Nucleic acid aptamer of transgenic corn CP4-EPSPS protein and molecular beacon fluorescent biosensor thereof

Abstract

The invention relates to the technical field of biosensors, in particular to a transgenic corn CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor. According to the invention, a systematic aptamer rational cutting and conformation adaptation strategy is constructed, the strategy obtains an initial aptamer through magnetic bead-SELEX screening, and the CP4-EPSPS protein aptamer is precisely cut and optimized by adopting the steps of loop region active core positioning, stem region structure optimization and base binding domain identification, so that the binding performance and the structural stability are remarkably improved. Subsequently, on the basis of stable conformational characteristics, a complementary chain competition sensing mechanism is designed, and a transgenic corn CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor is successfully constructed, so that amplification-free high-sensitivity rapid detection of CP4-EPSPS protein is realized, and a solution is provided for supervision of transgenic crops.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biosensors, and particularly relates to a transgenic corn CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor. BACKGROUND

[0002] Corn is the second largest commercialized genetically modified crop in the world. The 5-enolpyruvylshikimate-3-phosphate synthase gene (CP4-EPSPS) is widely used in transgenic corn varieties, and the protein expression amount thereof is a key marker for detecting transgenic components. Existing detection technologies (such as ELISA) rely on antibody recognition, but the antibody is high in cost and poor in stability, and nucleic acid detection methods (such as PCR) are high in sensitivity but rely on precise instruments, and cannot meet the demand for on-site rapid screening. Therefore, it is crucial to develop a simple, rapid, high-sensitivity and low-cost CP4-EPSPS protein screening method for detecting transgenic corn.

[0003] The nucleic acid aptamer technology obtains an oligonucleotide probe capable of replacing an antibody through the system evolution of ligands by exponential enrichment (SELEX) screening, and has advantages of high affinity, room temperature stability and low-cost synthesis. However, at present, there is still a significant gap in the field of transgenic protein detection: there is no public report on the aptamer for CP4-EPSPS protein in the world, and there is a lack of systematic study on the binding site and dissociation constant thereof, which limits the application of the aptamer sensor in the field of development.

[0004] Molecular beacon, as an important class of fluorescent biosensor, its core is composed of oligonucleotide probe with stem-loop structure, and the two ends are modified with fluorescent group (such as FAM) and quenching group (such as BHQ) respectively. The traditional working principle follows the "opening type" mechanism: the stem-loop structure is closed, the fluorescence is quenched, and the stem-loop structure is opened after the target is combined to release the fluorescence signal. This design is widely used in nucleic acid detection, but it faces severe challenges in protein detection - most proteins cannot directly open the stem-loop structure through base complementary pairing like nucleic acids. The existing technology tries to adapt to protein detection by two strategies: one is to construct an "opening type" aptamer, which requires the target to bind to actively dissociate the stem-loop structure; the second is to introduce a competitive binding element (such as a complementary strand), but this design relies on the conformational change of the aptamer after binding to the target. However, this study found that the CP4-EPSPS protein aptamer has a "stable" feature, that is, after binding to the target, not only does it not open the stem-loop, but it also strengthens the secondary structure through hydrogen bonding and hydrophobic interaction, making the fluorescence signal further quenched, which conflicts with the classical molecular beacon theory. In order to break through this limitation, the invention designs a complementary strand (cDNA) to hybridize with the 5' end of the aptamer, forcing the stem-loop to open and restore fluorescence; after adding the target protein, it competes with the cDNA to bind to the aptamer, prompting the cDNA to dissociate and restore the stable stem-loop structure, resulting in a second fluorescence quenching. This signal conversion mode based on conformational competition successfully converts the structural characteristics of the "stable" aptamer into a quantifiable detection signal, filling the technical gap of molecular beacons in the field of protein detection.

[0005] The present application constructs a systematic aptamer rational trimming and conformational adaptation strategy. The strategy obtains an initial aptamer through magnetic bead-SELEX screening, and uses the steps of ring region active core positioning, stem region structure optimization and base binding domain identification to precisely trim and optimize the CP4-EPSPS protein aptamer, significantly improving the binding performance and structural stability. Subsequently, based on the "stable" conformational characteristics, a complementary strand competition sensing mechanism is designed, and a transgenic corn CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor is successfully constructed, realizing the high-sensitivity rapid detection of CP4-EPSPS protein without amplification, and providing a solution for the supervision of transgenic crops. SUMMARY

[0006] In view of the shortcomings of the prior art, the present application aims to provide a transgenic corn CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor.

