Cyclic hybrid post-affinity enhanced nucleic acid aptamer, screening method and application thereof

Abstract

The application discloses a circular hybrid post-affinity enhanced nucleic acid aptamer and a screening method and application thereof, relates to the technical field of nucleic acid aptamer screening, and takes human alpha-thrombin as a research object. On the basis of compatibility of MB-SELEX, a CH-SELEX method is successfully developed under the condition of introducing circular hybridization. Through multiple rounds of positive screening under the condition of introducing circular hybridization and reverse screening under the condition of adding His-small peptide modified magnetic beads and human serum, a nucleic acid aptamer capable of specifically recognizing human alpha-thrombin and obviously improving the affinity after circular hybridization is screened. The affinity of the nucleic acid aptamer can be regulated by increasing the length of the hybrid complementary strand, which provides a reference for the diagnosis and treatment of diseases related to human alpha-thrombin and the development of sensors, and provides a conceptual verification and methodological reference for regulating other important biological processes, and fills the gap of such methods.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid aptamer screening technology, specifically to a nucleic acid aptamer with enhanced affinity after circular hybridization, its screening method, and its application. Background Technology

[0002] Nucleic acid aptamers, as a novel type of "receptor," have gradually attracted widespread attention in biology for their significance. Traditional receptors are typically large protein or polypeptide molecules, located on the cell membrane (e.g., G protein-coupled receptors, tyrosine kinase receptors) or intracellularly (e.g., nuclear receptors). After binding to their ligands, these receptors regulate cellular function through a series of complex biological signaling pathways. Nucleic acid aptamers are molecules composed of single-stranded DNA or RNA, usually small in size and simple in structure, but possessing specific binding capabilities. They can bind directly to their target molecules without requiring complex intracellular signaling mechanisms. Nucleic acid aptamers are mainly obtained through optimization during in vitro screening, using SELEX (systematic enrichment of ligands) to progressively select the nucleic acid molecules with the strongest binding to the target molecule. The affinity of these nucleic acid molecules can reach levels comparable to, or even stronger than, traditional receptors in some cases. However, in terms of the range of affinity regulation and applications, the affinity regulation of nucleic acid aptamers does not possess the complex biological regulatory mechanisms of protein receptors.

[0003] In previous work, we proposed an intuitively designed aptamer affinity modulation strategy. This involves adding clamp sequences to both ends of the aptamer and hybridizing them with allosteric inhibitor sequences to form double-stranded molecular clamps. By mechanically stretching and disrupting the aptamer's folding, we reversibly fine-tuned the aptamer's affinity. Using this method, we successfully reduced the affinity of the human α-thrombin aptamer HD22 by 65-fold. However, reducing the affinity of nucleic acid aptamers does not satisfy all applications. Currently, many researchers are dedicated to improving the affinity of screened aptamers, for example, through gene mutation, chemical modification, and structural tailoring. However, on the one hand, these mutagenesis-based methods are time-consuming and inefficient due to their trial-and-error nature, and because it is difficult to ensure that a given mutation will not only meaningfully alter the affinity but also achieve this by changing conformational equilibrium rather than the chemical interaction between the reagent and the analyte. On the other hand, these methods often rely on specific sequences and structures, lacking universal applicability. Summary of the Invention

[0004] In view of the technical problems existing in the above-mentioned existing research and technology, the purpose of this invention is to provide a nucleic acid aptamer with enhanced affinity after circular hybridization, a screening method (denoted as CH-SELEX), and its application.

[0005] The present invention achieves the above objectives through the following technical solutions: The first objective of this invention is to provide a method for screening nucleic acid aptamers with enhanced affinity after circular hybridization, comprising the following steps: (1) Synthesize random single-stranded DNA libraries and primer sequences; (2) First round of screening (2-1) Incubate random single-stranded DNA libraries with magnetic beads modified with target molecules to remove single-stranded libraries that can specifically bind to human α-thrombin and those that can non-specifically bind to magnetic beads. (2-2) Add complementary strands to the library obtained in step (2-1) to form a circular hybridization library; (2-3) Incubate the circular hybridization library with His-peptide-modified magnetic beads to remove the circular hybridization library that can bind to the His-peptide-modified magnetic beads; (2-4) Positive screening: Incubate the circular hybridization library obtained in step (2-3) with magnetic beads modified with target molecules, and recover the circular hybridization library that can bind to the target molecules; (2-5) Using the recovered circular hybridization library as a template, PCR amplification was performed using primer sequences to obtain PCR amplification products, and secondary libraries were prepared from the PCR amplification products. (3) Use the secondary library obtained in step (2-5) as the screening library, and perform multiple rounds of screening according to the process of the first round of screening in step (2); (4) In several single-round processes of multi-round screening, substrates that act on target molecules are added for reverse screening; (5) After multiple rounds of screening, the secondary libraries of the final round are subjected to high-throughput sequencing analysis. The sequences are selected for affinity verification under circular hybridization and non-circular hybridization conditions. Based on the affinity verification results, the sequences of nucleic acid aptamers with enhanced affinity after circular hybridization are selected.

