Thrombin-responsive circular DNA as well as preparation method and application thereof

Abstract

The invention discloses thrombin-responsive circular DNA (deoxyribonucleic acid) and a preparation method and application thereof, and belongs to the technical field of biology, the circular DNA comprises a thrombin nucleic acid aptamer, the nucleotide sequence of the thrombin nucleic acid aptamer is shown as SEQ ID NO: 1, on the basis, thiophosphate is used for modifying the specific position of the thrombin nucleic acid aptamer, and the specific position of the thrombin nucleic acid aptamer is modified to obtain the thrombin-responsive circular DNA. A short sequence ACCG is added to the 5'end of the DNA, a reverse complementary sequence CGGT is added to the 3 'end of the DNA, and the DNA is connected into a ring after annealing to obtain the circular DNA as shown in SEQ ID NO: 3. On the basis of not influencing the original capability of accurately combining thrombin, the thrombin-responsive circular DNA obviously enhances the anti-interference capability, and improves the structural stability of the thrombin-responsive circular DNA in a complex environment. The combination of cyclization and sulfo modification improves the structural stability and anti-interference ability, has strong targeting enrichment ability, and provides an innovative scheme for biomarker detection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a thrombin-responsive circular DNA and its preparation method and application. Background Art

[0002] Aptamers are single-stranded DNA or RNA molecules that can specifically bind to target molecules (such as proteins). Traditional linear aptamers are easily degraded by nucleases in complex biological environments, resulting in insufficient stability and limiting their application in clinical detection. In the prior art, the anti-interference ability can be partially enhanced through chemical modification (such as replacing phosphodiester bonds with phosphorothioate groups), but there are still problems such as inaccurate selection of modification sites and insufficient structural stability. In addition, linear aptamers also have limitations in terms of target enrichment efficiency.

[0003] Therefore, there is an urgent need to develop a nucleic acid aptamer structure with high stability, strong anti-interference ability, and high-efficiency target enrichment performance to meet the detection requirements in complex biological environments. Summary of the Invention

[0004] Object of the Invention: The present invention provides a thrombin-responsive circular DNA and its application. The circular DNA is a nucleic acid aptamer structure with high stability, strong anti-interference ability, and high-efficiency target enrichment performance. Through phosphorothioate modification and circularization design, the present invention significantly improves the anti-interference ability and structural stability of the nucleic acid aptamer, and can be used to prepare early diagnostic reagents or biomarkers for thrombin-related diseases (such as thrombotic diseases).

[0005] Technical Solution: A thrombin-responsive circular DNA is formed by complementary connection of the 5'-end and 3'-end of a single-stranded linear DNA; the nucleotide sequence of the linear DNA is as shown in SEQ ID NO:3, and the 22nd to 27th positions thereof are six consecutive thymines, and the phosphodiester bonds of the six thymines are all phosphorothiolated.

[0006] The preparation method of the thrombin-responsive circular DNA includes the following steps:

[0007] S1. Synthesize a thrombin nucleic acid aptamer, and its nucleotide sequence is as shown in SEQ ID NO:1;

[0008] S2. In the nucleotide sequence shown in SEQ ID NO:1, the 18th to 23rd positions are six consecutive thymines, and the phosphodiester bonds of the six thymines are phosphorothiolated to obtain a linear DNA as shown in SEQ ID NO:2;

[0009] S3. Add the sequence ACCG to the 5' end of the linear DNA and the sequence CGGT to the 3' end. Complementary pair and ligate the 5' and 3' ends of the linear DNA by annealing to form a circular DNA. The nucleotide sequence of the circular DNA is as shown in SEQ ID NO:3.

[0010] The circular DNA can be used to prepare a thrombin detection reagent. The circular DNA structure is contacted with the test sample, and targeted enrichment and quantitative analysis are achieved by detecting the binding signal between the DNA structure and thrombin. The binding signal is one or more of a fluorescent label, a colorimetric method, or an electrochemical signal.

[0011] Beneficial effects:

[0012] (1) The circular DNA prepared by the present invention has high stability: the sulfur modification and the circular structure act synergistically to keep its structure intact in complex environments such as serum.

[0013] Phosphorothioate modification: Located at the key cleavage site (TTTTTT region), it significantly improves the anti-interference ability.

[0014] Circularization design: Avoids the dissociation risk at the ends of linear DNA and further enhances the structural stability.

[0015] Targeted enrichment ability: Retains the high affinity of the original aptamer for thrombin and is suitable for target detection in complex samples.