[0007] To achieve the above-mentioned purpose, on the one hand, the present application provides a nucleic acid aptamer of a functionally enhanced transgenic maize CP4-EPSPS protein, and the sequence of the aptamer is shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 27.

[0008] On the other hand, the present application provides the application of the above-mentioned nucleic acid aptamer in the development of a CP4-EPSPS protein detection method.

[0009] On the other hand, the present application provides the application of the above-mentioned nucleic acid aptamer in a CP4-EPSPS protein detection kit.

[0010] On the other hand, the present application provides a transgenic maize CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor, which specifically comprises: 1. the principle of the biosensor; 2. the sequence of the biosensor; 3. the parameter adjustment of the CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor; and 4. the detection of the CP4-EPSPS protein. The principle of the above-mentioned biosensor is that the two ends of the aptamer are labeled with a fluorescence group and a quenching group, the complementary sequence is hybridized with the aptamer to open the stem loop structure, and the fluorescence signal is enhanced; when the target CP4-EPSPS protein exists, it competes with the cDNA to bind the aptamer, so that the aptamer restores the stem loop structure, the fluorescence is quenched, the signal decreases, the signal change amount is negatively correlated with the protein concentration, and quantitative detection is realized. The sequence of the above-mentioned biosensor is 5'-BHQ1-AAAGGGACGACCCATTCCCCTCGTTCCTTCCGTCGTCCCAAA-FAM-3', as shown in SEQ ID NO. 39.

[0011] The buffer pH of the above-mentioned biosensor is 6.5-8.5.

[0012] The target incubation time of the above-mentioned biosensor is 10-30 min.

[0013] The molecular beacon concentration of the above-mentioned biosensor is 0.8-1.2 μmol·L -1 .

[0014] The biosensor is applied to the development of a CP4-EPSPS protein detection method.

[0015] The biosensor is applied to a CP4-EPSPS protein detection kit.

[0016] Compared with the prior art, the application has the beneficial effects that: 1. The application proposes a systematic aptamer tailoring strategy, effectively simplifies the original aptamer structure, and accurately identifies the core binding site of the aptamer and the target, thereby providing a theoretical basis for aptamer structure optimization and sensor design. 2. The aptamer for the CP4-EPSPS protein is obtained based on tailoring, which effectively improves the affinity, simplifies the structure, and promotes the practicability in biosensing. 3. The application proposes a new type of CP4-EPSPS protein biosensing strategy, which uses a competitive molecular beacon to forcibly open the stem ring to emit fluorescence by using a complementary chain, and induces fluorescence quenching by target competition binding, thereby realizing label-free ratio-type sensing detection. 4. The CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor proposed by the application can realize simple, rapid, low-cost and high-sensitivity on-site detection of CP4-EPSPS protein, and has certain industrialization potential. 5. The CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor proposed by the application has a good linear relationship (R -1 ≥ 0.99) in the range of 200-1400nmol·L 2 . -1 BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 For preliminary screening of aptamer secondary structure prediction.

[0018] Figure 2 For preliminary screening of aptamer affinity comparison.

[0019] Figure 3 For preliminary screening of aptamer dissociation saturation curve.

[0020] Figure 4 For adjusting the secondary structure prediction of the aptamer by truncating the stem region.

[0021] Figure 5 ​Verification and optimization of AuNPs colorimetric method for truncated aptamer in stem region. (A) NaCl concentration optimization; (B) aptamer concentration optimization; (C) CP4-EPSPS protein concentration optimization; (D) verification of the absence of components in AuNPs colorimetric method; (E) absorbance ratio change amount of AuNPs colorimetric method with or without target.

[0022] Figure 6 Verification of SGI fluorescence method for truncated aptamer in stem region. (A) verification of the absence of components in SGI fluorescence method; (B) fluorescence intensity kinetics monitoring of adding aptamer and target in turn in buffer containing SGI; (C) fluorescence intensity of SGI system with or without CP4-EPSPS protein; (D) fluorescence intensity change amount of SGI system with or without CP4-EPSPS protein.