[0006] As a further optimization of the present invention, the target molecule is human α-thrombin, and the substrate on which the target molecule acts is human serum.

[0007] As a further optimization of the present invention, the sequence of the random single-stranded DNA library is 5'-ATGCTCGTGAGCAGC-30N-CAGCTCGATCGAGCT-3', where 30N represents a sequence composed of 30 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.

[0008] As a further optimization of the present invention, the primer sequences are: forward primer: 5'-ATGCTCGTGAGCAGC-3'; reverse primer: 5'-AGCTCGATCGAGCTG-3'. Furthermore, the 5' end of the forward primer sequence is modified with a FAM fluorescent group, and the 5' end of the reverse primer sequence is modified with poly-A.

[0009] As a further optimization of the present invention, the complementary chain is any one of the following: SAM-22: 5'-CTCACGAGCATAGCTCGATCGA-3'; SAM-24: 5'-GCTCACGAGCATAGCTCGATCGAG-3'; SAM-26: 5'-TGCTCACGAGCATAGCTCGATCGAGC-3'; SAM-28: 5'-CTGCTCACGAGCATAGCTCGATCGAGCT-3'.

[0010] As a further optimization of the present invention, in step (4), the multi-round screening is carried out in a total of 8 rounds, and 20%, 30%, and 50% human serum are added to the 6th, 7th, and 8th rounds of screening respectively for reverse screening.

[0011] A second objective of this invention is to provide an application of the screening method described in any of the above claims in screening nucleic acid aptamers with enhanced affinity after circular hybridization.

[0012] A third objective of this invention is to provide a nucleic acid aptamer with enhanced affinity after circular hybridization, obtained by screening using the screening method described in any one of the above claims. The nucleic acid aptamer with enhanced affinity after circular hybridization is a human α-thrombin nucleic acid aptamer, which can specifically bind to human α-thrombin, and the affinity for human α-thrombin increases with the increase of complementary chain length.

[0013] As a further optimization of the present invention, the nucleotide sequence of the human α-thrombin nucleic acid aptamer is shown in SEQ ID NO. 1.

[0014] The fourth objective of this invention is the application of a nucleic acid aptamer with enhanced affinity after circular hybridization as described in any of the above claims in the preparation of a product that specifically recognizes human α-thrombin. The product that specifically recognizes human α-thrombin can be further applied in the fields of tool development for disease diagnosis or treatment, intelligent drug delivery and targeted therapy, and development of biosensor devices.

[0015] The present invention has the following beneficial effects: This invention focuses on human α-thrombin. Based on compatibility with MB-SELEX (magnetic bead-phylogenetic enrichment ligand technology), a CH-SELEX method was successfully developed by introducing circular hybridization. Through multiple rounds of positive screening under circular hybridization and reverse screening with His-peptide-modified magnetic beads and human serum, nucleic acid aptamers that specifically recognize human α-thrombin and exhibit significantly increased affinity after circular hybridization were identified. Furthermore, the affinity of the nucleic acid aptamers can be modulated by increasing the length of the hybridization complementary strand. This provides a reference for the diagnosis and treatment of diseases related to human α-thrombin and for sensor development. It also provides proof of concept and methodological reference for regulating other important biological processes, filling a gap in this type of method. Attached Figure Description

[0016] Figure 1 A schematic diagram of the screening process for the method of screening human α-thrombin nucleic acid aptamers with enhanced affinity after circular hybridization provided by the present invention; Figure 2 The binding curves (a) and affinity constant Kd values ​​(b) of the cyclic hybrids with different complementary chain lengths that regulate human α-thrombin aptamers provided by the present invention are shown. Figure 3 The binding curves (a) and affinity constant Kd values ​​(b) of the cyclic hybrids with different notch lengths regulating human α-thrombin aptamers provided by the present invention are shown. Figure 4 This invention provides a prediction of the secondary structure of Thr-1. Detailed Implementation

[0017] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection 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.

