Magnetic field sensitive type metal ion nucleic acid aptamer as well as screening method and application thereof

Abstract

The invention discloses a magnetic field sensitive type metal ion nucleic acid aptamer as well as a screening method and application thereof, and relates to the technical field of nucleic acid aptamer screening. According to the invention, divalent cobalt ions with paramagnetism are taken as a research object, on the basis of compatibility of GO-SELEX, an HM-SELEX method is successfully developed in a 9Tesla steady-state strong magnetic field, efficient screening of magnetic sensitive metal ion aptamers is promoted, and nucleic acid aptamers with magnetic field adjustable binding characteristics are found for the first time, so that the method has the advantages of high efficiency, high sensitivity, high sensitivity and the like. The sequence of the cobalt ion nucleic acid aptamer is as shown in SEQ ID NO. 1-7. The invention also provides reference for the development of magnetic genetics, disease diagnosis and treatment and related sensors related to metal ions and magnetic fields, and fills the blank of such methods.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid aptamer screening, and in particular to a magnetic field-sensitive metal ion nucleic acid aptamer and a screening method and application thereof. Background Art

[0002] Magnetic fields are ubiquitous in nature. Existing research divides magnetic fields into steady-state magnetic fields and dynamic magnetic fields (alternating magnetic fields) according to how the magnetic field strength changes over time; and into weak magnetic fields (less than 1×10 - 3 Tesla), medium magnetic field (1×10 -3 To 1 Tesla), strong magnetic field (1 to 20 Tesla) and super strong magnetic field (above 20 Tesla). The earth we live on is itself a huge static weak magnet (magnetic field strength is about 5×10 -5 Tesla). The use of magnetic fields to modulate physical properties and chemical reactions is now widely used, but the effects of magnetic fields on biological processes remains an open and illuminating question.

[0003] The field of magnetogenetics is dedicated to the remote and non-invasive manipulation of neuronal functions. In 2015, Xie Can's team first published the magnetic responsive protein MagR, which sparked heated discussions in the scientific community. Although a large number of studies have explored the response of organisms to magnetic fields, another key dimension of this issue, the possibility of magnetic field-mediated regulation of biological recognition or affinity between receptors and their ligands, has rarely been reported.

[0004] Metal ions of the first transition series, including iron, cobalt, and nickel, often exhibit unusual behavior in magnetic fields due to their unpaired 3d orbital electrons. These metal ions are known to bind to proteins and are essential for sustaining life. They are cofactors for many enzymes, promoting their catalytic activity (e.g., vitamin B12), and are involved in the transport of important substances such as oxygen in organisms.

[0005] Compared to proteins, nucleic acids have been found to bind metal ions with greater affinity. This is due to the interaction between the metal cation and the negatively charged phosphate backbone, as well as coordination with the nucleobase donor atom. For example, some riboswitches can directly utilize Mn 2+ or Mg 2+Aptamers are widely used in the field of sequence-specific DNA-metal ion interactions due to their simple synthesis, ease of chemical modification, and cost-effectiveness compared to antibody production. The study of these metal ion-related aptamers is crucial for many applications such as disease diagnosis and treatment, catalyst engineering, and sensor device development. Summary of the invention

[0006] In view of the above existing research and technology, the purpose of the present invention is to provide a magnetic field-sensitive metal ion nucleic acid aptamer and a screening method and application thereof.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions: The first object of the present invention is to provide a method for screening magnetic field-sensitive metal ion nucleic acid aptamers, comprising the following steps: (1) Synthesize random single-stranded DNA library and primer sequences; (2) Performing denaturation treatment on the random single-stranded DNA library; (3) Forward screening, adding the target metal ion to the library that has undergone the renaturation treatment in step (2) and incubating under a steady-state strong magnetic field, and recovering the library that can bind to the target metal ion under a steady-state strong magnetic field; (4) using the primer sequences synthesized in step (1) and the library recovered in step (3) as a template, performing PCR amplification to obtain a PCR amplification product; (5) preparing a secondary library using the PCR amplification product obtained in step (4); (6) Using the secondary library prepared in step (5) as a screening library, and performing multiple rounds of screening according to the process from step (2) to step (5); (7) Introducing reverse screening in a single round of multi-round screening, the reverse screening specifically comprises: first, adding interfering metal ions to the secondary library obtained in step (5) for incubation under a steady-state strong magnetic field, removing the library that binds to the interfering metal ions under the steady-state strong magnetic field, then, continuing to add target metal ions for incubation under a normal magnetic field, removing the library that binds to the target metal ions under the normal magnetic field, and finally, continuing the retained library for the next round of screening; (8) After multiple rounds of screening, the secondary library of the final round is subjected to high-throughput sequencing analysis, and the selected sequences are subjected to affinity verification under a steady-state strong magnetic field and a normal geomagnetic field. Based on the affinity verification results, the sequences of magnetic field-sensitive metal ion nucleic acid aptamers are screened out.

