Nucleic acid aptamer for identifying D-serine

Abstract

The invention provides a nucleic acid aptamer used for identifying D-serine. The nucleic acid aptamer provided by the invention has the advantages of high affinity and strong specificity to D-serine, can be used for detecting D-serine, and is high in detection sensitivity and low in detection cost.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a nucleic acid aptamer for identifying D-serine. Background Art

[0002] Aptamers are nucleic acid molecules with special structures and functions obtained through in vitro screening technology (SELEX), which can bind to target substances with high specificity and selectivity. Compared with traditional recognition molecules, aptamers have many advantages such as high affinity, strong specificity, and good biocompatibility, and have received widespread attention in the fields of biomedicine and food safety.

[0003] As an important endogenous neuromodulator, D-serine plays a key role in the central nervous system and neurotransmission. The development of nucleic acid aptamers targeting D-serine can help researchers to more deeply study its function and activity in vivo. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a nucleic acid aptamer for recognizing D-serine.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] The first aspect of the present invention provides the use of a nucleic acid aptamer in detecting D-serine or in preparing a product for detecting D-serine, wherein the sequence of the nucleic acid aptamer has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:2.

[0007] Furthermore, the sequence of the nucleic acid aptamer is shown in SEQ ID NO:2.

[0008] Furthermore, the products include test strips and test kits.

[0009] Furthermore, the nucleic acid aptamer also includes an RNA sequence transcribed from its DNA sequence.

[0010] Furthermore, the nucleic acid aptamer also includes its derivatives.

[0011] Furthermore, the derivative is formed by connecting or modifying the nucleic acid aptamer with other substances.

[0012] Furthermore, the other substances include one or more of fluorescent labels, isotope labels, therapeutic substances, enzyme labels, and biotin labels.

[0013] Furthermore, the modification includes one or more modified nucleotides, and / or one or more substituted nucleotides.

[0014] Furthermore, the modified or substituted positions include one or more of a ribose position, a deoxyribose position, a phosphate position and a base position.

[0015] Furthermore, the detection of D-serine is for non-diagnostic purposes.

[0016] The second aspect of the present invention provides a nucleic acid aptamer, the sequence of which has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:2.

[0017] Furthermore, the sequence of the nucleic acid aptamer is shown in SEQ ID NO:2.

[0018] The third aspect of the present invention provides a method for detecting D-serine, comprising contacting a sample with the nucleic acid aptamer described in the first aspect or the second aspect of the present invention, thereby detecting D-serine in the sample.

[0019] Furthermore, the method is a method for non-diagnostic purposes.

[0020] The fourth aspect of the present invention provides use of the nucleic acid aptamer described in the first aspect or the second aspect of the present invention in inhibiting D-serine or in preparing a product that inhibits D-serine.

[0021] Furthermore, the inhibition of D-serine is inhibition of D-serine for non-therapeutic purposes.

[0022] Advantages and beneficial effects of the present invention:

[0023] The nucleic acid aptamer provided by the present invention has the advantages of high affinity and strong specificity for D-serine, and can be used for the detection of D-serine with high detection sensitivity and low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the specific binding diagram of candidate aptamer 6.7 and D-serine identified by qPCR method;

[0025] Figure 2 This is the specific binding diagram of aptamer 6.7 sequence and D-serine identified by electrochemical (electrical impedance method);

[0026] Figure 3 This is a graph showing the binding constant KD of aptamer 6.7 sequence and D-serine determined by the evanescent wave method. DETAILED DESCRIPTION

[0027] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] The present invention provides the use of a nucleic acid aptamer in detecting D-serine or in preparing a product for detecting D-serine. The sequence of the nucleic acid aptamer has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:2.

[0029] The nucleic acid aptamer also includes its derivatives.

[0030] The derivative is formed by connecting or modifying the nucleic acid aptamer with other substances.

[0031] The other substances include one or more of fluorescent labels, isotope labels, therapeutic substances, enzyme labels, and biotin labels.

[0032] Among them, the fluorescein labels include but are not limited to fluorescein, phycoerythrin (PE), Texas red (TR), rhodamine, free lanthanide salts, chelated lanthanide salts, CyDye, BODIPY and ALEXA.

[0033] Isotope labeling includes but is not limited to 2 H (deuterium labeled D), 13 C. 15 N. 17 O. 18 O. 34 S.

[0034] Therapeutic substances refer to substances with medicinal, pharmacological, psychosomatic and therapeutic effects, including but not limited to nanoparticles, RNA drugs, siRNA, synthetic or biologically produced drugs.

