A magnetic field-sensitive metal ion nucleic acid aptamer, its screening method and application

Abstract

The present invention discloses a magnetic field-sensitive metal ion nucleic acid aptamer, a screening method and an application thereof, relating to the technical field of nucleic acid aptamer screening. The present invention takes divalent cobalt ions with paramagnetism as the research object. On the basis of being compatible with GO-SELEX, the HM-SELEX method is successfully developed under a steady-state strong magnetic field of 9 Tesla, which promotes the efficient screening of magnetosensitive metal ion aptamers. Moreover, a nucleic acid aptamer with magnetic field-tunable binding characteristics is discovered for the first time, and the sequences of the cobalt ion nucleic acid aptamers are shown as SEQ ID NO.1-7. The present invention also provides a reference for the development of magnetogenetics, disease diagnosis and treatment related to metal ions and magnetic fields, as well as related sensors, filling the blank of such methods.

Description

Technical Field

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

[0002] Magnetic fields are ubiquitous in nature. Existing research classifies magnetic fields into static magnetic fields and dynamic magnetic fields (alternating magnetic fields) according to the variation of magnetic field intensity over time; and according to different magnetic induction intensities, they are further divided into weak magnetic fields (less than 1×10 - 3 Tesla), medium magnetic fields (1×10 -3 to 1 Tesla), strong magnetic fields (1 to 20 Tesla), and ultra-strong magnetic fields (above 20 Tesla). The Earth on which we live is itself a huge static weak magnet (magnetic field intensity is about 5×10 -5 Tesla). At present, the use of magnetic fields to regulate physical properties and chemical reactions has been widely applied, but the influence of magnetic fields on biological processes remains an open and inspiring issue.

[0003] The field of magnetogenetics is dedicated to the remote and non-invasive manipulation of neuronal functions. In 2015, the Xie Can team first announced the magnetic response protein MagR, which triggered a heated discussion 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 the biorecognition 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 behaviors in magnetic fields due to the unpaired electrons in their 3d orbitals. As is well known, these metal ions can bind to proteins and are essential for maintaining life. They are cofactors of many enzymes, promoting the catalytic activity of enzymes (such as vitamin B12), and participating in the transport of important substances such as oxygen in organisms.

[0005] Compared with proteins, it has been found that nucleic acids have a greater affinity for binding metal ions. This is due to the interaction between metal cations and the negatively charged phosphate backbone, as well as the coordination with nucleobase donor atoms. For example, certain riboswitches can directly utilize Mn 2+ or Mg 2+Metal ions such as... act as its specific ligands to induce structural changes, thereby regulating gene expression. In the field of the interaction between sequence-specific DNA and metal ions, aptamers obtained through in vitro screening have been widely used due to their simple synthesis, easy chemical modification, and cost-effective advantages compared to antibody production. These studies on metal ion-related aptamers are crucial for numerous applications such as disease diagnosis and treatment, catalyst engineering, and the development of sensor devices. Summary of the Invention

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

[0007] The present invention achieves the above object through the following technical solutions:

[0008] The first object of the present invention is to provide a screening method for a magnetic field-sensitive metal ion nucleic acid aptamer, comprising the following steps:

[0009] (1) Synthesize a random single-stranded DNA library and primer sequences;

[0010] (2) Perform denaturation-renaturation treatment on the random single-stranded DNA library;

[0011] (3) Positive screening: Under a steady-state strong magnetic field, add the target metal ions to the library that has undergone the denaturation-renaturation treatment in step (2) for incubation, and recover the library that can bind to the target metal ions under the steady-state strong magnetic field;

[0012] (4) Using the primer sequences synthesized in step (1), and taking the library recovered in step (3) as a template, perform PCR amplification to obtain a PCR amplification product;

[0013] (5) Prepare a secondary library from the PCR amplification product obtained in step (4);

[0014] (6) Take the secondary library prepared in step (5) as a screening library, and perform multiple rounds of screening according to the procedures of steps (2) to (5);

[0015] (7) Introduce reverse screening in a single round of the multiple rounds of screening. The reverse screening is specifically as follows: First, under a steady-state strong magnetic field, add interfering metal ions to the secondary library obtained in step (5) for incubation, remove the library that binds to the interfering metal ions under the steady-state strong magnetic field, then, under the normal geomagnetic field, continue to add the target metal ions for incubation, remove the library that binds to the target metal ions under the normal geomagnetic field, and finally, continue to perform the next round of screening on the remaining library;

[0016] After the multi-round screening is completed, the final-round secondary library is subjected to high-throughput sequencing analysis. Sequences are selected to verify the affinity under a steady-state strong magnetic field and the normal geomagnetic field respectively. According to the results of the affinity verification, sequences of the magnetic field-sensitive metal ion aptamers are screened out.