[0016] (2) The circular DNA prepared by the present invention has high detection accuracy: By combining fluorescent, colorimetric or electrochemical signals, high-sensitivity quantitative analysis of thrombin can be achieved.

[0017] (3) The circular DNA prepared by the present invention has wide applications: It can be used for the preparation of early diagnostic reagents for thrombin-related diseases such as thrombotic diseases and disseminated intravascular coagulation (DIC). Description of the drawings

[0018] Figure 1 For Example 1, the HPLC peak area integration shows a purity > 95%.

[0019] Figure 2 For the agarose gel electrophoresis characterization of the DNA structure in the example, the left side is linear DNA and the right side is circular DNA.

[0020] Figure 3 For the structural stability of different DNA structures under interference, the purified circularized product maintains the best conformation.

[0021] Figure 4To compare the fluorescence signals of the contrast structure at the thrombus and wound sites and verify its precise targeting ability, the left figure shows the fluorescence imaging at the thrombus, and the right figure shows the fluorescence imaging at the wound. The fluorescence signal of the wound tissue is significantly weaker than that of the thrombus site.

[0022] Figure 5 It is the response degree curve at different thrombin concentrations (linear range 0.1 - 50 U / mL). Specific embodiments

[0023] The technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to the described embodiments.

[0024] Example 1: Preparation of circular DNA structure

[0025] Step 1. Sequence design and synthesis:

[0026] Step 1.1: Synthesis of the original nucleic acid aptamer

[0027] Starting sequence: First, use the known thrombin nucleic acid aptamer (single-stranded DNA), and the nucleotide sequence is as shown in SEQ ID NO:1: 5'-GGTTGGTGTG GTTGGTGTTT TTTTTGTGGT TGGTGTGGTT GG-3'.

[0028] Step 1.2: Phosphorothioate modification

[0029] Modification site: For the continuous 6 thymines (TTTTTT region) at positions 18 - 23 in SEQ ID NO:1, replace its phosphodiester bond with a phosphorothioate bond.

[0030] Modification method:

[0031] 1) Solid-phase synthesis sulfurization step:

[0032] During the solid-phase synthesis of the thrombin nucleic acid aptamer, when synthesizing the thymine (T) at positions 18 - 23, use sulfurizing reagent (Sulfurizing Reagent, Glen Research) to replace the standard oxidation step. The concentration of the sulfurizing reagent is 0.1 M (acetonitrile solution), and the reaction temperature is 25°C.

[0033] 2) Specific operation process:

[0034] The sulfurization modification of each thymine nucleotide needs to be carried out in the following steps successively (repeated 6 times, corresponding to nucleotides 18 - 23):

[0035] Step 1 (Thiolation after coupling): After the coupling of the target nucleotide (T) is completed in a solid-phase synthesizer, immediately inject the phosphorothioamidite solution and react for 5 minutes to ensure that the oxygen atom in the phosphodiester bond is replaced by a sulfur atom.

[0036] Step 2 (Washing): Thoroughly wash the synthesis column with acetonitrile to remove the unreacted phosphorothioamidite.

[0037] Step 3 (Deprotection): Use trichloroacetic acid (TCA) solution to remove the dimethoxytrityl (DMT) protecting group on the 5'-hydroxyl group to prepare for the addition of the next round of nucleotides.

[0038] Verification: Confirm the molecular weight of the modified sequence (single-stranded DNA, SEQ ID NO: 2) by mass spectrometry (MALDI-TOF MS) (Theoretical value: 7320 Da, Measured value: 7322 Da).

[0039] SEQ ID NO: 2, the nucleotide sequence is: 5'-GGTTGGTGTG GTTGGTGnnn nnnTTGTGGTTGGTGTGGTT GG-3', where n represents thymine modified with phosphorothioate group, that is, the phosphodiester bond in thymine at positions 18 - 23 is phosphorothiolated.

[0040] Step 1.3: Addition of terminal sequence

[0041] Step 1.3.1, Addition of ACCG sequence at the 5' end

[0042] Synthesis direction and principle:

[0043] DNA chemical synthesis is based on the solid-phase phosphoramidite triester method, and the synthesis direction is 3'→5'. Therefore, nucleotides A, C, C, G need to be added sequentially to the 5' end of SEQ ID NO: 2 so that the final 5' end sequence is ACCG.

[0044] ① Preparation of the immobilized carrier: Fix SEQ ID NO: 2 obtained by thiophosphorylation modification in Step 1.2 to the solid-phase carrier (controlled pore glass, CPG) through its 3'-terminal hydroxyl group.

