A multivalent double lock-key system based on strand displacement reaction and its application in nucleotide polymorphism detection

Abstract

The present invention discloses a multivalent double lock and key system based on chain displacement reaction and its application in nucleotide polymorphism detection, which is used for highly specific and rapid nucleotide polymorphism determination. The system can only be defined as "On" state when the target is introduced as the only "key" and two matching operations are performed to activate the two fulcrum-mediated chain displacement reactions respectively, and the system remains in "Off" state in the absence of any step. Most importantly, this two-time matching strategy using two different target fragments as input provides an effective method for simultaneously distinguishing single nucleotide polymorphisms and double nucleotide polymorphisms. The mutant bases at different positions are even far apart from each other. This double lock design has stronger discrimination ability than the ordinary single lock system. The stable performance of the Multi-DLK system in serum and the final application of real sample determination in cancer cells show the potential of this platform in clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleotide polymorphism detection, and in particular to a multivalent double lock-key system based on strand displacement reaction and its application in nucleotide polymorphism detection. Background Art

[0002] DNA probes are an irreplaceable option for identifying nucleotide polymorphisms, which are widely recognized as important genetic biomarkers. Growing evidence indicates that certain polymorphisms are associated with various human diseases, particularly highly lethal diseases such as malignant tumors. Therefore, the development of practical DNA platforms for measuring tumor-associated nucleotide polymorphisms is crucial for disease diagnosis.

[0003] Generally speaking, the most direct approach is to perform specific enzyme-catalyzed reactions at key points of nucleotide polymorphisms, including primer extension, cleavage, and ligation. Traditional DNA probes are based on hybridization methods and are precisely designed to report hybridization differences between the target and its nucleotide polymorphism. Typical hairpin-shaped molecular beacon probes, known for their ease of design and practical application, have been widely used in various fields such as nucleic acid determination, molecular recognition, and fluorescence imaging. However, the negligible thermodynamic changes from one or two mutated bases limit the discrimination ability of molecular beacon probes and cannot clearly show the subtle differences between the target species and the mutant species.

[0004] In contrast, the strand displacement reaction (SDR) is a new hybridization-based technology that plays a vital role in dynamic DNA nanotechnology and has powerful functions in the development of DNA circuits, DNA nanocomputers, and biosensors. SDR is a process in which a single-stranded invader hybridizes with the pivot region and releases the chain through branch migration, thereby forming a more stable double-stranded structure. Considering the key role of the pivot in SDR and its much shorter length (typically 5-8 nt), the pivot becomes a structural domain for the recognition of single or multiple mutation points. Compared with typical hairpin-shaped molecular beacons, the proportion of mismatched bases is significantly increased, which enhances the discrimination ability and sharp thermodynamic changes, amplifying the small differences between the target species and the mutant species.

[0005] However, current SDR technology, when applied to nucleotide polymorphism detection, consists of only one chain and can therefore only detect nucleotide polymorphisms at a single target site. It cannot simultaneously distinguish nucleotide polymorphisms at different sites, reducing detection efficiency. Furthermore, the low specificity of a single chain sequence makes it susceptible to nonspecific unlocking by invading chains, resulting in false-positive results. This lack of target specificity can directly impact clinical diagnostic accuracy and lead to misdiagnosis. Therefore, existing single-chain SDR technology cannot meet the demands for high-precision diagnosis. Summary of the Invention

[0006] The technical problem to be solved by the present invention is as follows: The present invention discloses a multivalent double lock-key system based on SDR reaction, which solves the problems of low efficiency and poor specificity in the existing SDR detection of nucleotide polymorphisms.

[0007] In order to solve the above technical problems, the present invention provides a Multi-DLK system. When the Multi-DLK system detects the nucleotide polymorphism of the template (target gene), it first relies on T SDR1 and T The two consecutive branches of SDR2 mediate strand displacement reactions (which can be visualized as two locks), using the invasion of the single-stranded target sequence to sequentially displace the two locks (i.e., open the two locks simultaneously). This sequential displacement process of the locks provides two screenings for the target sequence and forms an orthogonal activation strategy, thereby exposing the palindromic ends in the linker as primers, and then initiating highly target-specific strand displacement amplification (KFP) for nucleotide polymorphism determination. TS SDA).

