A method for screening nucleic acid aptamers using lanthanide metal-induced in situ phase separation and its application

Abstract

The present invention discloses a method and application for screening nucleic acid aptamers by inducing in situ phase separation using lanthanide metals, and relates to the technical field of nucleic acid aptamer screening. 3+ The aptamers in the solution were effectively separated from the random single-stranded DNA library by phase separation, and the PS-SELEX method was successfully developed, which effectively improved the library fixation and efficiency in the aptamer screening process. 2+ and Fe 2+ The random single-stranded DNA library was used as the research object by PS-SELEX technology. 3+ The aptamer library was separated in situ by self-assembly into nanoparticles, and multiple rounds of forward screening and reverse screening of the corresponding interfering ions were performed to obtain aptamers that could specifically recognize Cd 2+ and Fe 2+ The two nucleic acid aptamer sequences provide a theoretical basis for the efficient screening of high-affinity nucleic acid aptamers.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid aptamer screening, and in particular to a nucleic acid aptamer screening method and application utilizing lanthanide metal-induced in-situ phase separation. Background Art

[0002] Aptamers are oligonucleotide fragments with targeted effects, primarily screened in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) technique. Due to their high affinity, ease of modification, and low immunogenicity, they have become an important alternative to antibodies. However, the major challenge currently hindering their application is the identification of aptamers with high affinity and selectivity.

[0003] Traditional screening methods, such as magnetic beads, rely on target immobilization, requiring complex modification of small molecules (especially metal ions) for attachment to the beads. This difficulty in target immobilization can lead to distortion of target structure and increased screening bias. Furthermore, random libraries may nonspecifically bind to the stationary phase through hydrophobic interactions and electrostatic adsorption, necessitating repeated washing to reduce background noise and resulting in loss of aptamer sequences. To circumvent this challenge of target immobilization, improved methods have been developed in recent years, focusing on library immobilization. The GO-SELEX technique, which utilizes graphene oxide (GO) to screen for differential π-π adsorption of ssDNA, has been developed. However, GO has significantly weaker adsorption capacity for double-stranded DNA. Localized duplex formation on stem-loop-rich ssDNA can reduce adsorption efficiency. Furthermore, the uneven oxidation degree and number of GO layers can lead to batch-to-batch fluctuations in adsorption performance. Therefore, a simple, stable, and efficient high-throughput aptamer screening method is urgently needed.

[0004] Metal ion coordination chemistry with DNA offers a novel and promising strategy for the design and synthesis of functional DNA nanomaterials. Direct coordination-driven self-assembly allows for the precise synthesis of metal-DNA nanostructures with tunable and controllable properties. Lanthanide metals, due to their high coordination numbers and flexibility, are considered excellent ligands for self-assembly with nucleic acids, with applications in drug delivery, bioimaging, and enhancing enzyme reactions. However, there is currently no precedent for utilizing this property of lanthanide metals with DNA for the screening of nucleic acid aptamers. Summary of the Invention

[0005] In view of the technical problems existing in the above existing research and technology, the purpose of the present invention is to provide a nucleic acid aptamer screening method using lanthanide metal induced in situ phase separation (denoted as PS-SELEX), specifically involving a lanthanide metal ion (such as Gd 3+ ) and nucleic acid coordination self-assembly to achieve rapid library immobilization, combined with the ion competition effect, to efficiently screen high-affinity and high-specificity nucleic acid aptamers.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The first object of the present invention is to provide a method for screening nucleic acid aptamers using lanthanide metal-induced in situ phase separation, comprising the following steps:

[0008] (1) Synthesize random single-stranded DNA library and primer sequences;

[0009] (2) Incubating the random single-stranded DNA library with lanthanide metal ions to form lanthanide metal-nucleic acid aptamer self-assembled nanoparticles, centrifuging to obtain the precipitate, and washing the formed self-assembled nanoparticles to remove excess lanthanide metal ions;

[0010] (3) Positive screening: incubate lanthanide metal-nucleic acid aptamer self-assembled nanoparticles with target metal ions and recover the library that can bind to the target metal ions;