[0023] Figure 7 Verification of SGI fluorescence method for core positioning aptamer in loop region. (A) aptamer base removal part (top) and secondary structure prediction of aptamer after removing bases (bottom); (B) fluorescence intensity of different truncated aptamers with or without CP4-EPSPS protein; (C) fluorescence intensity change amount of different truncated aptamers with or without CP4-EPSPS protein.

[0024] Figure 8 Verification of SGI fluorescence method for mutant aptamer. (A) secondary structure prediction of mutant aptamer; (B) fluorescence intensity of different mutant aptamers with or without CP4-EPSPS protein; (C) fluorescence intensity change amount of different mutant aptamers with or without CP4-EPSPS protein.

[0025] Figure 9 Verification of structure conversion of aptamer 8-1-8bp. (A) fluorescence intensity of aptamer 8-1-8bp combined with different concentrations of CP4-EPSPS protein verified by four kinds of dyes; (B) Tm temperature (left) and fluorescence intensity at different temperatures (right) of aptamer 8-1-8bp combined with different concentrations of CP4-EPSPS protein verified by qPCR; (C) fluorescence intensity (top) and signal-to-noise ratio (bottom) of modified molecular beacon of aptamer 8-1-8bp combined with different concentrations of CP4-EPSPS protein.

[0026] Figure 10 Schematic diagram of aptamer molecular beacon fluorescence biosensor.

[0027] Figure 11Optimization of the length of the complementary strand of the molecular beacon. (A) Agarose gel characterization of the hybridization of the molecular beacon MB-8bp with different complementary sequences; (B) fluorescence spectrophotometer determination of the fluorescence intensity of the hybridization of the molecular beacon MB-8bp with different complementary sequences; (C) qPCR determination of the fluorescence intensity at different temperatures of the hybridization of the molecular beacon MB-8bp with different complementary sequences.

[0028] Figure 12 Optimization of the conditions of the aptamer molecular beacon fluorescence biosensor. (A) Buffer pH optimization; (B) complementary sequence to molecular beacon ratio optimization; (C) molecular beacon complex concentration optimization; (D) molecular beacon incubation time optimization with CP4-EPSPS protein.

[0029] Figure 13 Performance evaluation of the aptamer molecular beacon fluorescence biosensor. (A) Aptamer molecular beacon fluorescence biosensor determination of the fluorescence intensity of different concentrations of CP4-EPSPS protein; (B) aptamer molecular beacon fluorescence biosensor determination of the signal-to-noise ratio of the fluorescence intensity of different concentrations of CP4-EPSPS protein; (C) aptamer molecular beacon fluorescence biosensor determination of the signal-to-noise ratio of CP4-EPSPS protein and other transgenic proteins. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0031] Example 1. SELEX screening of CP4-EPSPS protein aptamer The present application provides a method for screening CP4-EPSPS protein aptamer, comprising the following steps: (1) Preparation of magnetic bead-target protein complex ① Activation of carboxyl group The magnetic beads were mixed well, and then 100 μL was taken out for magnetic separation, and the supernatant was removed. 200 μL of MES (100 mmol·L -1 ) was added and washed twice, and the supernatant was removed. Freshly prepared EDC and NHS (10 mg·mL -1 ) were added at 100 μL each, vortexed to suspend well, and incubated at 25°C on a shaker for 30 min.

[0032] ② Coupling Remove the activation solution by magnetic separation, add 100 μL CP4-EPSPS protein solution (concentration 0.5 mg·mL -1 , the ratio of protein mass to magnetic bead mass is 1:1), mix evenly by shaking, and incubate at 25℃ for 2 h (or alternatively: couple at 25℃ for 1 h, then transfer to 4℃ for overnight incubation), and keep the suspension state during the whole process.

[0033] ③ Magnetic bead blocking and preservation Discard the supernatant by magnetic separation, add 250 μL TBS blocking solution, vortex for 20 s, discard the supernatant by magnetic separation, add 1 mL TBS buffer, and incubate at room temperature for 2 h by circumferential shaker to block the remaining binding sites, discard the supernatant by magnetic separation, and then wash twice with 200 μL PBS buffer, finally resuspend in protein buffer, and store at 4℃ to obtain the magnetic bead-target complex.

[0034] (2) DNA library screening process ① Library pretreatment Dissolve the single-stranded DNA library with ultrapure water, dilute with HEPES buffer, and immediately cool on an ice box for 10 min after heating in a metal bath at 95℃ for 5 min.