[0008] As a further optimization scheme of the present invention, in step (1), the sequence of the random single-stranded DNA library is 5'-TACACTGCACTGCGTTAGAG-40N-ATGGACGTGTCACAGTACTG-3', wherein 40N represents a sequence formed by connecting 40 arbitrary nucleotide bases. Furthermore, a FAM fluorescent group is added to the 5' end of the sequence of the random single-stranded DNA library for modification.

[0009] As a further optimization scheme of the present invention, in step (1), the primer sequence is: Forward primer: 5′-TACACTGCACTGCGTTAGAG-3′; Reverse primer: 5′-CAGTACTGTGACACGTCCAT-3′.

[0010] Furthermore, a FAM fluorescent group was added to the 5' end of the forward primer sequence for modification, and biotin was added to the 5' end of the reverse primer sequence for modification.

[0011] As a further optimization scheme of the present invention, the magnetic field strength of the steady-state strong magnetic field is 9 Tesla, and the magnetic field strength of the normal geomagnetic field is 5×10 -5 Tesla.

[0012] As a further optimization scheme of the present invention, the number of the multiple rounds of screening is 7 rounds in total, and the rounds in which reverse screening is introduced are the 4th and 6th rounds.

[0013] As a further optimization scheme of the present invention, the target metal ion is Co 2+ 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+ .

[0014] The second object of the present invention is to provide an application of any of the above-mentioned screening methods in screening magnetic field-sensitive metal ion nucleic acid aptamers.

[0015] The third object of the present invention is to provide a magnetic field-sensitive metal ion nucleic acid aptamer obtained by screening according to any of the screening methods described above, wherein the affinity of the magnetic field-sensitive metal ion nucleic acid aptamer to metal ions increases with the increase of magnetic field intensity.

[0016] As a further optimization scheme of the present invention, the magnetic field-sensitive metal ion nucleic acid aptamer is a magnetic field-sensitive cobalt ion nucleic acid aptamer, and the cobalt ion nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NO.1-7. The further optimization is that the cobalt ion nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO.3 and SEQ ID NO.5. The further optimization is that the cobalt ion nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO.3.

[0017] The fourth object of the present invention is to provide an application of any of the magnetic field-sensitive metal ion nucleic acid aptamers described above, specifically an application in at least one of (A) to (C): (A) development of diagnostic tools or therapeutic tools for diseases; (B) Catalyst engineering; (C)Sensor equipment development.

[0018] The present invention has the following beneficial effects: The present invention takes paramagnetic divalent cobalt ions as the research object, adopts HM-SELEX technology (high magnetic field exponential enrichment ligand systematic evolution technology) under a steady-state strong magnetic field, and screens magnetic-sensitive nucleic acid aptamers that can specifically recognize cobalt ions and have significantly improved affinity under a steady-state strong magnetic field through multiple rounds of positive screening under a strong magnetic field with a magnetic field strength of 9 Tesla and reverse screening under corresponding interfering ions and a normal geomagnetic field. The affinity of the nucleic acid aptamer can be regulated by increasing the intensity of the magnetic field, which provides a reference for magnetogenetics, disease diagnosis and treatment, and sensor development related to metal ions and magnetic fields, and fills the gap in such methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the screening process of an embodiment of the present invention; Figure 2 is the recovery efficiency of the screening library of HM-SELEX; Figure 3A Prediction of the secondary structure of Co-M1 in Co-family-1; Figure 3B Prediction of the secondary structure of Co-M2 in Co-family-1; Figure 3C Prediction of the secondary structure of Co-M3 in Co-family-1; Figure 3D Prediction of the secondary structure of Co-M4 in Co-family-1; Figure 3E Prediction of the secondary structure of Co-M9 in Co-family-1; Figure 4 The secondary structure prediction of Co-family-3; where a is Co-M8 in Co-family-3; b is Co-M10 in Co-family-3; Figure 5 is the affinity change of Co-family-1 under a steady-state strong magnetic field; where a is Co-M3 in Co-family-1; b is Co-M1 in Co-family-1; c is Co-M2 in Co-family-1; d is Co-M4 in Co-family-1; e is Co-M9 in Co-family-1; Figure 6 is the affinity change of Co-family-3 under steady-state strong magnetic field; where a is Co-M8 in Co-family-3; b is Co-M10 in Co-family-3; Figure 7 For the selected Co 2+ Verification of the specificity of the aptamer; where a is Co-M3 and b is Co-M8. DETAILED DESCRIPTION

