Nucleic acid aptamer for identifying D-serine and application thereof

Abstract

The invention discloses a nucleic acid aptamer for recognizing D-serine and application of the nucleic acid aptamer. The nucleic acid aptamer specifically bound with D-serine is found for the first time, the nucleic acid aptamer can specifically recognize D-serine and does not recognize L-serine, the detection time of D-serine is shortened, the effect of rapid detection can be achieved, and the nucleic acid aptamer has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a nucleic acid aptamer for recognizing D-serine and an application thereof. Background Art

[0002] D-serine is an endogenous brain information co-transmitter that can be used to prevent or treat brain damage caused by cerebral ischemia or hypoxia. D-serine is also an important intermediate of important pharmaceutical intermediates D-cycloserine and lacosamide. Cycloserine is a broad-spectrum antibiotic that has certain effects on tuberculosis, non-tuberculous respiratory infections, otitis media, diarrhea, etc. It is mainly used clinically to fight tuberculosis bacteria, especially for "multi-drug resistant" tuberculosis, and the market demand is large. In addition, cycloserine also has a large market space abroad as a psychotropic drug. There is currently no effective detection method for D-serine.

[0003] Aptamers are oligonucleotide fragments obtained by in vitro screening through exponential enrichment ligand evolution technology. They have biological properties such as low cost and modifiability. They can quickly and specifically bind to the corresponding targets and specifically recognize various targets. However, there are currently no reports on aptamer sequences for D-serine and their applications. 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 and its application.

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

[0006] A first aspect of the present invention provides a nucleic acid aptamer that specifically binds to D-serine, wherein the nucleotide 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 nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO:2.

[0008] The second aspect of the present invention provides use of the nucleic acid aptamer described in the first aspect of the present invention in detecting D-serine.

[0009] Furthermore, the nucleic acid aptamer is connected to a functional group.

[0010] Furthermore, the functional group includes a detectable agent.

[0011] Furthermore, the detectable agent includes a fluorescent label, a radioisotope, a chemiluminescent molecule, a paramagnetic ion or a spin-trapping agent.

[0012] Furthermore, the detection of D-serine is for non-diagnostic purposes.

[0013] The third aspect of the present invention provides use of the nucleic acid aptamer described in the first aspect of the present invention in the preparation of a product for detecting D-serine.

[0014] Furthermore, the product includes a test kit and a test paper.

[0015] The fourth aspect of the present invention provides a method for detecting D-serine, the method comprising contacting a sample with the nucleic acid aptamer described in the first aspect of the present invention, thereby detecting D-serine in the sample.

[0016] Furthermore, the method is a method for non-diagnostic purposes.

[0017] The fifth aspect of the present invention provides use of the nucleic acid aptamer described in the first aspect of the present invention in inhibiting D-serine.

[0018] Furthermore, the inhibition of D-serine is inhibition of D-serine for non-therapeutic purposes.

[0019] The sixth aspect of the present invention provides use of the nucleic acid aptamer described in the first aspect of the present invention in the preparation of a product that inhibits D-serine.

[0020] Advantages and beneficial effects of the present invention:

[0021] The present application discovered for the first time a nucleic acid aptamer that specifically binds to D-serine. The nucleic acid aptamer can specifically recognize D-serine but not L-serine, thereby shortening the detection time of D-serine, achieving a rapid detection effect, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the specific binding diagram of candidate aptamer 6.2 and D-serine identified by qPCR method;

[0023] Figure 2 This is the specific binding diagram of aptamer 6.2 sequence and D-serine identified by electrochemical (electrical impedance method);

[0024] Figure 3 This is a graph showing the binding constant KD of aptamer 6.2 sequence and D-serine determined by the evanescent wave method. DETAILED DESCRIPTION

[0025] The following provides definitions of some terms used in this specification. 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.

[0026] The present invention provides a nucleic acid aptamer that specifically binds to D-serine, wherein the nucleotide sequence of the nucleic acid aptamer has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:2.

[0027] In some embodiments, nucleic acid aptamers and aptamers, aptamers can be used interchangeably, and refer to nucleic acid molecules that can specifically bind to target molecules. In the embodiments of the present application, as long as nucleic acid aptamers have the function of specifically binding to D-serine, there is no upper limit to their length, and as long as the function of nucleic acid aptamers is not hindered, the same sequence can also be connected to different base sequences and nucleic acid aptamers.