[0035] ② Screening process Negative screening: pretreat the library and 20 μL protein-free carboxyl magnetic beads in 500 μL 1×HEPES buffer, and incubate by shaking for 30 min, and then retain the supernatant by magnetic separation to remove non-specific binding sequences.

[0036] Positive screening: add 100 μL magnetic bead-target complex to the supernatant of the negative screening, and incubate by shaking in 500 μL 1×HEPES buffer; after magnetic separation, wash three times with 1×HEPES, discard the unbound nucleic acid sequences; add 1×Taq buffer to the magnetic beads, heat at 90℃ for 10 min to dissociate the bound DNA, and collect the supernatant as the target template by magnetic separation.

[0037] Reverse screening: couple the non-target protein to the magnetic beads, incubate with the denatured library for 30 min, and then take the supernatant for positive screening to eliminate cross-reactive sequences.

[0038] (3) Library amplification and purification ① Real-time fluorescent quantitative PCR monitoring Take 1 μL of eluent as template, add SYBR Green Mix (10 μL), upstream and downstream primers (0.4 μL each), and ddH2O (8.2 μL), and the total system is 20 μL. The running program is 95°C pre-denaturation for 2 min; 95°C for 10 s → 60°C for 20 s, for a total of 30 cycles, repeated 4 times.

[0039] ② Mass amplification and enzyme digestion Conduct routine PCR on the single-stranded template, and the amplification procedure is the same as step (3) ①; take 264 μL of the amplification product, add 6 μL of lambda exonuclease and 30 μL of exonuclease buffer, mix uniformly, and perform enzyme digestion at 37°C; after enzyme digestion is completed, deactivate the enzyme at 75°C for 10 min.

[0040] ③ Alcohol precipitation purification Add 0.1 times the volume of sodium acetate (3 mol·L -1 ) and 2 times the volume of pre-cooled anhydrous ethanol to the enzyme digestion product, and precipitate overnight at -20°C; centrifuge at 4°C at 12000 rpm for 15 min, and discard the supernatant; wash the precipitate with 70% ethanol, dry, dissolve in 50 μL of ultrapure water, obtain the secondary library, and determine the concentration by Nanodrop.

[0041] (4) Screening termination and sequencing Repeat the screening process (steps 2-3) until the Ct value of qPCR is stable; after the last round of secondary library is amplified by PCR, verify the product purity and size by agarose gel electrophoresis; after meeting the requirements, send for sequencing, divide the top 20 aptamers with the highest abundance into five families according to sequence homology, and select 4 aptamers 1-L (SEQ ID NO. 1), 2-L (SEQ ID NO. 2), 4-L (SEQ ID NO. 3), and 8-L (SEQ ID NO. 4) as candidate aptamers for further research.

[0042] The nucleotide sequences used in the experiment are shown in Table 1.

[0043] Table 1 Summary of nucleotide sequences in Example 1 Example 2. Exploration of the minimum active structure of CP4-EPSPS protein nucleic acid aptamer 1. Preliminary screening and trimming optimization of CP4-EPSPS protein nucleic acid aptamer The secondary structures of four high-affinity aptamers 1-L (SEQ ID NO. 1), 2-L (SEQ ID NO. 2), 4-L (SEQ ID NO. 3), and 8-L (SEQ ID NO. 4) obtained by magnetic bead-based SELEX screening were analyzed, as shown in FIG. 1. The four candidate aptamers all have similar stem-loop structures, the stem contains a small bulge, and the stem is the primer binding region at both ends of the sequence, and the loop site is the 40 nt random region in the middle. First, the role of the random region, i.e., the loop site, in binding to the CP4-EPSPS protein was explored. The stem part was kept unchanged, and the loop stage of the four candidate aptamers was cut off by cutting, and 20 nt bases were retained on the left, middle, and right, respectively. The secondary structure prediction results are shown in FIG. 1. After qPCR binding capacity test, four truncated aptamers 1-1-L (SEQ ID NO. 5), 2-3-L (SEQ ID NO. 10), 4-3-L (SEQ ID NO. 13), and 8-1-L (SEQ ID NO. 14) with lower Ct values were screened out (FIG. 2). As shown in FIG. 3, 8-1-L (SEQ ID NO. 14) performed best (Ct = 13.41), and its Kd value was determined by the qPCR standard curve method to be 36.25 nmol·L -1 , which is 5.2 times lower than the Kd value of the original aptamer 8-L (SEQ ID NO. 4) of 189.6 nmol·L -1 , and was selected as the core research object.