[0020] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here 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. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] In the following examples, if no specific experimental methods are specified, all experimental methods can be carried out according to conventional methods. The materials and reagents used can be obtained through commercial channels unless otherwise specified.

[0022] Example 1: Screening of divalent cobalt ion nucleic acid aptamers with magnetic field-regulated affinity under a steady-state strong magnetic field The present embodiment provides a method for screening divalent cobalt ion nucleic acid aptamers whose affinity is regulated by the magnetic field under a steady-state strong magnetic field. The screening process diagram is shown in FIG. Figure 1 As shown, the specific steps include: 1. Synthesize random single-stranded DNA library and primer sequences The total length of the random single-stranded DNA library is 80 bp, including 20 bp of fixed sequence bases at both ends and 40 bp of random sequence bases in the middle. The sequence information is: 5'-TACACTGCACTGCGTTAGAG-40N-ATGGACGTGTCACAGTACTG-3' (SEQID NO.8), where 40N represents a sequence formed by connecting 40 arbitrary nucleotide bases. 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.

[0023] The primer sequence includes a forward primer: 5'-TACACTGCACTGCGTTAGAG-3' (SEQ ID NO.9); and a reverse primer: 5'-CAGTACTGTGACACGTCCAT-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.

[0024] 2. First round of screening 2.1. Library renaturation 500 pmol of DNA library was dissolved in binding buffer (50 mM HEPES, 100 mM NaCl, 0.025% tween-20, pH 7.5), denatured at 95°C for 10 min, cooled on ice for 10 min, and then placed at ambient temperature for 10 min.

[0025] 2.2. Positive screening Add 500 nmol of Co to the single-stranded DNA library solution that has been renatured in step 2.1. 2+ ions, incubated for 60 min in a steady-state strong magnetic field with a magnetic field strength of 9 Tesla, added a single layer of graphene oxide to the system after the incubation to adsorb the unbound library, and centrifuged at 15,000 rpm for 10 min to retain the supernatant to obtain the Co 2+ A library of ions that can bind under a steady-state strong magnetic field.

[0026] 2.3. Calculation of screening recovery rate The concentration of single-stranded DNA in the recovered supernatant was measured by ultra-micro-volume nucleic acid spectrophotometer, and the screening recovery rate of each round was calculated (screening recovery rate = recovery amount / input amount), see Figure 2 .

[0027] 2.4 PCR amplification The library in the supernatant obtained in step 2.2 was used as a template for PCR amplification. The forward primer was 5'-FAM-TACACTGCACTGCGTTAGAG-3', the reverse primer was 5'-biotin-CAGTACTGTGACACGTCCAT-3', and the total PCR system was 50 μL (including 1 μL of the recovered secondary library, 25 μL of 2×TaqPCRMix, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, and DEPC H2O to make up to a final volume of 50 μL). The amplification conditions were 94°C pre-denaturation for 4 min, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 60 s, a total of 20 cycles, 72°C renaturation for 3 min, and 4°C insulation.

[0028] 2.5. Secondary library preparation Pipette 50 μL of streptavidin magnetic beads and place them on the magnetic stand. Let it 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, 1 mg / mL BSA) to the centrifuge tube. Rotate and incubate for 60 minutes. Separate magnetically. Wash three times with 200 μL of 1× PBS buffer. Add the PCR product prepared in step 2.4. Add one-third of the volume of 4M NaCl solution. Place the centrifuge tube on a rotating mixer and incubate at room temperature for 60 minutes. Place the centrifuge tube back on the magnetic stand and let it stand for 1 minute. Remove the supernatant and wash the magnetic beads three times with 200 μL of 1× PBS buffer. Add 30 μL of 0.2M NaOH solution and mix for 1 minute to melt the double-stranded DNA. Place the centrifuge tube on a magnetic rack and let it stand for 1 min. 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.