[0011] (4) Using the recovered library as a template, PCR amplification is performed using primer sequences to obtain PCR amplification products;

[0012] (5) Prepare a secondary library from the PCR amplification products, use the secondary library as a screening library, and perform multiple rounds of screening according to the process from step (2) to step (5);

[0013] (6) Introducing interfering metal ions for counter-screening during a single round of multi-round screening;

[0014] (7) After multiple rounds of screening, the secondary library of the final round is subjected to high-throughput sequencing analysis, and the sequences are selected for affinity verification. Based on the affinity verification results, nucleic acid aptamer sequences with high affinity for the target metal ions are screened.

[0015] As a further optimization solution of the present invention, the lanthanide metal ion is Gd 3+ , using nucleic acid library and Gd 3+ Self-assembly formed nanoparticles (denoted as Gd-DNA nanoparticles), realizing in situ phase separation of the nucleic acid aptamer library. The diameter of the formed Gd-DNA nanoparticles was tens of nanometers.

[0016] As a further optimization scheme of the present invention, the interfering metal ions are a mixture of other metal ions other than the target metal ions. Further, the screening of Cd 2+ Aptamers and Fe 2+ When using nucleic acid aptamers, interfering metal ions include Mn 2 + 、Cu 2+ , Ca 2+ 、Al 3+ 、Ba 2+ 、Cd 2+ 、Ni 2+ 、Zn 2+ 、Ce 3+ , Pb 2+ Cr 3+ 、Fe 3+ and Hg 2+ mixture.

[0017] As a further optimization solution of the present invention, the target metal ion is Cd 2+ , screening Cd 2+ The sequence of the random single-stranded DNA library of nucleic acid aptamers is 5'-ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACTA-3', N 40 It represents a sequence composed of 40 random nucleotide bases. During screening, the 5' end of the random single-stranded DNA library sequence was further modified with a FAM fluorescent group to screen for Cd 2+ The primer sequences of the nucleic acid aptamer include a forward primer: 5'-ATGTCTACTAGCGTAGCATC-3' and a reverse primer: 5'-TAGTCCATGTAACGGTACGA-3'. During screening, the 5' end of the forward primer is further modified with a FAM fluorescent group, and the 5' end of the reverse primer is further modified with biotin.

[0018] As a further optimization scheme of the present invention, the target metal ion is Fe 2+ , screening Fe 2+ The sequence of the random single-stranded DNA library of nucleic acid aptamers is 5'-GACGTGATCTGACTCAAGCT-N 40 -GATTCGACAGGCTCTAGAGT-3', N 40 It represents a sequence composed of 40 random nucleotide bases. During screening, the 5' end of the random single-stranded DNA library sequence was further modified with a FAM fluorescent group to screen Fe 2+The primer sequences of the nucleic acid aptamer include a forward primer: 5'-GACGTGATCTGACTCAAGCT-3' and a reverse primer: 5'-ACTCTAGAGCCTGTCGAATC-3'. During screening, the 5' end of the forward primer is further modified with a FAM fluorescent group, and the 5' end of the reverse primer is further modified with biotin.

[0019] As a further optimization scheme of the present invention, screening Cd 2+ In the case of nucleic acid aptamers, the number of rounds of multi-round screening was 9, and the rounds for counter-screening with the introduction of interfering metal ions were the 4th, 6th and 8th rounds.

[0020] As a further optimization scheme of the present invention, screening Fe 2+ In the case of nucleic acid aptamers, the number of rounds of multi-round screening was 10, and the rounds for counter-screening with the introduction of interfering metal ions were the 4th, 6th and 9th rounds.

[0021] The second object of the present invention is to provide an application of any of the above-mentioned screening methods in screening metal ion nucleic acid aptamers.

[0022] As a further optimization solution of the present invention, the metal ion nucleic acid aptamer is Cd 2+ Nucleic acid aptamer, the Cd 2+ The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.11, and the Cd 2+ Aptamer and Cd 2+ The dissociation constant K of the binding d It is 141μM.

