NUCLEIC ACID APTAMER SCREENING METHOD BASED ON THE LOCALIZED SURFACE PLASMON RESONANCE TECHNOLOGY

Abstract

Provided is a nucleic acid aptamer screening method based on the localized surface plasmon resonance technology, falling within the fields of molecular recognition and nucleic acid aptamer screening. The method screens out a nucleic acid aptamer that can specifically bind to a target mainly with the aid of a localized surface plasmon resonance personal molecular interaction analyzer. The screening method comprises: taking a nano-gold chip as a medium, fixing the target on the medium, and then carrying out the visualized screening of the nucleic acid aptamer by taking the nucleic acid aptamer as a recognition element. With the aid of the LSPR-SELEX technique, the method does not require any marker during the process of detection by using a solid chip, and maintains the spatial structure and biological activity of the nucleic acid aptamer at the maximum. Compared to the traditional nucleic acid aptamer screening method, the LSPR-SELEX sensing technology is simple to operate and has a high sensitivity, and is less time-consuming (15 min) and has a quick response speed. The greatest advantage lies in that the interaction data is represented on-line in real time, and the affinity between molecules of each round can be acquired quickly and accurately.

Claims

1. A nucleic acid aptamer screening method based on the localized surface plasmon resonance technology, wherein, taking a nano-gold chip as a medium, fixing the target on the medium, and then carrying out the visualized screening of the nucleic acid aptamer by taking the nucleic acid aptamer as a recognition element.

2. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to claim 1, wherein, the target includes, but not limited to, a small molecule, a protein or a virus.

3. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to claim 1, wherein, firstly, the oligonucleotide library is diluted with PBS buffer solution as a sample, and then injected; secondly, the system is washed with PBS buffer solution to remove the weakly bound or unbound oligonucleotide molecules, then injection system is washed with water and evacuated with air to prevent the sample from adsorbing on the inner wall of the sample tube; finally, the NaOH regeneration buffer solution is injected, and the nucleotide molecule that specifically binds to a target is eluted and recovered for PCR amplification, so as to prepare a single strand, and obtain a single-chain secondary library; the single-chain secondary library is used for the next round of screening, screening and PCR amplification are repeated until the nucleic acid aptamer of interest is screened.

4. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to claim 3, wherein, prior to injection, the PBS buffer solution is run until the instrument reaches a stable signal baseline.

5. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to claim 3, wherein, the flow rate of injection is 20 L/min, and the time is 5 min.

6. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to claim 3, wherein, the flow rate of the PBS buffer solution during washing is 150 L/min, and the time is 5 min.

7. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to claim 3, wherein, the flow rate of the NaOH regeneration buffer solution during elution is 20 L/min.

8. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to any one of claims 3 to 7, wherein, the concentration of the NaOH regeneration buffer solution is 10 mM.

9. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to any one of claims 3 to 7, wherein, the concentration of the PBS buffer solution is 10 mM.

10. The nucleic acid aptamer screening method based on the localized surface plasmon resonance technology according to claim 1, wherein, the screened nucleic acid aptamer is used for quantitative and qualitative detection of the target.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1: results of affinity identification for aptamers and targets screened by LSPR-SELEX. Wherein, the ordinate represents the signal value detected by the sensor; the abscissa represents the time of interaction of the sample in the sensor.

[0021] Graph A shows the results of affinity identification for aptamers and targets screened in the first round. In the figure, the concentration of nucleic acid aptamers corresponding to the curves from top to bottom are gradually reduced (6 M, 3 M, 1.5 M, 0.75 M, and 0.375 M).

[0022] Graph B shows the results of affinity identification for aptamers and targets screened in the second round. In the figure, the concentration of the nucleic acid aptamers corresponding to the curves from top to bottom are gradually decreased (10 M, 5 M, 2.5 M, 1.25 M, and 0.625 M).

[0023] FIG. 2: shows the spatial structure of SBA.

[0024] FIG. 3: CE-SELEX characterization of SBA aptamers.

[0025] A: electrophoretic migration of SA (250 g/mL); B: electrophoretic migration of SBA (150 g/mL); C: electrophoretic migration of SBA (75 g/mL)+SA (125 g/mL); D: electrophoretic migration of BSA (250 g/mL); and E: electrophoretic migration of BSA (125 g/mL)+SBA (75 g/mL).

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

[0026] Specific embodiments of the present invention will be further described in detail below with reference to the Examples.

Example 1: Screening Method of LSPR-SELEX

[0027] (1) An oligonucleotide library (a fixed sequence containing 20 bp of upstream primers and 20 bp of downstream primers on both sides, and a random sequence of 80 nt in the middle) was centrifuged at high speed for 1 min. The result was further diluted with ddH.sub.2O to a 100 M stock solution. The oligonucleotide library was heat denatured (95 C. for 10 min) to destroy the polymer formed between the nucleic acid molecules, and subjected to ice bath for 5 min to avoid polymerization of the nucleic acid. The solution used in the test was filtered using a 0.22 m microporous filter before use.

[0028] (2) Experimental instruments: Open-SPR (Nicoya, Ca), using a nano-gold chip coated using streptavidin (SA), and 10 mM of PBS buffer solution was run at a flow rate of 150 L/min (4 g of NaCl, 1.45 g of Na.sub.2HPO.sub.4.12H.sub.2O, 0.1 g of KCl, 0.1 g of KH.sub.2PO4, dilute with ddH.sub.2O to a volume of 0.5 L, pH 7.4), until a stable signal baseline was reached.