[0044] 2. Stem region truncation adjustment of CP4-EPSPS protein nucleic acid aptamer The stem structure sequence 8-1-Loop (SEQ ID NO. 17) and the loop structure sequence 8-1-Stem (SEQ ID NO. 18) of the aptamer 8-1-L (SEQ ID NO. 14) were synthesized, and the aptamer 8-1-L (SEQ ID NO. 14) was subjected to stem region gradient truncation (from 12 bp to 5 bp) to obtain 8-1-12bp to 8-1-5bp series variants (SEQ ID NO. 19 to SEQ ID NO. 24), and the secondary structure was verified by RNAfold prediction as shown in FIG. 4.

[0045] The affinity change was verified by a double verification method: (1) AuNPs colorimetric method By electrostatic repulsion mechanism, AuNPs are red in dispersed state (absorption peak is obvious), salt ions induce aggregation leading to fading (absorption peak disappears); aptamer enhances the negative charge of AuNPs by non-specific binding, maintaining its dispersed state (absorption peak is restored); when the target protein exists, it competes with AuNPs for binding to aptamer, leading to weakened protection, and the solution turns blue, so the affinity of aptamer can be compared by visual observation or determination of the absorption peak values at 525 nm and 625 nm. To interfere with acidic target proteins (such as: CP4-EPSPS), by optimizing the salt concentration (0.55 mol·L -1 NaCl), aptamer concentration (50 nmol·L -1 ) and protein concentration (35 μg·mL -1 ), the anti-interference window is established (Fig. 5A, B, C, D), and the change in ΔA520 before and after the addition of target is used to represent the affinity. As shown in Fig. 5E, the affinity of 8-1-Loop (SEQ ID NO. 17) is significantly reduced, 8-1-Stem (SEQ ID NO. 18), 8-1-12bp (SEQ ID NO. 19) retains the original affinity of aptamer, 8-1-9bp (SEQ ID NO. 20), 8-1-8bp (SEQ ID NO. 21) has a certain improvement in affinity, 8-1-7bp (SEQ ID NO. 22), 8-1-6bp (SEQ ID NO. 23) affinity decreases, 8-1-5bp (SEQ ID NO. 24) affinity almost disappears.

[0046] (2) Sybr Green Ⅰ fluorescence method The specificity of Sybr Green I itself can interact with the small groove of double-stranded DNA to emit fluorescence, indicating the number of base pairs. The binding of target molecules (such as CP4-EPSPS) can change the structure of the aptamer, and these structural changes can change the number of base pairs of the aptamer. Therefore, the degree of opening of the double-stranded stem structure after the binding of CP4-EPSPS protein and aptamer is characterized by the amount of SG fluorescence change, further verifying the strength of the affinity of the aptamer. First, the feasibility of this method was verified by fluorescence intensity scanning at different wavelengths and molecular dynamics monitoring. In the absence of aptamer, the SG solution has no fluorescence signal output (Fig. 6A, B), and in the presence of Sybr Green I buffer, the addition of aptamer produces obvious fluorescence at 523 nm, and the addition of CP4-EPSPS protein lowers the absorption peak value (Fig. 6A). The fluorescence intensity decreases and tends to be stable within one minute (Fig. 6B), verifying that the aptamer 8-1-L (SEQ ID NO. 14) has a stem-loop structure, and the double-stranded structure is partially opened when it binds to the CP4-EPSPS protein, completing the structural transformation. As shown in Fig. 6C, as the length of the aptamer stem structure gradually shortens, the fluorescence intensity gradually decreases. 8-1-Loop (SEQ ID NO. 17) is a single strand and does not have a stem structure, so there is no fluorescence signal output. As shown in Fig. 6D, after calculating the amount of fluorescence intensity change in the presence and absence of CP4-EPSPS protein, it is found that the amount of change of aptamer 8-1-9bp (SEQ ID NO. 20) is the largest, and then as the length of the double-stranded structure gradually shortens, the amount of fluorescence intensity change also gradually decreases, and the affinity shows a downward trend. Aptamers 8-1-L (SEQ ID NO. 14), 8-1-Stem (SEQ ID NO. 18), and 8-1-12bp (SEQ ID NO. 19) have longer stem structures, making it difficult for their double-stranded structures to be opened by CP4-EPSPS protein, so the fluorescence change is not as significant as that of aptamers with shorter stem structures.