[0018] Unless otherwise specified, all experimental methods used in this study can be performed using standard procedures. All materials and reagents used are commercially available unless otherwise specified.

[0019] The schematic diagram of the screening process for affinity-enhanced human α-thrombin nucleic acid aptamers after circular hybridization provided in this study is shown below. Figure 1 As shown, the specific steps include: Synthesized random single-stranded DNA libraries and primer sequences The full length of the random single-stranded DNA library is 60 bp, including 15 bp of fixed sequence bases at each end and 30 bp of random sequence bases in the middle. The 5' end of this sequence is additionally modified with a FAM fluorescent group. The sequence information modified with the FAM fluorescent group is: 5'-FAM-ATGCTCGTGAGCAGC-30N-CAGCTCGATCGAGCT-3', where 30N represents a sequence composed of 30 arbitrary nucleotide bases.

[0020] The primer sequences include a forward primer modified with a FAM fluorescent group at the 5' end: 5'-FAM-ATGCTCGTGAGCAGC-3'; and a reverse primer modified with poly-A at the 5' end and linked by spacer 18: 5'-AAAAAAAAAAAAAAAAAAAAA-spacer 18-AGCTCGATCGAGCTG-3'.

[0021] First round of screening 2.1 First step reverse screening Using a synthesized random single-stranded DNA library as the initial library, 100 pmol of the random single-stranded DNA library was mixed with 10 μL of human α-thrombin-modified magnetic beads, and the volume was brought up to 100 μL with binding buffer. The mixture was then incubated on a rotating rack for 60 min. After incubation, the mixture was placed on a magnetic tube rack and allowed to stand for 2 min. The supernatant was then collected, and the magnetic beads were discarded to remove the random single-stranded DNA library that could bind to thrombin.

[0022] 2.2 The variability of a library The obtained supernatant was dissolved with 100 pmol of the complementary strand (5'-CTGCTCACGAGCATAGCTCGATCGAGCT-3', denoted as SAM-28) in binding buffer (1 mM MgCl2, 100 mM NaCl, 4 mM KCl, 0.025% Tween-20, pH 7.5). After denaturation at 95 °C for 10 min, cooling on ice for 10 min, and then incubation at ambient temperature for 60 min, a stable circular hybridization library was obtained.

[0023] 2.3 Second step: reverse screening Mix 10 μL of His-peptide (Angpu Topmai Biotechnology, catalog number SEP-HIS) modified magnetic beads with the incubated circular hybridization library and incubate on a rotating rack for 60 min. After incubation, place the mixture on a magnetic tube rack and let it stand for 2 min. Collect the supernatant and discard the magnetic beads to remove any circular hybridization library that can bind to the His-peptide.

[0024] 2.4. Positive screening Add 10 μL of human α-thrombin-modified magnetic beads to the supernatant obtained in step 2.2, and incubate on a rotating rack for 60 min. After incubation, place the tube on a magnetic tube rack and let it stand for 2 min. Discard the supernatant and wash the tube three times with 1×PBS solution to remove any library that does not specifically bind to thrombin. Add 50 μL of distilled water to the tube containing the magnetic beads and incubate at 95 °C for 5 min to elute the specifically bound circular hybridization library, obtaining the eluent.

[0025] 2.5 PCR Amplification The library obtained in step 2.4 was used as a template for PCR amplification. The forward primer was 5'-FAM-ATGCTCGTGAGCAGC-3'; the reverse primer was 5'-AAAAAAAAAAAAAAAAAAAA-spacer 18-AGCTCGATCGAGCTG-3'. The total PCR volume 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 DEPCH2O to a final volume of 50 μL). The amplification conditions were: 95 ℃ pre-denaturation for 3 min, 95 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 60 s, for a total of 30 cycles, 72 ℃ annealing for 2 min, and incubation at 4 ℃.

[0026] 2.6 Large-scale expansion After determining the optimal number of PCR amplification rounds, the elution buffer obtained in section 2.4 was used for large-scale amplification. A total of 20 tubes were amplified, with the solution system and amplification conditions in each tube as described in section 2.4 (50 μl system). A final library solution with a volume of 1 mL was obtained.