[0029] (3) 100 M oligonucleotide library stock solution was diluted with PBS buffer solution to a final concentration of 10 M as a sample. Firstly, 250 L of the oligonucleotide sample was injected at a flow rate of 20 L/min, and interacted with the sensor for 5 min. Once at the end of the interaction, the PBS buffer solution was immediately introduced at a flow rate of 150 L/min so as to wash the system at a high-speed for 5 min, and thereby removing the weakly bound or unbound oligonucleotide molecules; then the injection system is washed with water and evacuated with air to prevent the sample from adsorbing on the inner wall of the sample tube; finally, 250 L of NaOH regeneration buffer solution (weighing 0.4 g of NaOH, and diluting with ddH.sub.2O to a volume of 1 L) was injected, and interacted with the sensor chip for 5 min at a low flow rate of 20 L/min, and the nucleotide molecule that specifically binds to a target was eluted and recovered.

Example 2: PCR Amplification and Preparation of Single Strands

[0030] (1) The nucleotide molecule recovered by elution was used as a secondary library for PCR enrichment. The primer sequence was: P1: 5-TTGACTTGCCACTGACTACC-3 (SEQ ID NO. 1), P2: 5-GATGACGACCGACTGACTTC-3 (SEQ ID NO. 2). The reaction system and procedure for optimizing PCR were as follows: 50 L reaction systems were as follows: 2 L of 20 M P1, 2 L of 20 M P2, 25 L of 2Taq Master Mix, and 21 L of secondary library; PCR procedure was as follows: initial denaturation at 95 C. for 5 min; denaturation at 94 C. for 30 s, annealing at 61.7 C. for 30 s, and extending at 72 C. for 30 s (30 cycles); finally extending at 72 C. for 2 min, and storing at 4 C. The amplified product was identified by 2% agarose gel electrophoresis, and the purification was carried out after correct identification, and the product was used as a template for preparing a single-chain secondary library.

[0031] (2) Optimize the asymmetric PCR for preparing single-stranded secondary library. 50 L of reaction system were as follows: 4 L of 20 M P2, 25 L of 2Taq Master Mix, 19 L of ddH.sub.2O, and 2 L of template; PCR procedure was as follows: initial denaturation at 95 C. for 5 min; denaturation at 94 C. for 30 s, annealing at 61.7 C. for 30 s, and extending at 72 C. for 30 s (35 cycles); finally extending at 72 C. for 2 min, and storing at 4 C. The amplified product was electrophoresed by 2% agarose gel at 110 V for 30 min, and the PCR product was purified after correct identification, and the result was used for the next round of screening. At the same time, the PCR product was purified and renatured under the same conditions. After each round of screening, the TraceDrawer data processing and analysis software was used to calculate the KD.

[0032] The results showed that: KD value of the nucleic acid aptamers and target screened at the first round of LSPR-SELEX was 107 M (see FIG. 1A). KD value of the nucleic acid aptamers and target screened at the second round of LSPR-SELEX was 98 M (see FIG. 1B). This shows that a nucleic acid aptamer with an affinity of 98 M can be obtained by only two rounds of screening by using LSPR-SELEX technique.

Example 3: Cloning and Sequencing

[0033] The oligonucleotide sequence obtained by the second round of LSPR-SELEX screening was ligated to the T vector for cloning and sequencing, and thereby obtaining aptamer clones of different sequences. Finally, the sequence having the highest affinity and strongest specificity was used as the ideal aptamer for study, and was named as SBA (sequencing results were shown in SEQ ID NO. 3). The results were shown in FIG. 2.

Example 4: Characterization by Capillary Electrophoresis (CE)

[0034] (1) The LSPR-SELEX process was characterized by capillary electrophoresis. Experimental instruments: G7100A (Agilent Technologies, USA); fused silica capillary with an inner diameter of 50 M, and a length of 56 cm; buffer solution for running and sample diluent were PBS buffer solution (8.5 g of NaCl, 2.2 g of Na.sub.2HPO.sub.4, 0.1 g of NaH.sub.2PO.sub.4, dilute with ddH.sub.2O to a volume of 1 L, pH 7.6)

[0035] (2) Before use, 1M NaOH (weighing 4 g NaOH, and diluting with ddH.sub.2O to a volume of 0.1 L), and ddH.sub.2O were used for washing for 5 min and 20 min successively, then the PBS buffer solution was run once to check the baseline. After the baseline was stable, the samples were separated.

[0036] (3) Not less than 200 L per sample, SA, SBA, combination of SA and SBA (incubation at room temperature for 30 min), BSA, combination of BSA and SBA (incubation at room temperature for 30 min) were loaded respectively, the injection pressure was 50 mbar10 s, separation voltage was 20 KV; and samples were separated for 20 min.

[0037] The results of characterization by capillary electrophoresis showed that there was a complex peak after SA+SBA mixed incubation, indicating the interaction between SA and SBA (see FIG. 3A-C). There was no complex peak after BSA+SBA mixed incubation in the control group, indicating that BSA and SBA do not react each other (see FIGS. 3B and D-E).