[0045] ② Strand-by-strand extension: First step: Add guanine (G) phosphoramidite monomer and complete coupling, oxidation (or thiolation), and deprotection; Second step: Add cytosine (C) phosphoramidite monomer; Third step: Add cytosine (C) phosphoramidite monomer again; Fourth step: Add adenine (A) phosphoramidite monomer.

[0046] Synthesis conditions, coupling time: 30 seconds / step; oxidant: 0.02 M iodine solution (for standard phosphodiester bonds) or sulfurization reagent (for phosphorothioate bonds); deprotection: removal of DMT protecting group with trichloroacetic acid (TCA).

[0047] Step 1.3.2, addition of the CGGT sequence at the 3' end

[0048] Design logic: Since the DNA synthesis direction is 3'→5', the complementary sequence CGGT needs to be synthesized in reverse at the 3' end of SEQ ID NO:2 (i.e., the other end of the fixed carrier). During actual synthesis, the added sequence needs to be designed in the 5'→3' direction, that is, adding T, G, G, C in sequence, and finally forming CGGT at the 3' end (note: the actual sequence at the synthesized 3' end is CGGT, and the 5' end of the corresponding complementary strand is ACCG).

[0049] Specific steps:

[0050] ① Carrier switching: Transfer the 3' end of the sequence obtained in Step 1.3.1 to a new CPG carrier for extension from the 3' end.

[0051] ② Strand-by-strand extension: First step: Add C (cytosine) phosphoramidite monomer; Second step: Add G (guanine) phosphoramidite monomer; Third step: Add G (guanine) phosphoramidite monomer again; Fourth step: Add T (thymine) phosphoramidite monomer.

[0052] Synthesis conditions: The same as the steps for addition at the 5' end to ensure coupling efficiency and modification consistency.

[0053] (3) Post-synthesis treatment and verification

[0054] 1) Cleavage and deprotection: Treat with concentrated ammonia water (28% NH3·H2O) at 55°C for 16 hours to cleave the DNA strand from the carrier and remove the base protecting groups.

[0055] 2) Purification, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), conditions are as follows:

[0056] Chromatographic column: XBridge OST C18 (4.6×50 mm, 2.5 μm);

[0057] Mobile phase: Gradient elution (10% - 50% acetonitrile, 15 minutes);

[0058] Detection wavelength: 260 nm.

[0059] 3) Quality control:

[0060] Capillary electrophoresis (CE): Verify the sequence length (theoretical value: 56 nt, measured value: 55.8 nt);

[0061] Mass spectrometry (MALDI-TOF MS): Confirm the molecular weight (theoretical value: 7985 Da, measured value: 7987 Da);

[0062] Purity detection: HPLC peak area integration shows that the purity > 95% (as Figure 1 shown).

[0063] Step 1.4: Purification after synthesis

[0064] Purification was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) with the following conditions:

[0065] Column: XBridge OST C18 (4.6 × 50 mm, 2.5 μm);

[0066] Mobile phase: Phase A (0.1 M TEAA buffer, pH 7.0), Phase B (acetonitrile);

[0067] Gradient: 10% - 50% Phase B, 15 minutes;

[0068] Flow rate: 1 mL / min;

[0069] Detection wavelength: 260 nm;

[0070] Collection and lyophilization: Collect the main peak fraction and lyophilize it under vacuum to obtain the purified linear DNA (single-stranded DNA). The nucleotide sequence of the linear DNA is as shown in SEQ ID NO:3:

[0071] 5'-ACCGGGTTGGTGTGGTTGGTGnnnnnnTTGTGGTTGGTGTGGTTGGCGGT-3'

[0072] Step 2: Cyclization step

[0073] ① Dissolve the DNA shown in SEQ ID NO:3 in annealing buffer (10 mM Tris-HCl, 50 mM NaCl, pH 7.5).

[0074] ② Heat to 95°C and maintain for 5 minutes, then slowly cool to 25°C (rate: 1°C / min) to allow the 5'-end and 3'-end to pair complementarily.

[0075] ③ Add T4 DNA ligase and react at 25°C for 2 hours to form a covalently closed circular structure.

[0076] ④ Purification: Separate and purify the circular product by gel electrophoresis or high-performance liquid chromatography (HPLC).

[0077] ⑤ Verification: Analyze by agarose gel electrophoresis with the following operating steps:

[0078] Prepare a 1.5% agarose gel (containing 0.5 μg / mL ethidium bromide). Loading: The samples selected are the purified circular product, linear DNA (SEQ ID NO: 3), and DNA molecular weight standard (1 kb ladder). Buffer: 1×TAE buffer (containing 40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3). Electrophoresis conditions: 100 V voltage, running for 30 minutes.