[0029] 3. Reverse screening When the screening recovery rate becomes stable, reverse screening is introduced. 2+ Different from the positive screening in which the library is incubated, in the reverse screening, we add interfering ions (including 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+ The mixture of ions (all ions used were 100 nmol) was incubated with the library for 60 min, and a single layer of graphene oxide was added to the incubated system, and the supernatant was removed by centrifugation to remove the library bound to the interference. - 5 Tesla) added Co to the graphene oxide recycled above 2+ The ions were incubated for another 60 min, and the supernatant was centrifuged after the incubation was completed to remove the library that could bind to the target under a normal ambient magnetic field, and the graphene precipitate was retained to continue the positive screening under a steady-state strong magnetic field.

[0030] 4. Multiple rounds of screening The secondary library was used to replace 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, and counter-screening was introduced in the 4th and 6th rounds of screening. The specific screening conditions are shown in Table 1 below. The 7th round library was used for high-throughput sequencing.

[0031] Table 1. Co 2+ HM-SELEX screening conditions ; 5. Sequencing and sequence analysis The sequences with the highest number of repetitions obtained by high-throughput sequencing were clustered and divided into different families according to their homology and secondary structures simulated by Mfold, including Co-family-1 and Co-family-3. The sequences contained in Co-family-1 and Co-family-3 were named, and the sequence information is shown in Table 2.

[0032] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E and Figure 4 They are the secondary structure predictions of the sequences contained in Co-family-1 and Co-family-3 respectively.

[0033] Table 2. Sequence information contained in Co-family-1 and Co-family-3 ; 6. Affinity verification with magnetic field regulation First, the relevant sequences of Co-family-1 (including Co-M1, Co-M2, Co-M3, Co-M4 and Co-M9) and Co-family-3 (including Co-M8 and Co-M10) were synthesized and labeled with FAM fluorescent group at the 5' end. The selected aptamers were heated at 95℃ for 10min, cooled on ice for 10min, and then placed at ambient temperature for 10min.

[0034] Then, the selected candidate aptamers were subjected to binding affinity analysis under normal geomagnetic field (NM) or stable strong magnetic field conditions, that is, 10 pmol of aptamer was combined with (0 μM, 100 μM, 200 μM, 300 μM, 400 μM) target ions (Co 2+ ions) were incubated in a 400 μL system under a normal magnetic field or a strong magnetic field for 30 min.

[0035] Finally, 0.2 mg of graphene oxide was added to the centrifuge tube and incubated for 30 min. After incubation, the tube was centrifuged at 15,000 rpm for 3 min, and the fluorescence intensity of the supernatant was measured. In each affinity test, the fluorescence intensity was normalized with the maximum fluorescence intensity observed as the reference value 1. The fitting curve and dissociation constant K were obtained by nonlinear regression analysis using Origin software. d value.

[0036] See also Figure 5 as well as Figure 6 , the affinity of Co-family-1 and Co-family-3 gradually increased with the increase of magnetic field strength. The affinity curve of Co-M3 of Co-family-1 continued to move to the left with the increase of magnetic field strength, and the affinity increased by about 2 to 3 times with the increase of magnetic field. Compared with the negligible affinity in a weak magnetic field environment, the sequence Co-M8 in Co-family-3 began to show an affinity of about 200μM when exposed to a high magnetic field of 6Tesla and above.

[0037] 7. Specificity Verification Specificity verification was also performed using 10 pmol of the selected aptamers (Co-M3 and Co-M8 as representative sequences of the two families) and the target ion (300 μM Co 2+ ions) and 10 times the amount of interfering ions (including Mn 2+ , Cu 2+ , Ca 2+ 、Al 3+ , Ba 2+ 、Cd 2+ 、Ni 2+ 、Zn 2+ 、Ce3+ , Pb 2+ Cr 3+ , Fe 3+ and Hg 2+ ) were incubated in a 9 Tesla steady-state strong magnetic field and a normal geomagnetic field (5×10 -5 Tesla) conditions, after incubation for 30 min, 0.2 mg of graphene oxide was added to the system and incubated for another 30 min. The mixture was centrifuged and the fluorescence intensity of the supernatant was measured. The maximum fluorescence intensity observed was used as the reference value 1 for normalization.