[0008] According to the above reaction principle, T SDR1 and T Each detection unit of the SDR2 strand displacement reaction contains two single-stranded DNA chains. A positive indication is generated when both single-stranded DNA chains are simultaneously displaced by the target sequence. A positive indication in this application is the initiation of highly target-specific strand displacement amplification by Klenow polymerase (KFP) for nucleotide polymorphism detection, resulting in high-intensity fluorescence.

[0009] Preferably, the detection unit comprises a single-stranded linker sequence, and two single-stranded DNA chains are complementary paired with the linker sequence in sequence, wherein only the chain paired with the 3' end of the linker carries a fluorescent label, and the 3' end of the linker carries a quenching label.

[0010] Preferably, the 5' end of the linker is conjugated with biotin, and several detection units are connected to streptavidin via biotin. Furthermore, the performance of the biosensor can be further improved by the spatial confinement effect, as it increases the local concentration of DLK and shortens the distance between molecules compared to its free state. This allows the target to progressively move between the four DLKs centered on streptavidin, achieving a highly efficient strand displacement circuit. Finally, the target molecule hybridized to the linker can be released to continue initiating the reaction cycle. This circuit process can continue autonomously until all multiple DLKs are assembled into a grid-like DNA-protein complex (GDPH).

[0011] Preferably, the 5' end of the lock chain paired with the 3' end of the linker is coupled with a FAM fluorescent group for signal transduction, and the 3' end of the linker is coupled with BHQ-1 as a quencher.

[0012] Preferably, the complementary region between the linker and the target sequence contains at least two branch sequences.

[0013] As a verification of the above invention concept, the present invention selects the tumor suppressor gene p53 gene as the target model, divides the target p53 gene into two parts Ta and Tb and collectively refers to the target ST, respectively as T SDR1 and T Invaders of SDR2. To evaluate the application of Multi-DLK in nucleotide polymorphism determination. Specifically, the sequence of the linker is shown in SEQ ID No. 1, the sequence of the lock strand paired with the 3' end of the linker is shown in SEQ ID No. 2, and the sequence of the other lock strand is shown in SEQ ID No. 3.

[0014] A method for preparing the above-mentioned multivalent double lock-key system based on a strand displacement reaction comprises the following specific steps:

[0015] Step 1: Prepare reaction solution 1 at a volume ratio of 3:2:14.4 of single-stranded solution: 10× Blue buffer: H2O. Heat reaction solution 1 and gradually cool to room temperature. Then, add streptavidin solution and assemble the reaction to obtain the single-stranded solution containing three types of single-stranded DNA: linker, chain 1, and chain 2.

[0016] Preferably, the concentrations of the linker, chain 1, and chain 2 are all 10 μM; the concentration of the streptavidin solution is 9.6 μM, and the volume ratio of the streptavidin solution to the single-chain solution is 1:5.

[0017] Preferably, the reaction conditions of the reaction solution 1 are heating reaction at 95° C. for 5 minutes, and the streptavidin solution is added and reacted at 37° C. for 1 hour.

[0018] An application of a multivalent double lock-key system based on strand displacement reaction in nucleotide polymorphism detection comprises the following steps:

[0019] Step 1: Add the target sequence, Klenow polymerase (KFP), and dNTPs to the prepared multivalent double lock-key system and react at 37°C for 2 hours;

[0020] Step 2: The reaction solution was diluted 10-fold to the final volume with 1× Blue buffer, and excited and the fluorescence spectrum was measured by a fluorimeter.

[0021] The Multi-DLK system can be functionally divided into three parts: two of which are continuous branch-mediated strand displacement reactions, called T SDR1 and T SDR2 provides two screens for the target sequence and forms an orthogonal activation strategy to expose the palindromic ends in the linker as primers, which is the key to using Klenow polymerase to initiate highly target-specific strand displacement amplification (SDP) for nucleotide polymorphism determination in the third part by Klenow polymerase (KFP). TS SDA).

