The corresponding violin plot results are shown below. Figure 10 and Figure 11 The main text also clearly describes this point: ACROSS shows significant differences between groups with limited overlap in distribution, while ELISA shows more obvious overlap.

[0026] To further evaluate the overall discriminative performance of the two methods in clinical samples, ROC analysis was performed on the detection results. The results showed that ACROSS achieved a higher AUC than ELISA when used for the discrimination of serum breast cancer samples, indicating superior overall diagnostic efficacy. Figure 12 Based on this, confusion matrices were established according to optimized thresholds to visually demonstrate the consistency between test results and established clinical diagnoses. The results showed that ACROSS had a sensitivity of 89.4%, a specificity of 93.3%, an overall accuracy of 90.9%, and a positive predictive value of 95.5%. Figure 13 The sensitivity of ELISA was 76.6%, specificity was 90.0%, overall accuracy was 81.8%, and positive predictive value was 92.3%. Figure 14 The above results demonstrate that, under the same sample cohort and pretreatment conditions, the ACROSS method established in this invention exhibits superior discrimination ability and diagnostic efficacy compared to ELISA in clinical serum APE1 detection.

[0027] In summary, this invention establishes an APE1 detection method suitable for clinical serum sample analysis by constructing an APE1-responsive APS switch and combining it with the inverse cleavage amplification effect of CRISPR-LbuCas13a. This method forms a complete technical route from substrate design and in vitro validation to clinical sample testing, achieving stable detection in real serum samples and demonstrating better group discrimination ability and superior overall diagnostic performance compared to commercial ELISA. Attached Figure Description

[0028] Figure 1 : Schematic diagram of the APE1 responsive ACROSS detection system described in this invention.

[0029] Figure 2 PAGE characterization diagram of the APS assembly and APE1 triggering activation process described in this invention.

[0030] Figure 3 Real-time fluorescence kinetic characterization of the ACROSS system described in this invention under conditions with and without APE1.

[0031] Figure 4 Schematic diagrams of APS structure designs with different complementary lengths of blocking chains and corresponding fluorescence signal analysis diagrams.

[0032] Figure 5 Real-time fluorescence kinetics of the ACROSS system under different concentrations of APE1.

[0033] Figure 6 Standard curve and linear fitting plot of fluorescence response versus APE1 concentration.

[0034] Figure 7 Fluorescence changes in the ACROSS system under the action of APE1, UDG, FEN1, λ exonuclease, and Nt.BbvCI are shown in the diagram, which are used to evaluate the detection specificity of the method of the present invention for APE1.

[0035] Figure 8 A flowchart of clinical serum sample testing using ACROSS and ELISA.

[0036] Figure 9 Heatmap of clinical serum sample test results using ACROSS and ELISA.

[0037] Figure 10 Violin plot of normalized fluorescence signals obtained from serum samples of healthy donors and breast cancer patients detected by ACROSS.

[0038] Figure 11 Violin plot of normalized OD values ​​obtained from serum samples of healthy donors and breast cancer patients by ELISA.

[0039] Figure 12 ROC curves of ACROSS and ELISA for the diagnosis of breast cancer serum samples.

[0040] Figure 13 : ACROSS confusion matrix relative to established clinical diagnoses.

[0041] Figure 14 Confusion matrix diagram of ELISA relative to established clinical diagnostic results. Detailed Implementation

[0042] A one-pot detection method for CRISPR–LbuCas13a based on APE1 response is performed according to the following steps:

[0043] (1) Assembly of APS substrate

[0044] The target strand (TS) and blocking strand (BS) containing the AP site were dissolved separately in TEMg buffer, with a final concentration of 2 μM for P-AP-a14_o8 and 2.5 μM for P1-8. After mixing, the mixture was heated at 95 °C for 5 min, then slowly cooled to 25 °C at a rate of 0.1 °C / s, and incubated at 25 °C for 10 min to form a stable APS double-stranded structure for later use. This APS remains locked in the absence of APE1, making it difficult to directly activate LbuCas13a.

[0045] (2) APE1 cleavage reaction

[0046] Prepare a 20 μL digestion mixture in a 200 μL PCR tube, including 1× NEBuffer 4, 100 nM APS substrate, and 0.1 U / μL APE1. Make up the remaining volume with nuclease-free water. Incubate the reaction mixture at 37 °C for 30 min to allow APE1 to specifically cleave the AP site in APS. Then, terminate the reaction by heating at 65 °C for 10 min. The resulting digestion product is used for subsequent Cas13a detection.

[0047] (3) Construction of the ACROSS detection system

[0048] A 20 μL Cas13a detection system was prepared in a 200 μL PCR tube, comprising 50 nM LbuCas13a protein, 50 nM crRNA, 10 nM b14_o8 chain, 10 μL of APE1 cleavage product obtained in step (2), 1× Cas13a reaction buffer, and 500 nM fluorescence-quenched RNA reporter molecule. The Cas13a reaction buffer contained 40 mM Tris-HCl, 1 mM DTT, and 5 mM MgCl2, pH 7.5. After mixing, the mixture was incubated at 37 ℃, and the fluorescence signal changes were recorded in real time.