[0047] Based on the double verification results of AuNPs colorimetric method and Sybr Green I fluorescence method, the affinities of aptamer variants 8-1-8bp (SEQ ID NO. 21) and 8-1-9bp (SEQ ID NO. 20) are similar and significantly better than those of the original aptamer and other truncated variants. Under the premise of meeting the threshold of structural stability and functional equivalence, 8-1-8bp (SEQ ID NO. 21) with fewer bases is selected as the object of subsequent research.

[0048] 3. Core location of the loop region of the CP4-EPSPS protein nucleic acid aptamer According to the molecular docking results of aptamer 8-1-L (SEQ ID NO. 14), 26C~30G is the main binding site on the ring structure (Figure 7A), so by cutting off 5 bases of the main binding site and 5 bases at different positions of the ring structure, the affinities are compared. Since the stem structure sequence is not changed in this round of cutting, the fluorescence intensity of the aptamer is at the same level, and 8-1-8bp (SEQ ID NO. 21) removes the 5 bases at the end of the ring structure, opening the 1 base pair at the top of the stem structure, so its fluorescence intensity is slightly weaker than that of other aptamers (Figure 7B). The amount of fluorescence intensity change after adding CP4-EPSPS protein is shown in Figure 7C, and the affinities of the 6 cut aptamers are significantly weaker than that of aptamer 8-1-8bp (SEQ ID NO. 21), indicating that all the ring structures have binding effects; among them, 8-1-8bp-3 (SEQ ID NO. 27) has the largest decrease in affinity, indicating that 26C~30G is the core binding region, which is consistent with the previous molecular docking results.

[0049] According to the experimental results, it is shown that aptamer 8-1-L (SEQ ID NO. 14) mainly binds to CP4-EPSPS protein through the stem-loop structure, and the ring structure 26C~30G is the core binding region. The stem structure is responsible for stabilizing the properties of the ring part, and the 8bp base pairs on the upper part of the stem also have certain binding effects.

[0050] The nucleotide sequences used in the experiment are shown in Table 2.

[0051] Table 2 Summary of nucleotide sequences of Example 2 Example 3. Construction of CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor 1. Strategy proposal and verification analysis of CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor (1) Strategy proposal The aptamer 8-1-8bp (SEQ ID NO. 21) with better affinity after rational cutting optimization is used as the core element of the molecular beacon in the construction of the biosensor. The aptamer has a standard stem-loop structure, and the initial design is based on the hypothesis that the stem structure is opened when it binds to the target. It is designed as an open-type molecular beacon to complete the capture of CP4-EPSPS protein and signal output. The key to the fluorescence signal of the molecular beacon is the separation of the fluorescence group and the quenching group, so the stem structure of the aptamer needs to be more easily opened and converted to make the fluorescence quenching groups modified at both ends of the aptamer far enough apart, so as to produce a strong enough fluorescence signal and achieve visual result display.

[0052] Based on this purpose, 3 pairs of C-G bases with stable stem end binding were mismatched to weaken the stability of the stem structure. The sequence and naming of the trimmed aptamer are shown in Table 3, and the secondary structure prediction is shown in Figure 8A, in which the red bases represent the mutated bases.

[0053] (2) Verification analysis Using fluorescent dye SG I to determine the change in fluorescence intensity when there is no CP4-EPSPS protein (Figures 8B, C), it was found that 8-1-8bp-3T (SEQ ID NO. 37) had a more thorough change in stem-loop structure, and the stem structure was significantly shortened, resulting in lower fluorescence intensity than other aptamers; the fluorescence change of the aptamer after mutation of the 5' end base was significantly higher than that of the 3' end mutant. The mutation strategy failed to effectively open the stem structure, which was not consistent with the preset open conformation theory, and further analysis and exploration were needed.

[0054] The nucleotide sequences used in the above experiments are shown in Table 3.