[0023] As a further optimization solution of the present invention, the metal ion nucleic acid aptamer is Fe 2+ Nucleic acid aptamer, the Fe 2+ The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.12, and the Fc 2+ Aptamers and Fe 2+ The dissociation constant K of the binding d It is 91μM.

[0024] The third object of the present invention is to provide a use of the metal ion nucleic acid aptamer as described in any one of the above items in the preparation of a product that specifically recognizes the corresponding target metal ion.

[0025] The present invention has the following beneficial effects:

[0026] The present invention utilizes lanthanide metal ions Gd 3+The nucleic acid aptamers in the solution were effectively separated from the random single-stranded DNA library through phase separation (assembled into Gd-DNA nanoparticles), and the PS-SELEX method was successfully developed. This effectively improved the library fixation and efficiency during the nucleic acid aptamer screening process, providing a new solution for the efficient screening of nucleic acid aptamers.

[0027] Furthermore, the present invention uses metal ion Cd 2+ and Fe 2+ As the research object, Gd 3+ PS-SELEX technology is carried out in the presence of Gd 3+ The aptamer library was self-assembled into nanoparticles to achieve in situ phase separation. After multiple rounds of forward screening and reverse screening of the corresponding interfering ions, the aptamers that could specifically recognize Cd 2+ and Fe 2+ The two high-affinity nucleic acid aptamer sequences are screened by the above screening method, which provides an application basis for preparing products that specifically recognize corresponding target metal ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a SEM characterization image of the Gd-DNA nanoparticles provided in Example 1;

[0029] Figure 2 TEM characterization image of the Gd-DNA nanoparticles provided in Example 1;

[0030] Figure 3 This is a HAADF-STEM characterization image of the Gd-DNA nanoparticles provided in Example 1;

[0031] Figure 4 The adsorption isotherms of the interaction between GO and ssDNA of different structures provided in Example 2;

[0032] Figure 5 The kinetic curves of the interaction between GO and ssDNA of different structures provided in Example 2;

[0033] Figure 6 is the Gd provided in Example 2 3+ Adsorption isotherms of self-assembly with ssDNA of different structures;

[0034] Figure 7 is the Gd provided in Example 2 3+ Kinetic curves of self-assembly with ssDNA of different structures;

[0035] Figure 8 This is a schematic diagram of the screening process provided in Example 3;

[0036] Figure 9 The Cd-1 and Cd 2+ Affinity determination of

[0037] Figure 10 The secondary structure prediction of Cd-1 provided in Example 3;

[0038] Figure 11 The Fe-1 and Fe 2+ Affinity determination of

[0039] Figure 12 The secondary structure prediction of Fe-1 provided in Example 3;

[0040] Figure 13 The cDNA length and ratio optimization provided in Example 4, in the figure, ab corresponds to Cd 2+ Ion detection; cd corresponds to Fe 2+ Ion detection;

[0041] Figure 14 The Cd-1 and different Cd 2+ Fluorescence detection curves at different concentrations;

[0042] Figure 15 The Fe-1 and different Fe 2+ Fluorescence detection curves at different concentrations;

[0043] Figure 16 The selected Cd provided in Example 4 2+ Validation of aptamer specificity;

[0044] Figure 17 The selected Fe provided in Example 4 2+ Aptamer specificity test. DETAILED DESCRIPTION

[0045] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0046] Unless otherwise specified, all materials and reagents used in the following examples can be obtained from commercial sources.

[0047] Example 1: Using Gd 3+ Self-assembly of ions and DNA to form nanoparticles

[0048] This embodiment provides the use of Gd 3+ The method of self-assembly of ions and DNA to form nanoparticles and characterize the morphology of the nanoparticles is as follows:

[0049] 1. Preparation of Gd-DNA Nanoparticles

[0050] Synthesize a DNA library with a sequence length of 80 nt, the sequence of the DNA library is ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACT (SEQ ID NO. 5), 300 nmol Gd 3+ Mix with 100 pmol ssDNA library in 100 μL binding buffer (Gd 3+ Gd-DNA nanoparticles were prepared by incubating Gd-DNA with a molar ratio of 3000:1 to ssDNA. The binding buffer contained 50 mM HEPES, 100 mM NaCl, 2 mM MgCl2, and 0.025% Tween-20, adjusted to pH 7.5. After incubation at room temperature for 3 h, the Gd-DNA nanoparticles were washed several times to remove excess Gd. 3+ , which were subsequently used for further characterization.