[0017] 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’, where 40N represents a sequence composed of 40 arbitrary nucleobases. Further, the 5’ end of the sequence of the random single-stranded DNA library is additionally modified with a FAM fluorescent group.

[0018] As a further optimization scheme of the present invention, in step (1), the primer sequences are as follows:

[0019] Forward primer: 5’-TACACTGCACTGCGTTAGAG-3’;

[0020] Reverse primer: 5’-CAGTACTGTGACACGTCCAT-3’.

[0021] Further, the 5’ end of the forward primer sequence is additionally modified with a FAM fluorescent group, and the 5’ end of the reverse primer sequence is additionally modified with a biotin Biotin.

[0022] As a further optimization scheme of the present invention, the magnetic field intensity of the steady-state strong magnetic field is 9 Tesla, and the magnetic field intensity of the normal geomagnetic field is 5×10 -5 Tesla.

[0023] As a further optimization scheme of the present invention, the total number of rounds of the multi-round screening is 7 rounds, and the rounds of introducing reverse screening are the 4th round and the 6th round.

[0024] As a further optimization scheme of the present invention, the target metal ion is Co 2+ , and the interfering metal ions are a mixture of other metal ions other than the target metal ion. 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 Hg2+ 。

[0025] The second object of the present invention is to further provide an application of the screening method as described in any one of the above in screening magnetic field-sensitive metal ion nucleic acid aptamers.

[0026] The third object of the present invention is to further provide a magnetic field-sensitive metal ion nucleic acid aptamer, which is obtained by screening through the screening method as described in any one of the above, and the affinity of the magnetic field-sensitive metal ion nucleic acid aptamer for the metal ion is enhanced as the magnetic field strength increases.

[0027] As a further optimized solution 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 shown in any one of SEQ ID NO.1-7. Further optimization lies in that the cobalt ion nucleic acid aptamer has nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.5. Further still more optimization lies in that the cobalt ion nucleic acid aptamer has a nucleotide sequence shown in SEQ ID NO.3.

[0028] The fourth object of the present invention is to further provide an application of the magnetic field-sensitive metal ion nucleic acid aptamer as described in any one of the above, specifically an application in at least one of (A)-(C):

[0029] (A) Development of disease diagnosis tools or treatment tools;

[0030] (B) Catalyst engineering;

[0031] (C) Development of sensor devices.

[0032] The present invention has the following beneficial effects:

[0033] The present invention takes divalent cobalt ions with paramagnetism as the research object, adopts the HM-SELEX technology (high magnetic field index enrichment ligand system evolution technology) carried out under a steady-state strong magnetic field, and through multiple rounds of positive screening under a strong magnetic field with a magnetic field strength of 9 Tesla and corresponding interference ions and negative screening under the normal geomagnetic field, a magnetic field-sensitive nucleic acid aptamer that can specifically recognize cobalt ions and has a significantly improved affinity under a steady-state strong magnetic field is screened, and the affinity of the nucleic acid aptamer can be regulated by increasing the magnetic field strength, which provides a reference for magnetogenetics, disease diagnosis and treatment, and sensor development related to metal ions and magnetic fields, and makes up for the blank of such methods. Description of the Drawings

[0034] Figure 1 It is a schematic diagram of the screening process of the embodiment of the present invention;

[0035] Figure 2For the recovery efficiency of the screening library of HM-SELEX;

[0036] Figure 3A For the secondary structure prediction of Co-M1 in Co-family-1;

[0037] Figure 3B For the secondary structure prediction of Co-M2 in Co-family-1;

[0038] Figure 3C For the secondary structure prediction of Co-M3 in Co-family-1;

[0039] Figure 3D For the secondary structure prediction of Co-M4 in Co-family-1;

[0040] Figure 3E For the secondary structure prediction of Co-M9 in Co-family-1;

[0041] Figure 4 For 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;

[0042] Figure 5 For 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;

[0043] Figure 6 For the affinity change of Co-family-3 under a steady-state strong magnetic field; where a is Co-M8 in Co-family-3; b is Co-M10 in Co-family-3;

[0044] Figure 7 For the selected Co 2+ Specificity verification of the aptamer; where a is Co-M3; b is Co-M8. Detailed implementation manners

[0045] The present application will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following specific implementation manners are only used to further illustrate the present application and cannot be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0046] In the following examples, if the specific experimental methods are not indicated, they can all be carried out according to conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained through commercial channels.