[0079] Imaging and analysis: The results are as Figure 2 shown. The circular DNA migrates faster than the linear DNA, presenting a band closer to the bottom of the gel, indicating a high rate of DNA structure circularization.

[0080] Example 2: Comparative test by testing the anti-interference ability of ionic strength

[0081] 1) Test samples:

[0082] Group 1: Unmodified thrombin nucleic acid aptamer (SEQ ID NO: 1), final concentration 100 nM;

[0083] Group 2: Linear DNA modified with phosphorothioate groups (SEQ ID NO: 2), final concentration 100 nM;

[0084] Group 3: Circular DNA prepared in Example 1 (SEQ ID NO: 3), final concentration 100 nM;

[0085] 2) Thrombin solution: Concentration gradients are 0.1, 0.5, 1, 10, 50 U / mL (prepared with PBS buffer)

[0086] 3) Reaction system: Each tube contains 50 μL DNA sample (100 nM) + 50 μL thrombin solution (different concentrations), with a total volume of 100 μL.

[0087] The experimental steps are as follows:

[0088] 1) Sample pretreatment: Dissolve each group of DNA samples (Group 1 - Group 3) in a simulated physiological buffer (containing 137 mM NaCl, 10 mM PBS, 5 mg / mL BSA, 1 mM MgCl2, pH 7.4). Dilute the thrombin solution in concentration gradients for standby.

[0089] Among them, for the buffer system, 137 mM NaCl (used to simulate plasma ionic strength); 10 mM PBS buffer (pH 7.4, used to simulate physiological pH); 5 mg / mL bovine serum albumin (BSA, used to simulate serum protein interference).

[0090] 2) Mixing and Incubation: Take 50 μL of DNA sample and mix it with 50 μL of thrombin solutions at different concentrations. Vortex for 10 seconds. Incubate at 37 °C for 60 minutes to ensure sufficient binding.

[0091] 3) Stability Detection: Add 5 μL of Proteinase K (1 U / μL) to the reaction system and treat at 37 °C for 30 minutes to degrade unbound DNA. Analyze the residual amount of DNA by 1.5% agarose gel electrophoresis (100 V, 30 minutes) to evaluate the anti-interference ability.

[0092] 4) Data Collection and Analysis: Use a gel imaging system (Bio-Rad ChemiDoc) to quantify the gray value of the bands.

[0093] Relative Stability (%) = (Band intensity of the treatment group / Band intensity of the untreated group) × 100%.

[0094] Curve Plotting: The abscissa is the thrombin concentration (U / mL, logarithmic coordinate), and the ordinate is the relative DNA stability (%). Group 1 (unmodified aptamer): When the thrombin concentration ≥ 1 U / mL, the stability decreases significantly (<40%), indicating easy enzymatic degradation. Group 2 (thio-modified linear DNA): The stability is increased to 60% - 70%, but still decreases with the increase of thrombin concentration. Group 3 (circular DNA): In the range of 0.1 - 50 U / mL, the stability is always >90%, proving that its anti-interference ability is significantly better than that of the control group. (As Figure 3 shown).

[0095] Conclusion: In a simulated physiological environment, due to the synergistic effect of thio-modification and circular design, the circular DNA structure of the present invention can effectively resist nuclease degradation and protein interference, and is suitable for the detection of complex biological samples.

[0096] Example 3: Target Enrichment Verification

[0097] Fluorescence Detection: Label the circular DNA with a fluorophore (FAM) and a quencher (Dab) to form a molecular beacon. After adding the sample containing thrombin, the DNA structure binds to thrombin, and the conformational change leads to fluorescence recovery. Detect the concentration of circular DNA by fluorescence intensity.

[0098] Select a thrombus rat model as the animal model, and the experimental design method is as follows:

[0099] 1) Inject a molecular beacon formed by labeling the circular DNA with a fluorophore (FAM) and a quencher (Dab) into the tail vein (dose: 10 mg / kg).

[0100] 2) Sacrifice after 24 hours and take the thrombus tissue and wound tissue

[0101] 3) Detect the concentration of circular DNA in response to thrombin according to the fluorescence intensity

[0102] Results: The drug concentration at the thrombus site was 15 times that at the wound site (as Figure 4 shown), and the results indicated that the circular DNA could achieve targeted enrichment through specific binding to thrombin and produce a significant signal difference at the thrombus site, verifying its feasibility as a highly sensitive and specific detection tool and providing a new method for assisting in the accurate diagnosis and treatment judgment of thrombotic diseases.