[0051] 2. Characterization of Gd-DNA Nanoparticles

[0052] like Figure 1 and Figure 2 As shown in the figure, the morphology of the Gd-DNA nanoparticles, which are about tens of nanometers in size, was characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Energy dispersive X-ray spectroscopy (EDX) elemental mapping confirmed the uniform distribution of phosphorus (P) and nitrogen (N) from the DNA library and gadolinium (Gd) within the nanoparticles, as shown in the figure. Figure 3 shown.

[0053] Example 2, Gd 3+ Comparison of DNA immobilization efficiency between induced DNA self-assembly and GO-mediated

[0054] This example provides a method for systematically comparing the performance of PS-SELEX and GO-SELEX, comparing the immobilization efficiency of the two methods for DNA libraries with different secondary structures, specifically comprising the following steps:

[0055] 1. Synthesis of DNA libraries with different secondary structures

[0056] Four DNA libraries with 80-nt length but different secondary structures were synthesized, as shown in Table 1.

[0057] Table 1. Sequence information of DNA libraries with different secondary structures

[0058] ;

[0059] From structure 1 to structure 4, the bases gradually form complementary pairs along the nucleotide chain, and the hybridization process significantly increases the rigidity of the DNA structure.

[0060] 2. Test GO and Gd 3+ Adsorption capacity of ssDNA with different structures

[0061] GO and Gd were further evaluated 3+ Adsorption capacity for 100 pmol of DNA chains with different secondary structures. These two reagents were used to bind to the DNA library at different concentrations and exposure times. After incubation at room temperature, the supernatant was centrifuged to separate the unbound DNA chains. Subsequently, 1× SYBR Gold dye was introduced, and the fluorescence intensity (F) and initial value (F0) were measured. The adsorption capacity was calculated using the following formula: Adsorption capacity = (F0-F) / F0×100%. The test results are shown in Figure 2. Figure 4-7 shown.

[0062] As the bases gradually form complementary pairs along the nucleotide chain, the hybridization process significantly increases the rigidity of the DNA structure. The enhanced rigidity reduces the adsorption capacity of GO on the DNA chain from 99.8% to 69.6% ( Figure 5 ), which is due to the reduced availability of flexible sites for π-π stacking interactions, leading to biased adsorption and separation of enriched sequences during GO-SELEX. In contrast, due to a different mechanism of immobilization, Gd 3+ The self-assembly efficiency with DNA chains is not affected by the secondary structure of the nucleic acid chains ( Figure 6 ).

[0063] By Gd 3+ Adsorption isotherm curves of various immobilized DNA chains ( Figure 7 ) showed that all chains achieved near-complete adsorption at higher concentrations, although the adsorption efficiency decreased at lower Gd 3+ The concentration of GO changes slightly, and this behavior is significantly different from the adsorption curve of GO to nucleic acid ( Figure 4 ). Gd 3+ The kinetic curves of Gd-mediated DNA chain immobilization demonstrate the rapid and efficient binding of different DNA chains in just a few minutes. These results confirm that Gd-mediated DNA immobilization is more efficient than conventional substrates such as magnetic beads or graphene. 3+ Fixed pools are significantly more efficient and exhibit less batch variation.