[0047] Example 1. Screening of divalent cobalt ion nucleic acid aptamers with magnetic field-regulated affinity under steady-state strong magnetic field

[0048] The method for screening divalent cobalt ion nucleic acid aptamers with magnetic field-regulated affinity under steady-state strong magnetic field provided in this example has a screening process schematic diagram as Figure 1 shown, and specifically includes the following steps:

[0049] 1. Synthesis of random single-stranded DNA library and primer sequences

[0050] The full 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’ (SEQ ID NO.8), where 40N represents a sequence composed of 40 arbitrary nucleotide bases. When performing the following screening method, a FAM fluorescent group was further modified at the 5’ end of the random single-stranded DNA library sequence.

[0051] The primer sequences include 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, a FAM fluorescent group was further modified at the 5’ end of the forward primer, and a biotin Biotin was further modified at the 5’ end of the reverse primer.

[0052] 2. First round of screening

[0053] 2.1. Library denaturation and renaturation

[0054] Dissolve 500 pmol of the DNA library in binding buffer (50 mM HEPES, 100 mM NaCl, 0.025% tween-20, pH 7.5). Denature at 95 °C for 10 min, cool on ice for 10 min, and then place at ambient temperature for 10 min.

[0055] 2.2. Positive screening

[0056] Add 500 nmol of Co to the single-stranded DNA library solution that has completed denaturation and renaturation in step 2.1 2+Ions were incubated for 60 min under a steady-state strong magnetic field with a magnetic field strength of 9 Tesla. Monolayer graphene oxide was added to the incubated system to adsorb the unbound library. The supernatant was retained by centrifugation at 15,000 rpm for 10 min to obtain the library that could bind to Co 2+ ions under a steady-state strong magnetic field.

[0057] 2.3. Calculation of screening recovery rate

[0058] The concentration of single-stranded DNA in the recovered supernatant was measured by a ultra-micro nucleic acid spectrophotometer, and the screening recovery rate for each round was calculated (screening recovery rate = amount recovered / amount input), see Figure 2 .

[0059] 2.4. PCR amplification

[0060] 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’, and the reverse primer was 5’-biotin-CAGTACTGTGACACGTCCAT-3’. The total volume of the PCR system was 50 μL (including 1 μL of the recovered secondary library, 25 μL of 2× Taq PCR Mix, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, and made up to a final volume of 50 μL with DEPC H 2 2O). The amplification conditions were pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 60 s, for a total of 20 cycles, followed by post-extension at 72 °C for 3 min and incubation at 4 °C.

[0061] 2.5. Preparation of secondary library

[0062] 50 μL of streptavidin magnetic beads were pipetted onto a magnetic rack and allowed to stand for 1 min. The supernatant was removed, and 200 μL of 1× PBS buffer (containing 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na 2 HPO 4 , 1.76 mM KH 2 PO 4, 1 mg / mL BSA) into a centrifuge tube, rotate and incubate for 60 min, and perform magnetic separation. Wash three times with 200 μL of 1×PBS buffer, add the PCR product prepared in step 2.4, add one-third volume of 4 M NaCl solution, place the centrifuge tube on a rotary mixer, and incubate with shaking at room temperature for 60 min. Place the centrifuge tube back on the magnetic rack, let it stand for 1 min, aspirate and discard the supernatant, and wash the magnetic beads three times with 200 μL of 1×PBS buffer. Add 30 μL of 0.2 M NaOH solution, mix for 1 min to denature the double-stranded DNA. Place the centrifuge tube on the magnetic rack, let it stand for 1 min, aspirate the supernatant into a new centrifuge tube, adjust the pH of the library to neutral with hydrochloric acid, and use it as the secondary library for the next round of screening and measure its concentration.

[0063] 3. Counter-screening

[0064] When the screening recovery rate tends to be stable, counter-screening is introduced. Different from the positive screening in the first round where the target Co was incubated with the library under a steady-state strong magnetic field of 9 Tesla. In counter-screening, we add interfering ions (including Mn 2+ , Cu 2+ , Ca 2+ , Al 2+ , Ba 3+ , Cd 2+ , Ni 2+ , Zn 2+ , Ce 2+ , Pb 3+ , Cr 2+ , Fe 3+ , and Hg 3+ (the dosage of all the above ions is 100 nmol) to the single-stranded library and incubate for 60 min under a steady-state strong magnetic field. Add monolayer graphene oxide to the incubated system, centrifuge to remove the supernatant to remove the library bound to the interfering substances. Then, under the normal geomagnetic field (magnetic field strength is about 5×10 2+ Tesla), add Co - 5 ions to the recovered graphene oxide above and continue to incubate for 60 min. After incubation, centrifuge to remove the supernatant to remove the library that can bind to the target under the normal environmental magnetic field, and retain the graphene precipitate to continue the positive screening under the steady-state strong magnetic field. 2+ ions to the recovered graphene oxide above and continue to incubate for 60 min. After incubation, centrifuge to remove the supernatant to remove the library that can bind to the target under the normal environmental magnetic field, and retain the graphene precipitate to continue the positive screening under the steady-state strong magnetic field.

