Nucleic acid aptamer for the identification of d-serine

Abstract

The application provides a nucleic acid aptamer for identifying D-serine. The nucleic acid aptamer has high affinity and high specificity for D-serine, and can be used for detecting D-serine, has high detection sensitivity, and has low detection cost.

Description

Technical Field

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

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

[0003] D-serine is an important endogenous neuromodulator that plays a key role in the central nervous system and neurotransmission. Developing nucleic acid aptamers targeting D-serine can help researchers delve deeper into its functions and activities in vivo. Summary of the Invention

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

[0005] To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] A first aspect of the present invention provides 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] A second aspect of the present invention provides a nucleic acid aptamer, wherein the sequence of the nucleic acid aptamer 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 according to 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 according to the first or 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 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 graph of candidate aptamer 6.7 and D-serine identified by qPCR method;

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

[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 following provides definitions of some terms used in this specification. Unless defined otherwise, 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 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 label includes but is 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 standard D), 13 C. 15 N. 17 O. 18 O. 34 S.

[0034] Therapeutic substances refer to substances with medicinal, pharmacological, psychosocial 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 generally 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 the oligonucleotide is an analog or ester of a naturally occurring nucleotide. In some embodiments, modified nucleotides can make the oligonucleotide resistant to nucleases. In some embodiments, the modified nucleotides mainly cause the hydrophobic interaction of the aptamer with its protein target, 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., α-anomeric nucleic acids, etc.). In addition, any of the hydroxyl groups typically present on the sugar of a nucleotide can be replaced with a phosphonate or phosphate group; protected with standard protecting groups; or activated to form additional bonds with additional nucleotides or a solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with an amine, an organic end-capping moiety having from about 1 to about 20 carbon atoms, a polyethylene glycol (PEG) polymer in the range of from about 10 kDa to about 80 kDa in some embodiments, a PEG polymer in the range of from 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 direct amide linkage at the C-5 position or by other types of linkages.

[0038] The present invention will be further described below with reference to 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 present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0039] Example

[0040] 1. Experimental Methods

[0041] 1. SELEX screening

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

[0043] 2) Screening for specific 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 adding counter-screening steps, thereby obtaining aptamers that recognize D-serine.

[0044] a. Incubate D-serine with the ssDNA library at room temperature for 1 hour. Then, capture the D-serine and its bound ssDNA using activated carboxyl magnetic beads. Wash the beads four times with buffer. Finally, elute the oligonucleotide ligands that specifically bind to the target protein. A secondary library is prepared by PCR amplification for the next round of screening.

[0045] b. The eluted ssDNA obtained during the fourth and sixth rounds of library identification 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] 20 pmol of the identified aptamer and 60 pmol of D-serine were added to the screening buffer and incubated at room temperature for 1 hour. Carboxyl-activated magnetic beads were then added and incubated for another 30 minutes. The beads were discarded, and only the supernatant was used as a qPCR template. The CT value was recorded to determine whether the aptamer bound 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 immobilization is used for detection, when the nucleic acid aptamer immobilized 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 electrode electrical impedance.

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

[0053] 3) Identification of the specificity of nucleic acid aptamers binding to D-serine using the Epstein-Barr method

[0054] Based on an evanescent wave sensor immobilized with aptamers, the in situ enrichment of D-serine on the optical fiber surface and the in situ purification of the target D-serine by the modified aptamer on the optical fiber are utilized to detect the target D-serine by the aptamer. 500 nM D-serine was immobilized on the optical fiber, and aptamer 6.7 labeled with Cy5.5 was prepared at a concentration of 0-160 nM in a 300 μL sample volume. The fluorescently modified aptamer in the sample specifically bound to the D-serine immobilized on the optical fiber surface. After the fluorescent group entered the evanescent wave field, it was excited by the evanescent wave to produce fluorescence. As the aptamer concentration in the sample increased, the number of aptamers bound to Cy on the optical fiber increased, and the fluorescence signal increased. A curve of fluorescence intensity versus aptamer concentration was plotted and fitted using 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 targeting D-serine

[0059] Using SELEX technology, D-serine was used as a target for specific screening. After six rounds of screening, a ssDNA library specifically binding to D-serine was obtained. The enriched libraries were then subjected to high-throughput sequencing in rounds 4 and 6. RNA structure software was used to analyze the primary and secondary structures of the sequences and identify seven candidate sequences. These candidate sequences were further evaluated for affinity and specificity.

[0060] 2. Experimental Results

[0061] An irrelevant sequence was selected as the control sequence. The magnetic bead adsorption subtraction experiment showed 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 in 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 concentration of 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM and L-serine was diluted to the corresponding concentration of 10 nM, 100 nM, 1 μM, 10 μM, 100 μM with binding buffer solution, respectively, and incubated with the blocked aptamer 6.7 electrode for 5 min. After washing three times with binding buffer solution, impedance test was performed. The results showed that aptamer 6.7 could specifically recognize D-serine and did not recognize L-serine. Figure 2

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

[0066] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.​​

Claims

1. Use of a nucleic acid aptamer for detecting D-serine for non-diagnostic purposes or in preparing a product for detecting D-serine, wherein the sequence of the nucleic acid aptamer is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The products include test strips and test kits.

3. The use according to claim 1, characterized in that The nucleic acid aptamer also includes its derivatives; The derivative is formed by connecting or modifying the nucleic acid aptamer with other substances; The other substances include one or more of fluorescent labels, isotope labels, and biotin labels.

4. A nucleic acid aptamer, characterized in that The sequence of the nucleic acid aptamer is shown in SEQ ID NO:

2.

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

Images