[0103] Example 4: Thrombin-responsive detection

[0104] Incubate the circular DNA structure of the present invention with thrombin solutions at different concentrations, and verify its response sensitivity and linear range through changes in fluorescence signals.

[0105] 1) Reagents and instruments:

[0106] The circular DNA structure prepared according to the method of Example 1, with a final concentration of 100 nM (dissolved in annealing buffer: 10 mM Tris-HCl, 50 mM NaCl, pH 7.5);

[0107] Thrombin standard solutions with concentration gradients of 0.1, 1, 5, 10, 20, 50 U / mL (prepared with PBS buffer);

[0108] The 5' end of the circular DNA was labeled with a FAM fluorophore and the 3' end was labeled with a Dabcyl quencher group to form a fluorescence label of a molecular beacon.

[0109] A fluorescence spectrophotometer was selected (excitation wavelength: 494 nm, emission wavelength: 518 nm).

[0110] 2) Experimental procedures

[0111] ① Sample preparation: Take 20 μL of the circular DNA solution (100 nM) and mix it with 80 μL of thrombin standard solutions at different concentrations, with a final volume of 100 μL, and the thrombin concentration range is 0.1 - 50 U / mL.

[0112] ② Incubation conditions: Incubate at a constant temperature of 37 °C for 30 minutes to ensure sufficient binding.

[0113] ③ Signal detection: Transfer the mixture to a 96-well black fluorescence plate and measure the fluorescence intensity in a fluorescence spectrophotometer (take the average value of three replicates).

[0114] ④ Blank control: Use PBS buffer without thrombin as a negative control (baseline signal).

[0115] 3) Data analysis

[0116] Calculate the fluorescence recovery rate: Recovery rate (%) = (F - F0) / (F max - F0)*100, where F is the fluorescence intensity of the sample, F0 is the intensity of the blank control, and F max is the maximum fluorescence intensity at complete dissociation (measured after adding an excess of thrombin).

[0117] Plot the standard curve: Use the thrombin concentration (logarithmic coordinate) as the abscissa and the fluorescence recovery rate as the ordinate to fit a linear equation (as Figure 5 shown). The fluorescence recovery rate increases with the increase in thrombin concentration, and the fluorescence recovery rate of circular DNA is the highest, indicating that compared with linear DNA, circular DNA can more effectively bind thrombin and generate a detectable signal.

[0118] 4) Experimental results

[0119] Linear range: 0.1 - 50 U / mL, correlation coefficient (R 2 ) ≥ 0.99.

[0120] Limit of detection (LOD): 0.05 U / mL (signal-to-noise ratio S / N = 3).

[0121] This example confirms that the circular DNA structure has high sensitivity and a wide linear response range to thrombin (as Figure 5 shown), is suitable for the quantitative detection of thrombin in complex biological samples, and provides a reliable technical solution for the preparation of early diagnostic reagents for thrombotic diseases.

[0122] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes can be made in its form and details without departing from the spirit and scope of the present invention.

Claims

1. A thrombin-responsive circular DNA, characterized in that, The circular DNA is formed by complementary ligation of the 5'-end and 3'-end of single-stranded linear DNA; the nucleotide sequence of the linear DNA is as shown in SEQ ID NO:3, with 22-27 being six consecutive thymines, and the phosphodiester bonds of the six thymines are all phosphorothioated.

2. A method for preparing a thrombin-responsive circular DNA as described in claim 1, characterized in that, It includes the following steps: S1. Synthesize a thrombin nucleic acid aptamer, whose nucleotide sequence is as shown in SEQ ID NO:1; S2. In the nucleotide sequence shown in SEQ ID NO:1, positions 18-23 are six consecutive thymines, and the phosphodiester bonds of these six thymines are phosphorothioated to obtain linear DNA as shown in SEQ ID NO:2; S3. Add the sequence ACCG to the 5'-end of the linear DNA and the sequence CGGT to the 3'-end, and anneal to complementarily pair and ligate the 5'-end and 3'-end of the linear DNA into circular DNA, whose nucleotide sequence is as shown in SEQ ID NO:

3.

3. Use of the circular DNA according to claim 1 in the preparation of a thrombin detection reagent.

4. The application according to claim 3, wherein The structure of the circular DNA is contacted with a test sample, and targeted enrichment and quantitative analysis are achieved by detecting the binding signal between the DNA structure and thrombin.

5. According to the use described in claim 4, the binding signal is one or more of a fluorescent label, a colorimetric method, or an electrochemical signal.

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