[0055] Table 3 Summary of nucleotide sequences of Example 3 2. Model verification and design optimization of CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor (1) Model verification Three double-stranded fluorescent dyes (SG I, EVA Green and SYTO 9) and one single-stranded fluorescent dye (SG II) were used to verify whether the aptamer was an open aptamer after binding to the target. The results showed that with the increase of target concentration, the fluorescence intensity of the four dyes showed a downward trend (Figure 9A), confirming that the target binding occupied the fluorescent group binding site of the stem-loop structure. This result was consistent with the conclusion that the stem and loop of aptamer 8-1-8bp (SEQ ID NO. 21) had a binding effect, but the evidence for the opening of the double strand was weak. Further analysis of the melting temperature (Tm) found that the Tm value increased from 30°C to 68°C in the presence of the target, and the derivative peak decreased (Figure 9B), clearly indicating that the binding mode of the aptamer was stable, not open.

[0056] (2) Design optimization Based on this, it is decided to still choose aptamer 8-1-8bp (SEQ ID NO. 21) to develop molecular beacon MB-8bp (SEQ ID NO. 39), the design includes: 5' end modification FAM fluorescence group, 3' end modification BHQ1 quenching group, total length 36 nt (each end increases 3 nt A base to avoid FAM-GGG quenching interference); functional test shows that with the increase of target concentration, the fluorescence intensity increases slightly to 67.9 A.U. (Figure 9C), indicating that the distance between the fluorescence group and the quenching group increases slightly. It is speculated that the target should be that the aptamer stem structure is not opened, but the 3 nt A base on each end is pulled away, causing partial recovery of fluorescence. The above design corrects the initial open conformation assumption through experimental verification, establishes a stable signal conversion mechanism, and constructs a core detection element based on competitive fluorescence quenching.

[0057] To amplify the above weak signal, a competitive fluorescence quenching strategy is designed for the molecular beacon (Figure 10), the core principle is: the complementary strand (cDNA) hybridizes with the 5' end of the aptamer to open the stem-loop structure, FAM and BHQ1 are separated to emit fluorescence signal; after adding target CP4-EPSPS protein, it competes with the complementary strand to bind to the aptamer, prompting the complementary strand to dissociate and restore the stem-loop structure, resulting in fluorescence quenching, and the amount of fluorescence intensity decrease is used as the detection signal. To optimize this mechanism, the hybridization efficiency of different lengths of complementary strands (11-14 nt) is verified by agarose gel electrophoresis and fluorescence intensity analysis, as shown in the results of Figure 11: 11-12 nt complementary strands have almost no hybridization; 13 nt partially hybridizes; 14 nt hybridizes completely and has the maximum fluorescence intensity, indicating that 14 nt is the optimal complementary strand length, which can maximize the signal and improve the response sensitivity of competitive dissociation.

[0058] 3. Parameter adjustment of CP4-EPSPS protein aptamer molecular beacon fluorescence biosensor To improve the performance of the sensor, the key detection parameters are optimized (Figure 12): first, optimize the buffer pH environment, the results show that when pH is 7.5, the fluorescence intensity of the molecular beacon without target reaches saturation, and the fluorescence decrease is the most significant after adding the target; pH that is too high or too low will result in a decrease in response; second, verify the ratio of complementary strand to molecular beacon concentration, when the ratio is 1:1, the fluorescence intensity is the largest, indicating that the aptamer stem-loop structure is fully opened; then test the molecular beacon concentration (0.8-1.2 μmol·L -1 ), when the concentration is 1 μmol·L -1 , the fluorescence decrease induced by the target is the largest; finally, observe the fluorescence decrease under different incubation times, and determine that 25 min is the best incubation time, at which the fluorescence intensity tends to be stable, indicating that the complex binding has reached saturation.

[0059] 4. Performance evaluation of CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor (1) Sensitivity evaluation of CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor The MB-8bp (SEQ ID NO. 39) was used to detect the known concentration of CP4-EPSPS protein, and a standard curve was prepared according to the concentration of target CP4-EPSPS protein in the solution and the signal-to-noise ratio (F0-F) / F0 (F0 is the fluorescence intensity without EPSPS protein, and F is the fluorescence intensity after adding EPSPS protein) calculated from the FAM fluorescence intensity. 1 μmol·L -1 The beacon aptamer MB-8bp (SEQ ID NO. 39) was mixed with 1 μmol·L -1 The cDNA was placed in a HEPES buffer system with a pH of 7.5, and after annealing and hybridization, 0 nmol·L -1 , 200 nmol·L -1 , 400 nmol·L -1 , 600 nmol·L -1 , 800 nmol·L -1 , 1000 nmol·L -1 , 1200 nmol·L -1 , 1400 nmol·L -1 , 1600 nmol·L -1 EPSPS protein was added, and after incubation for 25 min, the fluorescence intensity of the FAM fluorescence group was measured, with an excitation wavelength of 492 nm and an emission wavelength of 517 nm.