[0027] 2.7 Preparation of Secondary Libraries Add 5 mL of n-butanol to the library solution obtained from large-scale amplification, vortex to precipitate, and after the supernatant becomes clear, centrifuge at 7500 rpm for 3 min, remove the supernatant, and add the resulting library concentrate to a 12% denaturing gel, run at 35 mA for 1.5 h, cut out the target band, crush it, and add nucleic acid extraction buffer (2 mL 5M NaCl, 0.5 mL 1M Tris HCl, pH 7.5, 0.1 mL 0.5M EDTA, pH 8.0, and distilled water to 50 L). Mix well and heat at 60 °C and 1000 rpm for 1 h, then rotate overnight on a rotating rack. Centrifuge to collect the supernatant, add sodium acetate and anhydrous ethanol to the supernatant, and incubate at -20 °C for 2 h. Then, centrifuge at 15000 rpm for 20 min to precipitate the nucleic acid, and carefully remove the supernatant. The resulting solution was heated in a metal bath until the water evaporated. After the water evaporated, the library was dissolved in a binding buffer solution in a test tube, vortexed, and centrifuged. This process was repeated three times to obtain the secondary library.

[0028] Multiple rounds of screening The secondary library was used to replace the initial library in the first round of screening. The screening process was repeated, and 20%, 30%, and 50% human serum were added for reverse screening in the 6th, 7th, and 8th rounds of screening, respectively. The specific screening conditions are shown in Table 1 below. The library in the 8th round was used for high-throughput sequencing.

[0029] Table 1 CH-SELEX screening criteria for human α-thrombin ; 4. Sequencing and Sequence Analysis After the final round of screening, the enriched ssDNA library was sent for cloning and sequencing. Finally, the sequence with the highest enrichment level was selected as the aptamer sequence (denoted as Thr-1). The secondary structure of aptamer Thr-1 was simulated using the mfold webpage. Figure 4 The information of the Thr-1 sequences obtained by screening is as follows: Thr-1: 5'-ATGCTCGTGAGCAGCCATGGGATGGGATACTGGTAGGCGTGGTAGCAGCTCGATCGAGCT-3' (SEQ ID NO. 1).

[0030] 5. Verify that aptamer affinity is regulated by circular hybridization. First, the Thr-1 sequence was synthesized and labeled with a FAM fluorescent group at its 5' end. The selected aptamer was then added to complementary strands of different lengths, followed by heating at 95 °C for 10 min, cooling on ice for 10 min, and then incubating at ambient temperature for 60 min. The sequences of complementary strands of different lengths are shown in Table 2. Table 2 Complementary strand sequences of different lengths ; Note: Figure 3 The sequence indicated by gap 0 in the sequence is the complementary strand of SAM-28.

[0031] Then, the selected aptamers were subjected to binding affinity analysis, and the specific steps are as follows: (1) 3 μL of magnetic beads conjugated with thrombin protein were mixed with different concentrations (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1000 nM, 3000 nM, 6000 nM) of circular hybridization aptamers (with complementary strands SAM-22, SAM-24, SAM-26, and SAM-28) in a 50 μL system and incubated at room temperature for 40 min by rotation. At the same time, a control solution was prepared by mixing 3 μL of magnetic beads conjugated with thrombin protein with different concentrations (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1000 nM, 3000 nM, 6000 nM) of aptamers without complementary strands in a 50 μL system and incubating at room temperature by rotation for the same time.

[0032] (2) After rotation, place each test tube on a magnetic test tube rack for 2 min to remove the supernatant, and then wash the magnetic beads three times with 1×PBS. After the last wash, add 300 μL of 1×PBS to each test tube and mix well with a pipette.

[0033] (3) Add the samples to the ELISA plate respectively, measure the fluorescence intensity of the samples with an ELISA reader, and normalize the fluorescence intensity by taking the observed maximum fluorescence intensity as the reference value 1 in each affinity test. Obtain the fitting curve and dissociation constant Kd value by using Origin software for nonlinear regression analysis.

[0034] The results are as follows Figure 2 As shown, compared with a single linear aptamer, the addition of complementary strands of different lengths all improved the affinity of the original aptamer to varying degrees, and the results showed a strong regularity. That is, as the length of the complementary strand increased, the affinity curve continued to shift to the left, and the affinity changed by up to about 10 times with the increase of the length of the complementary strand.