[0022] Beneficial effects obtained by the present invention:

[0023] The present invention relates to a multivalent double lock-key (Multi-DLK) system based on SDR reaction, which can be precisely activated to perform branch-mediated orthogonal DNA circuit (ODC) to achieve highly target-specific strand displacement amplification ( TS SDA) is used for accurate clinical diagnosis. The specificity can be improved by the strategy of orthogonal activation, and two matches can be achieved based on the use of two different target fragments as input, which provides a method for simultaneously distinguishing nucleotide polymorphisms at different positions. The present invention also demonstrates the stable performance of the Multi-DLK system in fetal bovine serum and the application of real sample determination in cancer cells, which shows the great potential of this platform in clinical diagnosis. This detection method provides a new method for distinguishing multiple nucleotide polymorphism detections, opens up new insights, and is easy to promote and apply in disease diagnosis, drug development and cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The schematic diagram of the Multi-DLK system for detecting targets. Figure 1 A is a schematic diagram of the Multi-DLK detector assembly; Figure 1 B represents the sequences of the P53 gene as an example. Multi-DLK is the detector, which forms a grid-like DNA-protein complex (GDPHs) with the target. In the figure, F represents the FAM label, Q represents the BHQ-1 quencher label, B- represents biotin, toehold 1 represents the pivot 1 sequence, toehold 2 represents the pivot 2 sequence, and FMR1 and FMR2 represent the sequences complementary to pivot 1 and pivot 2 on the p53 gene.

[0025] Figure 2 The Multi-DLK system was characterized by polyacrylamide gel electrophoresis.

[0026] Figure 3 Fluorescence spectra and linear fitting equations of the Multi-DLK system for different target concentrations.

[0027] Figure 4 Comparison of the SNP and DNP detection performance between the Multi-DLK system and Multi-SLK.

[0028] Figure 5 The ability of the Multi-DLK system to distinguish various mutation types.

[0029] Figure 6 To evaluate the performance of the Multi-DLK system for nucleotide polymorphism detection under physiological conditions using 10% FBS (fetal bovine serum).

[0030] Figure 7 The feasibility of the Multi-DLK system for detecting the p53 tumor suppressor gene in real samples was demonstrated. DETAILED DESCRIPTION

[0031] The specific implementation methods of the present invention are further explained in detail below through the description of embodiments, so as to help those skilled in the art to have a more complete, accurate and in-depth understanding of the inventive concept and technical solution of the present invention.

[0032] Example 1: Preparation of a Multivalent Double Lock-Key (Multi-DLK) System Based on SDR Reaction:

[0033] The experimental method is as follows:

[0034] Step 1: Assembly of the Multi-DLK system was performed in a 20 μL volume containing equal amounts (1 μL, 10 μM) of Linker, Lock 1, and Lock 2, 2 μL of 10× Blue buffer, and 14.4 μL of H O. After heating at 95°C for 5 minutes, the solution was gradually cooled to room temperature.

[0035] Step 2: Then, streptavidin (SAV) (0.6 μL, 9.6 μM) was added and reacted at 37°C for 1 hour.

[0036] Step 3: Characterize the stepwise assembly of the Multi-DLK system using 8% polyacrylamide gel electrophoresis. The gradually slow-moving bands (lanes ac) and the construction of the multi-DLK system by capturing four DLKs attached to streptavidin (SAV) are demonstrated by the appearance of an even slower-moving band in lane d. Lane a is the linker, lane b is the linker and Lock2, lane c is the linker, Lock 1, and Lock 2, and lane d is the linker, Lock 1, Lock 2, and SAV (see results). Figure 2 ).

[0037] Example 2: Sensitivity of the Multi-DLK system to different target concentrations

[0038] The concentrations of the detected targets Ta and Tb were set to 0 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, and 50 nM, and fluorescence measurement was performed according to the method in step 2. Figure 3 A is the fluorescence emission spectra of the Multi-DLK system in the presence of various target concentrations ranging from 10 fM to 50 nM. Figure 3 B is the dependence of peak fluorescence intensity on target concentration in the range of 10fM to 50nM. Figure 3 C) and high ( Figure 3 D) Linear response over the target concentration range.