[0065] 4. Multiple rounds of screening

[0066] Use the secondary library to replace the initial library in the first round of screening process, repeat the screening process, adjust the screening pressure according to the screening recovery rate of each round, and introduce counter-screening in the 4th and 6th rounds of screening. The specific screening conditions are shown in Table 1 below. Use the library of the 7th round for high-throughput sequencing.

[0067] Table 1. Screening conditions for Co 2+ in HM-SELEX

[0068] ;

[0069] 5. Sequencing and sequence analysis

[0070] Cluster analysis was performed on the sequences with the top-ranked number of repeats obtained by high-throughput sequencing. According to their homology and the secondary structures simulated by Mfold, the sequences were divided into different families, including Co-family-1 and Co-family-3. The sequences included in Co-family-1 and Co-family-3 were named, and the sequence information is shown in Table 2.

[0071] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E and Figure 4 are the secondary structure predictions of the sequences included in Co-family-1 and Co-family-3, respectively.

[0072] Table 2. Sequence information of the sequences included in Co-family-1 and Co-family-3

[0073] ;

[0074] 6. Verification of affinity regulated by magnetic field

[0075] 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 groups at the 5' end. The above-selected aptamers were heated at 95°C for 10 min, cooled on ice for 10 min, and then placed at ambient temperature for 10 min.

[0076] Then, the binding affinity of the screened candidate aptamers was analyzed under normal geomagnetic field (NM) or stable strong magnetic field conditions, that is, 10 pmol of aptamer was incubated with (0 μM, 100 μM, 200 μM, 300 μM, 400 μM) target ions (Co 2+ ions) in a 400 μL system under normal geomagnetic field or strong magnetic field for 30 min.

[0077] Finally, add 0.2 mg of graphene oxide into the centrifuge tube and incubate for 30 min. After incubation, centrifuge at 15,000 rpm for 3 min, and measure the fluorescence intensity of the supernatant. In each affinity detection, the fluorescence intensity is normalized with the maximum observed fluorescence intensity as the reference value 1, and the fitting curve and dissociation constant K d value are obtained through non-linear regression analysis using Origin software.

[0078] See Figure 5 and Figure 6 , the affinities of Co-family-1 and Co-family-3 both gradually increase with the increase of magnetic field strength. For Co-M3 in Co-family-1, the affinity curve continuously shifts to the left with the increase of magnetic field strength, and the affinity increases by about 2 to 3 times with the increase of the magnetic field. Compared with the negligible affinity in a weak magnetic field environment, the sequence Co-M8 in Co-family-3 starts to show an affinity of about 200 μM when exposed to a high magnetic field of 6 Tesla and above.

[0079] 7. Specificity verification

[0080] Specificity verification is also carried out in a 400 μL binding buffer system using 10 pmol of the selected aptamers (using Co-M3 and Co-M8 as the representative sequences of the two families) and the target ion (300 μM Co 2+ ion) and 10-fold amounts of 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+ ) for incubation. Under two conditions of a steady-state strong magnetic field of 9 Tesla and the normal geomagnetic field (5×10 -5 Tesla), after incubating for 30 min, add 0.2 mg of graphene oxide into the system and continue to incubate for 30 min, then centrifuge the mixture and measure the fluorescence intensity of the supernatant, and normalize it with the maximum observed fluorescence intensity as the reference value 1.

[0081] The specificity test shows that as Figure 7As shown, Co-M3 has significant selectivity in both steady-state strong magnetic fields and normal geomagnetic fields. In contrast, Co-M8 exhibits excellent selectivity only under steady-state strong magnetic field conditions. These findings further validate the effectiveness of the selection strategy we employed.

[0082] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

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

2. Use of the magnetic field-sensitive metal ion nucleic acid aptamer according to claim 1 in the preparation of a product that specifically recognizes metal cobalt ions, characterized in that: The affinity of the magnetic field-sensitive metal ion nucleic acid aptamer to metal cobalt ions increases with the increase of magnetic field intensity.

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