[0064] Example 3: Using Gd 3+ Induced in situ phase separation screening of Cd 2+ions and Fe 2+ Ionic aptamers

[0065] This embodiment provides the use of Gd 3+ Induced in situ phase separation screening of Cd 2+ ions and Fe 2+ The method of ionic nucleic acid aptamer, the screening process diagram is as follows Figure 8 As shown, the specific steps include:

[0066] 1. Synthesis of random single-stranded DNA library and primer sequences

[0067] The random single-stranded DNA library is 80 bp in length, including 20 bp of fixed sequence bases at both ends and 40 bp of random sequence bases in the middle. When performing the following screening method, the 5' end of the random single-stranded DNA library sequence is further modified with a FAM fluorescent group.

[0068] (1) For screening Cd 2+ Aptamer: Initial library: 5'-ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACTA-3' (denoted as library-1, SEQ ID NO.5), where N 40 It represents a sequence consisting of 40 random nucleotide bases.

[0069] The primer sequences include a forward primer: 5'-ATGTCTACTAGCGTAGCATC-3' (SEQ ID NO. 6); and a reverse primer: 5'-TAGTCCATGTAACGGTACGA-3' (SEQ ID NO. 7). When performing the following screening method, the 5' end of the forward primer is further modified with a FAM fluorescent group, and the 5' end of the reverse primer is further modified with biotin.

[0070] (2) For screening Fe 2+ Aptamer: Initial library: 5'-GACGTGATCTGACTCAAGCT-N 40 -GATTCGACAGGCTCTAGAGT-3' (denoted as library-2, SEQ ID NO.8), where N 40 It represents a sequence consisting of 40 random nucleotide bases.

[0071] The primer sequences include a forward primer: 5'-GACGTGATCTGACTCAAGCT-3' (SEQ ID NO. 9); and a reverse primer: 5'-ACTCTAGAGCCTGTCGAATC-3' (SEQ ID NO. 10). When performing the following screening method, the 5' end of the forward primer is further modified with a FAM fluorescent group, and the 5' end of the reverse primer is further modified with biotin.

[0072] 2. First round of screening

[0073] 2.1. Preparation of Gd-DNA Nanoparticles

[0074] 500 pmol of DNA library and 300 nmol of Gd 3+ Dissolved in binding buffer (50 mM HEPES, 100 mM NaCl, 2 mM Mg 2+ , 0.025% Tween-20, pH 7.5), and incubated with rotation at room temperature for 60 min to obtain Gd-DNA self-assembled nanoparticles.

[0075] 2.2. Positive screening

[0076] Add 500 nmol Cd to the Gd-DNA nanoparticle suspension formed in step 2.1. 2+ ions or Fe 2 + ions, and incubate with rotation at room temperature for 60 min to allow Cd 2+ ions or Fe 2+ Ions compete for binding to Cd from Gd-DNA self-assembled nanoparticles 2+ ions or Fe 2+ The nucleic acid aptamer with affinity to Cd was centrifuged at 15000 rpm for 10 min and the supernatant was retained to obtain the nucleic acid aptamer that can bind to Cd. 2+ ions or Fe 2+ ions can bind to the library.

[0077] 2.3 PCR amplification

[0078] The library in the supernatant obtained in step 2.2 was used as a template for PCR amplification in a 50 μL PCR system (including 1 μL of the recovered secondary library, 25 μL of 2× TaqPCR Mix, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, and 50 μL of DEPC HO). Amplification conditions were as follows: 94°C denaturation for 4 min, 94°C denaturation for 30 s, 50°C annealing for 30 s, and 72°C extension for 60 s, for a total of 20 cycles, followed by 72°C annealing for 3 min, and a 4°C incubation period.

[0079] 2.4 Secondary Library Preparation

[0080] Pipette 50 μL of streptavidin magnetic beads onto a magnetic rack and let stand for 1 minute. Remove the supernatant and add 200 μL of 1× PBS buffer (containing 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na₂HPO₄, 1.76 mM KH₂PO₄, and 1 mg / mL BSA) to a centrifuge tube. Incubate with rotation for 60 minutes and then separate magnetically. Wash three times with 200 μL of 1× PBS buffer. Add the PCR product prepared in step 2.3 and one-third of the volume of 4 M NaCl solution. Place the centrifuge tube on a rotating mixer and incubate at room temperature with shaking for 60 minutes. Return the centrifuge tube to the magnetic rack and let stand for 1 minute. Remove the supernatant and wash the beads three times with 200 μL of 1× PBS buffer. Add 30 μL of 0.2 M NaOH solution and mix for 1 minute to melt the double-stranded DNA. Place the centrifuge tube on a magnetic stand and let it stand for 1 minute. Pipette the supernatant into a new centrifuge tube, add hydrochloric acid to adjust the pH of the library to neutral, and use it as a secondary library for the next round of screening, and measure its concentration.