[0035] Enzyme label refers to a label with enzyme activity. A typical and preferred example is horseradish peroxidase (HRP).

[0036] The modification includes one or more modified nucleotides, and / or one or more substituted nucleotides.

[0037] In some embodiments, modified nucleotides refer to a nucleotide that is not naturally occurring, but is an analog or ester of a naturally occurring nucleotide. When used in the context of an oligonucleotide or nucleic acid molecule (e.g., nucleic acid aptamer), it generally means that at least one of the four constituent nucleotides (i.e., A, G, T / U, and C) of an oligonucleotide is an analog or ester of a naturally occurring nucleotide. In some embodiments, modified nucleotides can make an oligonucleotide resistant to nucleases. In some embodiments, modified nucleotides mainly cause hydrophobic interactions between aptamers and their protein targets, resulting in high binding efficiency and a stable co-crystallized complex. A pyrimidine having a substitution at the C-5 position is an example of a modified nucleotide. Modifications may include backbone modifications, methylation, rare base pairing combinations (e.g., isobases, isocytidine, and isoguanidine), etc. Modifications may also include 3' and 5' modifications, such as capping. Other modifications may include substitution of one or more natural nucleotides with analogs, internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications comprising chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), modifications comprising alkylating agents, and modifications with modified bonds (e.g., alpha anomeric nucleic acids, etc.). In addition, any of the hydroxyl groups typically present on the sugar of a nucleotide may be replaced by a phosphonate or phosphate group; protected with a standard protecting group; or activated to form additional bonds with another nucleotide or solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with an amine, an organic end-capping group moiety having about 1 to about 20 carbon atoms, a polyethylene glycol (PEG) polymer in the range of about 10 kDa to about 80 kDa in some embodiments, a PEG polymer in the range of about 20 kDa to about 60 kDa in some embodiments, or other hydrophilic or hydrophobic biological or synthetic polymers. In some embodiments, the modification can be at the C-5 position of the pyrimidine. These modifications can be generated by amide linkages directly at the C-5 position or by other types of linkages.

[0038] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0039] Example

[0040] 1. Experimental Methods

[0041] 1. SELEX screening

[0042] 1) Design a random oligonucleotide library and deliver it to a company for synthesis.

[0043] 2) Screening of specific nucleic acid aptamers is performed through incubation, washing, elution, amplification and other steps. As the number of screening rounds increases, the screening pressure is gradually increased by reducing the amount of ssDNA input and appropriately increasing the number of counter-screening steps, thereby obtaining nucleic acid aptamers that recognize D-serine.

[0044] a. Incubate D-serine with the ssDNA library at room temperature for 1 hour, then capture D-serine and its bound ssDNA with activated carboxyl magnetic beads, wash them 4 times with buffer, and finally elute the oligonucleotide ligands that specifically bind to the target protein. Prepare the secondary library by PCR amplification for the next round of screening.

[0045] b. The eluted ssDNA obtained during the identification of the 4th round library and the 6th round library was amplified by PCR into a double-stranded DNA (dsDNA) library for sequencing.

[0046] c. Analyze the sequencing results to obtain enriched DNA aptamer sequence information; select several sequences as candidate aptamers through aptamer primary structure homology and secondary structure analysis.

[0047] 2. Identification of specificity and affinity of nucleic acid aptamers

[0048] 1) Preliminary identification of aptamers binding to D-serine using qPCR

[0049] Take 20 pmol of the identified aptamer and 60 pmol of D-serine and add them to the screening buffer and incubate at room temperature for 1 hour. Then add carboxyl-activated magnetic beads and continue incubation for 30 minutes. Discard the magnetic beads and take only the supernatant as a qPCR template to record the CT value to determine whether the aptamer binds to D-serine.

[0050] 2) Electrochemical method to identify the specificity of nucleic acid aptamer binding to D-serine

[0051] When the D-serine electrical impedance biosensor based on nucleic acid aptamer fixed on the electrode surface specifically binds to D-serine, it will affect the secondary structure of the nucleic acid aptamer, thereby affecting the impedance of the electrode. The detection of D-serine can be achieved based on the change in the electrical impedance of the electrode.

[0052] 0.1μM thiol-modified nucleic acid aptamer 6.7 was bound to the disk electrode. Using L-serine as a control, D-serine and L-serine were prepared into 10pM-1mM concentration solutions for specificity testing of the electrical impedance biosensor. The electrode was incubated with serine solutions of different concentrations for 3-5min, and then the electrical impedance was measured.