[0039] The measurement results show that the present invention has a wide linear range for the detected target, and the linear fitting equation is made by using the fluorescence signal F and the target concentration C, with a detection limit of 10fM and good sensitivity (the results are shown in FIG. Figure 3 shown).

[0040] The experimental method is as follows:

[0041] Step 1: Prepare the Multi-DLK system (15.5 μL). Then, add equal amounts of Ta and Tb (0.5 μL each), 0.5 μL of KFP (5 U / μL), and 3 μL of dNTPs (10 mM) to a total reaction volume of 20 μL. Incubate at 37°C for 2 hours.

[0042] Step 2: For fluorescence measurement, 20 μL of the reaction solution was diluted with 1× Blue buffer to a final volume of 200 μL so that the final concentrations of Ta and Tb were the set concentrations, and the emission spectra were recorded by F-7000 fluorimeter (Hitachi, Japan) at a set excitation wavelength of 492 nm, an emission wavelength range of 500 to 600 nm, a photomultiplier tube detector voltage of 600 V, and both the incident and emission slits of 5 nm.

[0043] Example 3: High specificity of the Multi-DLK system for nucleotide polymorphism detection

[0044] Experiment 1: The present invention detected different single nucleotide polymorphisms (SNPs) and double nucleotide polymorphisms (DNPs). Figure 4As shown, SNPs and DNPs can be effectively identified simultaneously (relative fluorescence intensity is less than 50% of that when detecting the target ST). For comparison, a multivalent single lock and key (Multi-SLK) system equipped with only one lock (Lock 2) was designed. This system was unable to effectively identify SNPs and DNPs, demonstrating the superior performance of the dual lock and key strategy and its potential as a powerful tool for nucleotide polymorphism detection. The target p53 gene was divided into two regions, Ta and Tb, and single-base and double-base mutations in these two regions were used to evaluate the application of Multi-DLK in nucleotide polymorphism detection. Figure 4 A is the performance of the Multi-DLK system equipped with two locks for different single nucleotide polymorphisms (SNPs) and double nucleotide polymorphisms (DNPs). Figure 4 B is a Multi-DLK system equipped with one lock.

[0045] The experimental method is as follows:

[0046] Step 1: The Multi-DLK system was assembled according to the assembly method of Example 1 in a 20 μL solution containing equal amounts (1 μL, 10 μM) of Linker and Lock 2, 2 μL of 10× Blue buffer, and 15.4 μL of HO. After heating at 95°C for 5 minutes, the solution was gradually cooled to room temperature. Streptavidin (SAV) (0.6 μL, 9.6 μM) was then added and allowed to react for 1 hour. In this example, the Linker was linked to biotin at its 5' end and a BHQ-1 quencher at its 3' end. Lock 2 was labeled with a FAM fluorescent marker at its 5' end.

[0047] Step 2: The target sequence used in Example 2 was replaced with a single-base mutation sequence and a double-base mutation sequence. The remaining reaction system and conditions were the same as in Example 2. After the reaction was completed, the fluorescence signal was measured. The specific sequence is shown in Table 1.

[0048] Experiment 2: The applicant also studied the discrimination ability of the Multi-DLK system using mutation types such as substitutions (mA, mT, mC), insertions (iA, iT, iC, iG), and deletions (dG). Only the matching target (ST) could trigger the double-lock system to enter the open state, while the system remained in the locked state when detecting all other systems. Figure 5 A and Figure 5 B is the ability to identify various single-base mutation types in the target gene region a (FMR1) and b (FMR2). Figure 5 C and Figure 5 D. Detection of double base mutation types.

[0049] The experimental method is as follows:

[0050] According to the targets Ta and Tb detected in Example 2, the sequences of various base mutation types were replaced. The rest of the reaction system and conditions were the same as in Example 2. After the reaction was completed, fluorescence measurement was performed. The specific sequences are shown in Table 1.