[0081] 3. Counter-screening

[0082] In the first round of screening, Cd 2+ ions or Fe 2+ The difference between incubation with Gd-DNA nanoparticles is that when counter-screening, interfering ions (including Mn 2+ 、Cu 2+ , Ca 2+ 、Al 3+ 、Ba 2+ 、Ni 2+ 、Zn 2 + 、Ce 3+ , Pb 2+ Cr 3+ 、Fe 3+ and Hg 2+ The mixture was incubated with the library for 60 min, and the supernatant was removed by centrifugation to remove the library bound to the interfering substances. The remaining Gd-DNA nanoparticles were continued to the positive screening step.

[0083] 4. Multiple rounds of screening

[0084] (1) For Cd 2+ Ionic aptamer screening:

[0085] The secondary library replaced the initial library in the first round of screening, and the screening process was repeated. The screening pressure was adjusted according to the recovery rate of each round of screening. Counter-screening was introduced in the 4th, 6th, and 8th rounds of screening. The specific screening conditions are shown in Table 2 below. The library from the 9th round was used for high-throughput sequencing.

[0086] (2) For Fe 2+ Ionic aptamer screening:

[0087] The secondary library replaced the initial library in the first round of screening, and the screening process was repeated. The screening pressure was adjusted according to the recovery rate of each round of screening. Counter-screening was introduced in the 4th, 6th, and 9th rounds of screening. The specific screening conditions are shown in Table 3 below. The library from the 10th round was used for high-throughput sequencing.

[0088] Table 2. Cd 2+ PS-SELEX screening conditions

[0089] ;

[0090] Table 3. Fe 2+ PS-SELEX screening conditions

[0091] ;

[0092] 5. Sequencing and sequence analysis

[0093] After the final round of screening, the enriched ssDNA library was sent for cloning and sequencing. Finally, we selected the sequence with the highest enrichment as the representative for subsequent characterization. The two sequences screened were:

[0094] Cd-1: 5'-ATGTCTACTAGCGTAGCATCCAACTGGATGCAGATTACAATCTCGAAAACTAACTATCATTTCGTACCGTTACATGGACTA-3' (SEQ ID NO. 11);

[0095] Fe-1: 5'-GACGTGATCTGACTCAAGCTTACCTCTTCAGTTCCTAGGTATTCTAATCTATTTCCTCCCGATTCGACAGGCTCTAGAGT-3' (SEQ ID NO. 12).

[0096] 6. Verification of affinity of aptamers screened by PS-SELEX for target ions

[0097] (1) For the screened Cd 2+ Aptamer to Cd 2+ Ion affinity verification:

[0098] ITC was used to quantitatively assess the binding affinity between the selected aptamers and the target metal ion. Titrations were performed using a MicroCal PEAQ-ITC system with 20 μM Cd-1 added to the reaction cell and 2 mM CdCl2 added to the syringe. Experiments were performed at a stirring speed of 750 rpm and maintained at 25°C. The titration experiment consisted of 19 injections, each delivering 2 μL of metal solution, except for the first injection, which was 0.4 μL. A 120-second interval was maintained between each injection to allow for complete thermal equilibrium. Data were integrated and analyzed using MicroCal PEAQ-ITC Analysis software.

[0099] The final result is as follows Figure 9 As shown, the dissociation constant Kd = 141 μM, the stoichiometric ratio of the reaction is about 8.25, and the enthalpy change ΔH is -4.48 kcal·mol -1 , the entropy change ΔS is 2.60 cal·mol -1 ·K -1 (Free energy change -TΔS = -0.775 kcal·mol -1 , 298K), using M-fold to simulate the secondary structure of Cd-1, as Figure 10 shown.