[0038] Specificity tests have shown that Figure 7 As shown in Figure 2, Co-M3 has remarkable selectivity in both steady-state strong magnetic field and normal geomagnetic field. In contrast, Co-M8 only exhibits excellent selectivity under steady-state strong magnetic field conditions. These findings further verify the effectiveness of the selection strategy we adopted.

[0039] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for screening magnetic field-sensitive metal ion nucleic acid aptamers, characterized in that: The following steps are involved: (1) Synthesize random single-stranded DNA library and primer sequences; (2) Performing denaturation treatment on the random single-stranded DNA library; (3) Forward screening, adding the target metal ion to the library that has undergone the renaturation treatment in step (2) and incubating under a steady-state strong magnetic field, and recovering the library that can bind to the target metal ion under a steady-state strong magnetic field; (4) using the primer sequences synthesized in step (1) and the library recovered in step (3) as a template, performing PCR amplification to obtain a PCR amplification product; (5) preparing a secondary library using the PCR amplification product obtained in step (4); (6) Using the secondary library prepared in step (5) as a screening library, and performing multiple rounds of screening according to the process from step (2) to step (5); (7) Introducing reverse screening in a single round of multi-round screening, the reverse screening specifically comprises: first, adding interfering metal ions to the secondary library obtained in step (5) for incubation under a steady-state strong magnetic field, removing the library that binds to the interfering metal ions under the steady-state strong magnetic field, then, continuing to add target metal ions for incubation under a normal magnetic field, removing the library that binds to the target metal ions under the normal magnetic field, and finally, continuing the retained library for the next round of screening; (8) After multiple rounds of screening, the secondary library of the final round is subjected to high-throughput sequencing analysis, and the selected sequences are subjected to affinity verification under a steady-state strong magnetic field and a normal geomagnetic field. Based on the affinity verification results, the sequences of magnetic field-sensitive metal ion nucleic acid aptamers are screened out.

2. The method for screening magnetic field-sensitive metal ion nucleic acid aptamers according to claim 1, characterized in that: In step (1), the sequence of the random single-stranded DNA library is 5'-TACACTGCACTGCGTTAGAG-40N-ATGGACGTGTCACAGTACTG-3', wherein 40N represents a sequence formed by connecting 40 arbitrary nucleotide bases.

3. The method for screening magnetic field-sensitive metal ion nucleic acid aptamers according to claim 1, characterized in that: In step (1), the primer sequence is: Forward primer: 5′-TACACTGCACTGCGTTAGAG-3′; Reverse primer: 5′-CAGTACTGTGACACGTCCAT-3′.

4. The method for screening magnetic field-sensitive metal ion nucleic acid aptamers according to claim 1, characterized in that: The magnetic field strength of the steady-state strong magnetic field is 9 Tesla, and the magnetic field strength of the normal geomagnetic field is 5×10 -5 Tesla.

5. The method for screening magnetic field-sensitive metal ion nucleic acid aptamers according to claim 1, characterized in that: The number of rounds of the multiple screenings is 7 in total, and the rounds in which reverse screening is introduced are the 4th and 6th rounds.

6. The method for screening magnetic field-sensitive metal ion nucleic acid aptamers according to claim 1, characterized in that: The target metal ion is Co 2+ 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+ .

7. Use of the screening method according to any one of claims 1 to 6 in screening magnetic field-sensitive metal ion nucleic acid aptamers.

8. A magnetic field-sensitive metal ion nucleic acid aptamer, characterized in that: The magnetic field-sensitive metal ion nucleic acid aptamer is obtained by screening according to any one of the screening methods described in claims 1 to 6, and the affinity of the magnetic field-sensitive metal ion nucleic acid aptamer to the metal ion increases with the increase of the magnetic field intensity.

9. The magnetic field-sensitive metal ion nucleic acid aptamer according to claim 8, characterized in that: The magnetic field-sensitive metal ion nucleic acid aptamer is a magnetic field-sensitive cobalt ion nucleic acid aptamer, and the cobalt ion nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NOs. 1-7.

10. A use of the magnetic field-sensitive metal ion nucleic acid aptamer according to any one of claims 8 to 9, characterized in that: Specifically, the application in at least one of (A)-(C): (A) development of diagnostic tools or therapeutic tools for diseases; (B) Catalyst engineering; (C)Sensor equipment development.

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