[0051] Experiment 3: To explore the performance of the Multi-DLK system in nucleotide polymorphism determination under physiological conditions. This experiment used 10% FBS (fetal bovine serum) for simulation. The above results show that the Multi-DLK system has high specificity and versatility in nucleotide polymorphism determination. (Results are shown in Figure 6 shown).

[0052] The experimental method is as follows:

[0053] Step 1: Prepare the Multi-DLK system (13.5 μL). Then, add equal amounts of Ta and Tb (0.5 μL each), 0.5 μL of KFP (5 U / μL), 3 μL of dNTPs (10 mM), and 2 μL of FBS, for a total reaction volume of 20 μL. Incubate at 37°C for 2 hours.

[0054] Step 2: After the reaction is completed, the fluorescence signal is measured using the method of step 2 of Example 2.

[0055] Table 1 Target sequences used in each experimental group

[0056]

[0057]

[0058] Example 4: Real sample measurement

[0059] To evaluate the feasibility of the Multi-DLK system for detecting the p53 tumor suppressor gene in a clinical setting, specific detection of PCR products from genomic DNA in tumor cells was performed. As described in the experimental section, genomic DNA was extracted from two cell lines (HeLa and A549) and asymmetric PCR ( a PCR) amplification to prepare p53PCR products, while symmetric PCR ( s The p53 PCR product prepared by PCR was used as a control. Figure 7 As shown, obviously, s The low response signal induced by PCR products was almost the same as that of the system without any PCR products (blank group). a A significant signal enhancement was detected in the absence of PCR products, confirming the assay specificity of the Multi-DLK system. Even more strikingly, when more extracted genomic DNA was used as aWhen the PCR template was used, the fluorescence intensity of both groups (HeLa and A549) increased, demonstrating its potential application in clinical sample analysis.

[0060] Step 1: Cells in 10cm 2 When the culture dish grew to 80%, genomic DNA was extracted using a whole genome extraction kit (TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0) according to the kit instructions.

[0061] Step 2: The symmetric PCR mixture consisted of a forward primer (2 μL, 10 μM), a reverse primer (2 μL, 10 μM), 2 μL of genomic DNA (A549 180 ng / μL, HeLa 110 ng / μL), 25 μL of 2× Taq Plus Master Mix, and 19 μL of ultrapure water. After mixing thoroughly, PCR amplification was performed according to the following protocol: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds, followed by a final extension at 72°C for 5 minutes. The product from this step is double-stranded DNA and cannot undergo single-strand invasion. The symmetric PCR product serves only as a control.

[0062] Step 3: The asymmetric PCR mixture consists of a forward primer (5 μL, 10 μM), a reverse primer (0.5 μL, 2 μM), 2 μL of genomic DNA (A549 180 ng / μL, Hela 110 ng / μL), 25 μL of 2× Taq Plus Master Mix, and 17.5 μL of ultrapure water. The thermal cycling procedure is the same as for symmetric PCR. Due to the unequal amounts of forward and reverse primers, asymmetric PCR products contain a large amount of single-stranded target DNA. This single-stranded target DNA can act as an invading strand to displace strands 1 and 2. When the invading strand successfully displaces strands 1 and 2, the liner sequence exposes a palindromic sequence that acts as a primer. This highly target-specific strand displacement mediated by KFP generates fluorescence, which can be measured to rapidly determine the nucleotide polymorphism of the target gene.

[0063] Step 4: 5 μL of each of the symmetric PCR product and the asymmetric PCR product were then added to the reaction solution prepared in Example 2 with a final volume of 20 μL, and the reaction was carried out according to the method in Example 2 and the fluorescence was measured.