[0100] (2) For the screened Fe 2+ Aptamer to Fe 2+ Ion affinity verification:

[0101] ITC technology was used to quantitatively evaluate the binding affinity between the selected aptamers and the target metal ions. Titrations were performed using MicroCal PEAQ-ITC, with 50 μM Fe-1 added to the reaction cell and 5 mM FeCl2 added to the syringe. The experiments were performed at a stirring speed of 750 rpm and maintained at 25°C. The titration experiment consisted of 19 injections, each delivering 2 μL of metal solution, except for the first injection, which was 0.4 μL. An interval of 120 seconds was maintained between each injection to allow complete thermal equilibrium. The data were integrated and analyzed using MicroCal PEAQ-ITC Analysis software. The final results are shown in Figure 2. Figure 11 As shown, the dissociation constant Kd = 91 μM, the stoichiometric ratio of the reaction is about 7.31, and the enthalpy change ΔH is -0.351 kcal·mol -1 , the entropy change ΔS is 17.3 cal·mol -1 ·K -1 (Free energy change -TΔS = -5.16 kcal·mol -1 , 298K), using M-fold to simulate the secondary structure of Fe-1, such as Figure 12 shown.

[0102] Example 4: Using the screened aptamers to construct a specific detection system for Cd 2+ and Fe 2+ Standard shift fluorescence analysis

[0103] This example provides a method for constructing a specific detection system for Cd in solution by using the aptamers Cd-1 and Fe-1 obtained by screening. 2+ and Fe 2+ The method specifically comprises the following steps:

[0104] 1. Modify the aptamer and construct complementary chains of different lengths

[0105] The 5' ends of the two aptamers were modified with a FAM fluorescent group. In addition, complementary cDNA chains of 14-nt, 16-nt, 18-nt, 20-nt, and 22-nt lengths were constructed, and the 3' ends of the complementary cDNA chains of different lengths were modified with Dabcyl.

[0106] (1) For the Cd-1 aptamer, the complementary cDNA chain sequences of different lengths are shown in Table 4:

[0107] Table 4. Sequence information of complementary cDNA chains constructed for Cd-1 aptamers

[0108] ;

[0109] (2) For Fe-1 aptamers, the complementary cDNA chain sequences of different lengths are shown in Table 5:

[0110] Table 5. Sequence information of complementary cDNA chains constructed for Fe-1 aptamers

[0111] ;

[0112] 2. Optimized for Cd 2+ or Fe 2+ Length and amount of cDNA for fluorescence detection

[0113] 0.1 μM FAM-aptamer was mixed with target ions (Cd 2+ 100μM, Fe 2+ The cells were incubated with aptamers at a concentration of 60 μM for 30 minutes. Subsequently, Dabcyl-modified complementary chains of varying lengths (14, 16, 18, 20, and 22 nucleotides) were introduced at a concentration of 0.5 μM. Fluorescence intensity was measured after a 20-minute incubation. In addition to optimizing the length of the complementary chains, the present invention also optimizes the ratio of aptamers to their corresponding complementary chains.

[0114] Finally, the present invention determines the scheme used as follows Figure 13 As shown, for Cd 2+ ion detection, the optimal cDNA strand length was 20-nt, and the optimal ratio of quenching strand to FAM-aptamer was determined to be 5:1; 2+ ion detection, the optimal cDNA strand length was 18-nt, and the optimal quencher strand to FAM-aptamer ratio was determined to be 3:1.