[0053] 3) Identification of the specificity of nucleic acid aptamers binding to D-serine by the swirl method

[0054] Based on the evanescent wave sensor with aptamer immobilization, the in situ enrichment of D-serine on the optical fiber surface and the in situ purification of target D-serine by the modified aptamer on the optical fiber are used to detect the target D-serine by the aptamer. 500nM D-serine is immobilized on the optical fiber, and the Cy5.5-labeled aptamer 6.7 is configured with a concentration of 0-160nM. The sample volume is 300μL. The fluorescently modified aptamer in the sample specifically binds to the D-serine immobilized on the optical fiber surface. After the fluorescent group enters the evanescent wave field, it is excited by the evanescent wave to produce fluorescence. As the concentration of aptamer in the sample increases, the number of aptamers bound to Hcy on the optical fiber increases, and the fluorescence signal increases. The curve of fluorescence intensity changing with the concentration of aptamer is plotted and fitted with formula (1) to obtain KD.

[0055]

[0056] Where Bmax is the maximum fluorescence signal value obtained by fitting, and KD is the dissociation constant.

[0057] 3. Identification of specificity and affinity of nucleic acid aptamers

[0058] 1) Screening and identification of DNA aptamers for D-serine

[0059] The SELEX technology was used to perform specific screening with D-serine as the target. After 6 rounds of screening, a ssDNA library that specifically binds to D-serine was obtained. Subsequently, the 4th and 6th rounds of enrichment libraries were subjected to high-throughput sequencing. The RNA structure software was used to analyze the primary and secondary structures of the sequences and 7 candidate sequences were selected. The candidate sequences were further identified for affinity and specificity.

[0060] 2. Experimental Results

[0061] An irrelevant sequence was selected as the control sequence. The magnetic bead adsorption subtraction experiment found that compared with the control sequence, the CT value of the supernatant after incubation of aptamer 6.7 (sequence shown in Table 1) with D-serine was higher during qPCR, while the control sequence did not change, indicating that aptamer 6.7 can specifically bind to D-serine ( Figure 1 ).

[0062] Table 1. Aptamer 6.7 sequence

[0063]

[0064] D-serine was diluted to the corresponding concentrations of 100pM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, and 1mM with binding buffer solution, and L-serine was diluted to the corresponding concentrations of 10nM, 100nM, 1μM, 10μM, and 100μM, and incubated with the blocked aptamer 6.7 electrode for 5 minutes. After washing three times with binding buffer solution, the electrical impedance test was performed. The results showed that aptamer 6.7 can specifically recognize D-serine, but not L-serine ( Figure 2 ).

[0065] The results of the test showed that as the concentration of specific aptamers in the sample increased, the number of aptamers bound to D-serine on the optical fiber increased, and the fluorescence signal increased. The curve of fluorescence intensity versus aptamer concentration was plotted and fitted with formula (1) to obtain KD. The results showed that the dissociation constant KD value of aptamer 6.7 and D-serine was about 339nM ( Figure 3 ).

[0066] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a nucleic acid aptamer for detecting D-serine or in preparing a product for detecting D-serine, wherein the sequence of the nucleic acid aptamer has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2; Preferably, the sequence of the nucleic acid aptamer is as shown in SEQ ID NO: 2; Preferably, the product includes a test strip or a test kit.

2. The use according to claim 1, characterized in that: The nucleic acid aptamer also includes an RNA sequence transcribed from its DNA sequence.

3. The use according to claim 1, characterized in that: The nucleic acid aptamer also includes its derivatives.

4. The use according to claim 3, characterized in that: The derivative is formed by connecting or modifying the nucleic acid aptamer with other substances.

5. The use according to claim 4, characterized in that: The other substances include one or more of fluorescent labels, isotope labels, therapeutic substances, enzyme labels, and biotin labels.

6. The use according to claim 4, characterized in that: The modification includes one or more modified nucleotides, and / or one or more substituted nucleotides.

7. The use according to claim 6, characterized in that: The modified or substituted positions include one or more of a ribose position, a deoxyribose position, a phosphate position and a base position.

8. A nucleic acid aptamer, characterized in that The sequence of the nucleic acid aptamer has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2; Preferably, the sequence of the nucleic acid aptamer is as shown in SEQ ID NO:

2.

9. A method for detecting D-serine, characterized in that: The method comprises contacting a sample with the nucleic acid aptamer according to claim 1 or the nucleic acid aptamer according to claim 8, thereby detecting D-serine in the sample.

10. Use of the nucleic acid aptamer according to claim 1 or 8 in inhibiting D-serine or in preparing a product for inhibiting D-serine.

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