[0060] As shown in FIGS. 13A and B, the detection limit of the CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor for CP4-EPSPS protein was 200 nmol·L -1 , and the linear response range was 200-1400 nmol·L -1 , and the linear regression equation was y = 0.0006087 x - 0.1187 (R 2 = 0.9962).

[0061] (2) Specificity evaluation of CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor The MB-8bp (SEQ ID NO. 39) was used to detect the known concentrations of CP4-EPSPS protein, Bar protein, Cry1A protein and VIP3 protein, and the signal-to-noise ratio (F0-F) / F0 (F0 is the fluorescence intensity without target protein, and F is the fluorescence intensity after adding target protein) was calculated according to the concentration of target protein in the solution. 1 μmol·L -1 The beacon aptamer MB-8bp (SEQ ID NO. 39) was used to detect the known concentrations of CP4-EPSPS protein, Bar protein, Cry1A protein and VIP3 protein, and the signal-to-noise ratio (F0-F) / F0 (F0 is the fluorescence intensity without target protein, and F is the fluorescence intensity after adding target protein) was calculated according to the concentration of target protein in the solution. 1 μmol·L -1 The cDNA was placed in a HEPES buffer system with a pH of 7.5, and after annealing and hybridization, it was mixed with 400 nmol·L -1 The CP4-EPSPS protein, Bar protein, VIP3 protein and Cry1A protein were mixed uniformly and incubated for 25 min, and the fluorescence intensity of the FAM fluorescence group was measured using a fluorescence spectrophotometer with an excitation wavelength of 492 nm and an emission wavelength of 517 nm.

[0062] As shown in FIG. 13C, 1 μmol·L -1 The signal-to-noise ratio of the CP4-EPSPS protein was 0.49, while the signal-to-noise ratios of the other three non-target proteins (phosphinothricin acetyltransferase - Bar protein, Cry1A class insecticidal crystal protein - Cry1A protein, and vegetative insecticidal protein 3 - VIP3 protein) were not more than 0.2, confirming that the sensor has high selectivity and specificity.

Claims

1. A nucleic acid aptamer for enhanced transgenic maize CP4-EPSPS protein, characterized in that, The aptamer sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.

27.

2. The application of the nucleic acid aptamer according to claim 1 in the development of a method for detecting CP4-EPSPS protein.

3. The use of the nucleic acid aptamer according to claim 1 in the CP4-EPSPS protein detection kit.

4. A transgenic maize CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor, characterized in that, I. Principles of Biosensors; II. Sequences of Biosensors; III. Parameter Adjustment of CP4-EPSPS Protein Aptamer Molecular Beacon Fluorescent Biosensor; IV. Detection of CP4-EPSPS Protein; The principle of the biosensor is that the aptamer is labeled with a fluorescent group and a quencher group at both ends. The complementary sequence hybridizes with the aptamer to open the stem-loop structure and enhance the fluorescence signal. When the target CP4-EPSPS protein is present, it competes with cDNA to bind to the aptamer, causing the aptamer to restore the stem-loop structure, quench the fluorescence, and decrease the signal. The amount of signal change is negatively correlated with the protein concentration, thus achieving quantitative detection. The sequence of the biosensor is: 5'-BHQ1-AAAGGGACGACCCATTCCCCTCGTTCCTTCCGTCGTCCCAAA-FAM-3', as shown in SEQ ID NO.

39.

5. The biosensor according to claim 4, characterized in that, The buffer solution of the biosensor has a pH of 6.5 to 8.

5.

6. The biosensor according to claim 4, characterized in that, The target incubation time for the biosensor is 10-30 min.

7. The biosensor according to claim 4, characterized in that, The molecular beacon concentration of the biosensor is 0.8–1.2 μmol·L⁻¹. -1 .

8. The application of the biosensor according to any one of claims 3 to 7 in the development of a method for detecting CP4-EPSPS protein.

9. The application of the biosensor according to any one of claims 3 to 7 in the CP4-EPSPS protein detection kit.

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