[0115] 3. Detection of targets at different concentrations using standard shift fluorescence analysis

[0116] Using optimized conditions, FAM-labeled aptamers were incubated with varying concentrations of the target ion for 30 minutes. Fluorescence intensity (denoted as F) was then measured 20 minutes later in the presence of a quencher strand. Following hybridization of the aptamer to the complementary DNA strand, baseline fluorescence intensity was measured, denoted as F0. To determine the binding affinity and detection limit of the assay, the change in fluorescence intensity was calculated using the formula (F-F0) / F0. This value was plotted against the concentration of the target ion, allowing calculation of the Kd of the aptamer. Furthermore, based on this data, the detection limit of the assay was established, providing insight into the sensitivity and performance of the aptamer-based detection system for the target ion.

[0117] The final result is as follows Figure 14 and Figure 15 As shown in the figure, similar to ITC, the Kd values for Cd-1 and Fe-1 were 122 μM and 116 μM, respectively. The detection limits for both target ions were 0.3 μM.

[0118] 4. Specificity evaluation of detection methods

[0119] The FAM-labeled aptamer was exposed to 12 potential interfering ions, each at a concentration of 100 μM. Figure 16 and Figure 17 As shown in the figure, both Cd-1 and Fe-1 aptamers showed the highest fluorescence signals in the presence of their respective target ions, proving that they are sensitive to their target metal ions (i.e., Cd 2+ 、Fe 2+ ) with high specificity and affinity.

[0120] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for screening nucleic acid aptamers using lanthanide metal-induced in situ phase separation, characterized in that: The following steps are involved: (1) Synthesize random single-stranded DNA library and primer sequences; (2) Incubating the random single-stranded DNA library with lanthanide metal ions to form lanthanide metal-nucleic acid aptamer self-assembled nanoparticles, wherein the lanthanide metal ions are Gd 3+ ; (3) Positive screening: incubate lanthanide metal-nucleic acid aptamer self-assembled nanoparticles with target metal ions and recover the library that can bind to the target metal ions; (4) Using the recovered library as a template, PCR amplification is performed using primer sequences to obtain PCR amplification products; (5) Prepare a secondary library from the PCR amplification products, use the secondary library as a screening library, and perform multiple rounds of screening according to the process from step (2) to step (5); (6) Introducing interfering metal ions for counter-screening during a single round of multi-round screening; (7) After multiple rounds of screening, the secondary library of the final round is subjected to high-throughput sequencing analysis, and the sequences are selected for affinity verification. Based on the affinity verification results, nucleic acid aptamer sequences with high affinity for the target metal ions are screened.

2. The method for screening nucleic acid aptamers using lanthanide metal-induced in situ phase separation according to claim 1, characterized in that: The interfering metal ions are a mixture of other metal ions other than the target metal ions, and the other metal ions include Mn 2+ 、Cu 2+ , Ca 2+ 、Al 3+ 、Ba 2+ 、Cd 2+ 、Ni 2+ 、Zn 2+ 、Ce 3+ , Pb 2+ Cr 3+ 、Fe 3+ and Hg 2+ .

3. The method for screening nucleic acid aptamers using lanthanide metal-induced in situ phase separation according to claim 1, characterized in that: The target metal ion is Cd 2+ , screening Cd 2+ The sequence of the random single-stranded DNA library of nucleic acid aptamers is 5'-ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACTA-3', N 40 It represents a sequence consisting of 40 random nucleotide bases.

4. The method for screening nucleic acid aptamers using lanthanide metal-induced in-situ phase separation according to claim 1, characterized in that: The target metal ion is Fe 2+ , screening Fe 2+ The sequence of the random single-stranded DNA library of nucleic acid aptamers is 5'-GACGTGATCTGACTCAAGCT-N 40 -GATTCGACAGGCTCTAGAGT-3', N 40 It represents a sequence consisting of 40 random nucleotide bases.

5. Use of the screening method according to any one of claims 1 to 4 in screening metal ion aptamers.

6. A metal ion nucleic acid aptamer, characterized in that The metal ion nucleic acid aptamer is Cd 2+ Aptamer or Fe 2+ Nucleic acid aptamer, the Cd 2+ The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.11, and the Fc 2+ The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.

12.

7. Use of the metal ion nucleic acid aptamer according to claim 6 in preparing a product that specifically recognizes the corresponding target metal ion.

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