[0064] In summary, the present invention relates to a multivalent double lock and key (Multi-DLK) system for highly specific and rapid nucleotide polymorphism determination. The principle is that only when the target is introduced as a unique "key" and two matching operations are performed to activate two branch-mediated chain displacement reactions respectively,T SDR1 and T The system is defined as “On” only when SDR2 is present, while the system remains in “Off” state in the absence of any step. Most importantly, this two-matching strategy using two different target fragments as input provides an effective method for simultaneously distinguishing single nucleotide polymorphisms and double nucleotide polymorphisms, even when the mutated bases at different positions are far apart from each other. TS The SDA combination acts as a signal amplifier, enabling detection limits as low as 10 fM. Comparative analysis demonstrates that this dual-lock design offers superior discrimination capabilities compared to conventional single-lock systems. Furthermore, the robust performance of the Multi-DLK system in serum and its ultimate application in real-world cancer cell samples demonstrates the platform's potential for clinical diagnostics. In summary, this invention provides a new, rapid and efficient method for distinguishing multiple nucleotide polymorphisms, with significant implications for disease diagnosis, drug development, and cancer treatment.

[0065] The above embodiments are only for illustrating the technical ideas of the present invention and cannot be used to limit the scope of protection of the present invention. Any changes made on the basis of the technical solutions in accordance with the technical ideas proposed by the present invention fall within the scope of protection of the present invention; any technologies not involved in the present invention can be implemented by existing technologies.

Claims

1. A polyvalent double lock-key system based on strand displacement reaction, characterized in that: Each detection unit of the strand displacement reaction contains two single-stranded DNA chains, and a positive indication is generated when the two single-stranded DNA chains are simultaneously displaced by the target sequence; The detection unit includes a single-stranded linker sequence, and two single-stranded DNA chains are complementary paired with the linker sequence in sequence, wherein only the chain paired with the 3' end of the linker carries a fluorescent label, and the 3' end of the linker carries a quenching label; The sequence of the linker is shown in SEQ ID No. 1, the sequence of the lock chain paired with the 3' end of the linker is shown in SEQ ID No. 2, and the sequence of the other lock chain is shown in SEQ ID No.

3.

2. A polyvalent double lock-key system based on a strand displacement reaction according to claim 1, characterized in that: The 5' end of the linker is coupled with biotin, and several detection units are connected to streptavidin via biotin.

3. The polyvalent double lock-key system based on strand displacement reaction according to claim 1, characterized in that: The 5' end of the chain paired with the 3' end of the linker is coupled with FAM, and the 3' end of the linker is coupled with BHQ-1.

4. The polyvalent double lock-key system based on strand displacement reaction according to claim 1, characterized in that: The complementary region between the linker and the target sequence contains at least two branch sequences.

5. A method for preparing a multivalent double lock-key system based on a strand displacement reaction according to any one of claims 1 to 4, characterized in that: The specific steps are as follows: Step 1: Prepare reaction solution 1 at a volume ratio of 3:2:14.4 of single-stranded solution: 10× Blue buffer: H2O. Heat reaction solution 1 and gradually cool to room temperature. Then, add streptavidin solution and assemble the reaction to obtain the single-stranded solution containing three types of single-stranded DNA: linker, chain 1, and chain 2.

6. The method for preparing a polyvalent double lock-key system based on a strand displacement reaction according to claim 5, characterized in that: The concentrations of the linker, chain 1, and chain 2 were all 10 μM; the concentration of the streptavidin solution was 9.6 μM, and the volume ratio of the streptavidin solution to the single-chain solution was 1:

5.

7. The method for preparing a polyvalent double lock-key system based on a strand displacement reaction according to claim 5, characterized in that: The reaction conditions of the reaction solution 1 are heating reaction at 95° C. for 5 minutes, and the streptavidin solution is added and reacted at 37° C. for 1 hour.

8. Use of the multivalent double lock-key system based on strand displacement reaction according to any one of claims 1 to 4 in nucleotide polymorphism detection, characterized in that: Step 1: Add the target sequence, KFP, and dNTP to the prepared multivalent double lock-key system and react at 37°C for 2 hours; Step 2: The reaction solution was diluted 10-fold to the final volume with 1× Blue buffer, and excited and the fluorescence spectrum was measured by a fluorimeter.

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