[0049] (4) Optimization of complementary length of blocking chain

[0050] After establishing the above system, the complementary length between BS and TS was optimized. The results show that as the complementary length increases, the signal-to-noise ratio of the system first increases and then decreases, reaching a better level at 8 nt. Therefore, subsequent experiments preferably use a blocking structure with a complementary length of 8 nt.

[0051] (5) Quantitative detection of APE1

[0052] Under optimized conditions, different concentrations of APE1 were added to the above system for detection. The results showed that the ACROSS system at 1.0 × 10⁻ 5 It exhibits good linear response within the range of –1.0 × 10⁻¹ U mL⁻¹, and can be used for the quantitative analysis of APE1.

[0053] (6) Evaluation of detection specificity

[0054] To evaluate the specificity of the method of the present invention, UDG, FEN1, λ exonuclease, and Nt.BbvCI were selected as control enzymes for detection. The results showed that only APE1 could induce a significant fluorescence response, while the other enzymes produced low background signals, indicating that the method of the present invention has good detection specificity.

[0055] (7) Clinical serum sample testing

[0056] A total of 77 clinical serum samples were collected, including 47 serum samples from breast cancer patients and 30 serum samples from healthy donors, all from the First Affiliated Hospital of Chongqing Medical University. After centrifugation, the serum samples were stored at −80 ℃ and diluted 1×PBS at a ratio of 1:9 to prepare a 10% serum working solution before testing.

[0057] Add 2 μL of the diluted serum sample to a 20 μL ACROSS mixture containing 50 nM LbuCas13a, 50 nM crRNA, 100 nM APS, 10 nM b14_o8, 500 nM FQ-reporter, and 1× Cas13a reaction buffer. Incubate at 37 °C for 1 h, recording the fluorescence kinetic signal every 20 s. Perform triple replicates for each sample, using PBS as a negative control instead of serum.

[0058] (8) ELISA control test

[0059] To compare the detection performance of the method of this invention, a commercially available human APE1 ELISA kit was used to detect serum samples from the same batch. The kit was operated according to the instructions, and the endpoint signal was obtained by measuring the absorbance at 450 nm. The results were then compared with those of ACROSS. The results showed that ACROSS exhibited better differentiation between the breast cancer group and the healthy group, and its ROC analysis and confusion matrix results were superior to those of ELISA.

Claims

1. A method and system for detecting APE1 based on discontinuous DNA activation of LbuCas13a mediated by a pendant structure, characterized in that, Includes the following steps: S1, hybridize the target strand and the blocking strand to construct a switchable double-stranded DNA substrate APS containing AP sites; S2, the sample to be tested is brought into contact with the APS. When APE1 is present in the sample, APE1 cuts the AP site in the APS and releases the activation chain. S3, add the cleavage product obtained in step S2 to the CRISPR-LbuCas13a detection system, the detection system including LbuCas13a protein, crRNA, pre-set DNA strand and RNA reporter molecule; S4, the activation strand and the pre-placed DNA strand work together on the LbuCas13a / crRNA complex to activate the trans-cleavage activity of LbuCas13a, thereby cleaving the RNA reporter molecule and generating a detectable signal. S5, perform qualitative or quantitative analysis on APE1 in the sample to be tested based on the detectable signal.

2. The detection method according to claim 1, characterized in that, The activation strand is a DNA fragment with a pendant structure released after APE1 cleaves APS, and the pre-placed DNA strand is a DNA strand that can co-activate LbuCas13a with the activation strand. The APS is a bi-stranded structure formed by annealing a target strand containing AP sites and a blocking strand.

3. The detection method according to claim 2, characterized in that, The APS is prepared by dissolving the target chain and the blocking chain in a buffer solution, mixing them, heating to denature them, and then slowly cooling and annealing them to form a stable double-stranded structure.

4. The detection method according to claim 3, characterized in that, The APE1 cleavage reaction in step S2 is carried out in a buffer system, and the cleavage is terminated by heating after the reaction is completed.

5. The detection method according to claim 4, characterized in that, The CRISPR–LbuCas13a detection system in step S3 includes LbuCas13a protein, crRNA, pre-prepared DNA strand, RNA reporter molecule and reaction buffer.

6. The detection method according to claim 5, characterized in that, The RNA reporter molecule is an RNA molecule labeled with a fluorescent group at one end and a quencher group at the other end; the detectable signal is a fluorescence signal, and APE1 in the test sample is analyzed by detecting fluorescence intensity, fluorescence growth rate or endpoint fluorescence value.

7. The detection method according to claim 6, characterized in that, The sample to be tested is a serum sample.

8. The detection method according to claim 7, characterized in that, The serum sample was diluted with buffer before being added to the CRISPR–LbuCas13a detection system for reaction.

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