[0035] like Figure 3As shown, this study designed a series of complementary strands based on the screened circular hybrid aptamers. One group placed several thymine nucleotides at the center of the complementary strand. In principle, widening this gap would release the mechanical tension applied to the aptamer body. Experimental results showed that as the gap length increased from 0 nt to 3 nt, the curve gradually shifted to the right, and the aptamer Kd gradually decreased, proving that the affinity of the circular hybrid aptamer is indeed related to this rigid structure.

[0036] The embodiments described above are merely examples of several implementations of the present invention, and 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 those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer with enhanced affinity after circular hybridization, characterized in that, The nucleic acid aptamer with enhanced affinity after circular hybridization is a human α-thrombin nucleic acid aptamer. The human α-thrombin nucleic acid aptamer can specifically bind to human α-thrombin, and the affinity for human α-thrombin increases with the increase of complementary chain length. The nucleotide sequence of the human α-thrombin nucleic acid aptamer is shown in SEQ ID NO.

1.

2. The nucleic acid aptamer with enhanced affinity after circular hybridization according to claim 1, characterized in that, The method for screening human α-thrombin nucleic acid aptamers includes the following steps: (1) Synthesize random single-stranded DNA libraries and primer sequences; (2) First round of screening (2-1) Incubate a random single-stranded DNA library with magnetic beads modified with a target molecule to remove single-stranded libraries that can bind to the target molecule and the magnetic beads, wherein the target molecule is human α-thrombin; (2-2) Add complementary strands to the library obtained in step (2-1) to form a circular hybridization library; (2-3) Incubate the circular hybridization library with His-peptide-modified magnetic beads to remove the circular hybridization library that can bind to the His-peptide-modified magnetic beads; (2-4) Positive screening: Incubate the circular hybridization library obtained in step (2-3) with magnetic beads modified with target molecules, and recover the circular hybridization library that can bind to the target molecules; (2-5) Using the recovered circular hybridization library as a template, PCR amplification was performed using primer sequences to obtain PCR amplification products, and secondary libraries were prepared from the PCR amplification products. (3) Use the secondary library obtained in step (2-5) as the screening library, and perform multiple rounds of screening according to the process of the first round of screening in step (2); (4) In several single-round processes of multi-round screening, the substrate of the target molecule is added for reverse screening, wherein the substrate of the target molecule is human serum; (5) After multiple rounds of screening, the secondary libraries of the final round are subjected to high-throughput sequencing analysis. The sequences are selected for affinity verification under circular hybridization and non-circular hybridization conditions. Based on the affinity verification results, the sequences of nucleic acid aptamers with enhanced affinity after circular hybridization are selected.

3. The nucleic acid aptamer with enhanced affinity after circular hybridization according to claim 2, characterized in that: The sequence of the random single-stranded DNA library is 5'-ATGCTCGTGAGCAGC-30N-CAGCTCGATCGAGCT-3', where 30N represents a sequence composed of 30 arbitrary nucleotide bases linked together.

4. The nucleic acid aptamer with enhanced affinity after circular hybridization according to claim 2, characterized in that, The primer sequences are: forward primer: 5'-ATGCTCGTGAGCAGC-3'; reverse primer: 5'-AGCTCGATCGAGCTG-3'.

5. The nucleic acid aptamer with enhanced affinity after circular hybridization according to claim 2, characterized in that, The complementary chain is any one of the following: SAM-22: 5'-CTCACGAGCATAGCTCGATCGA-3'; SAM-24: 5'-GCTCACGAGCATAGCTCGATCGAG-3'; SAM-26: 5'-TGCTCACGAGCATAGCTCGATCGAGC-3'; SAM-28: 5'-CTGCTCACGAGCATAGCTCGATCGAGCT-3'.

6. The nucleic acid aptamer with enhanced affinity after circular hybridization according to claim 2, characterized in that, In step (4), the multi-round screening is carried out in a total of 8 rounds. In the 6th, 7th and 8th rounds of the 8 rounds of screening, 20%, 30% and 50% of human serum are added respectively for reverse screening.

7. The use of a nucleic acid aptamer with enhanced affinity after circular hybridization as described in claim 1 in the preparation of a product that specifically recognizes human α-thrombin.

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