[0028] In some embodiments, specificity refers to the ability of the nucleic acid aptamer involved in the present application to bind to D-serine but not to other serines.

[0029] In some embodiments, a nucleotide refers to a glycoside comprising a sugar portion, a base portion and a covalently linked group (linker), such as a phosphate or phosphorothioate internucleotide linker, and includes naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and / or base portions.

[0030] In some embodiments, identity or homology refers to the sequence similarity between two nucleic acid aptamers. Identity can be determined by comparing the positions in each sequence that can be compared for comparison purposes. When the equivalent positions in the compared sequences are occupied by the same bases, the molecules are identical at that position; when the equivalent sites are occupied by the same or similar nucleic acid residues (e.g., similar in space and / or electronic properties), the molecules can be referred to as being homologous at that position.

[0031] The present invention provides application of the nucleic acid aptamer in detecting D-serine.

[0032] The nucleic acid aptamer is connected to a functional group.

[0033] In some embodiments, functional groups refer to compounds suitable for performing at least one function. The functions include, but are not limited to, the ability to specifically bind D-serine or other receptor D-serine, the ability to inhibit D-serine or other receptor D-serine, the ability to detect directly and indirectly, etc. It will be understood by those skilled in the art that functional groups can be associated with one or more functions. Non-limiting examples of functional groups include detectable agents.

[0034] The detectable agents include fluorescent labels, radioisotopes, chemiluminescent molecules, paramagnetic ions or spin-trapping agents.

[0035] Among them, the fluorescent label includes but is not limited to one or more of Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, OregonGreen 514, Pacific Blue, REG, rhodamine green, rhodamine red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine or Texas Red.

[0036] Radioactive isotopes include but are not limited to astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 Eu, Gallium 67 , 3 Hydrogen, iodine 123 ,iodine 125 ,iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, technetium 99m (technicium) or yttrium 90 One or more of the .

[0037] Paramagnetic ions include, but are not limited to, ions of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III).

[0038] The invention provides application of nucleic acid aptamers in preparing products for detecting D-serine.

[0039] The products include test kits and test strips.

[0040] In some embodiments, the kit includes one or more reagents for various detection assays, including, for example, immunoassays such as ELISA (sandwich or competitive formats). The components of the kit can be pre-attached to a solid support, or when the kit is used, can be applied to the surface of a solid support. In some embodiments, the signal generation means can be pre-bound to the nucleic acid aptamer of the present application, or may need to be combined with one or more components such as buffer, antibody-enzyme conjugate, enzyme substrate, etc. before use. The kit can also include additional reagents, such as blocking reagents, washing reagents, enzyme substrates, etc. for reducing non-specific binding to the solid surface. The solid surface can be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materials suitable for fixing proteins, peptides, or polypeptides. In particular aspects, an enzyme that catalyzes chemiluminescence or color development product formation or chemiluminescence or color development substrate reduction is a component of the signal generation means. Such enzymes are well known in the art. The kit can include any capture reagent and detection reagent described in the present application. Optionally, the kit can also include instructions for carrying out the method of the present invention.

[0041] 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.

[0042] Example

[0043] 1. Experimental Methods

[0044] 1. SELEX screening

[0045] 1) Design a random oligonucleotide library and deliver it to a company for synthesis.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 2. Identification of specificity and affinity of nucleic acid aptamers

[0051] 1) Preliminary identification of aptamers binding to D-serine using qPCR

[0052] 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.

[0053] 2) Electrochemical method to identify the specificity of nucleic acid aptamer binding to D-serine

[0054] 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.

[0055] 0.1 μM thiol-modified nucleic acid aptamer 6.2 was bound to the disk electrode. Using L-serine as a control, D-serine and L-serine were prepared into 10 pM-1 mM concentration solutions for specificity testing of the electrical impedance biosensor. The electrode was incubated with serine solutions of different concentrations for 3-5 minutes, and then the electrical impedance was measured.

[0056] 3) Identification of the specificity of nucleic acid aptamers binding to D-serine by the swirl method

[0057] 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 6.2 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 versus aptamer concentration is drawn and fitted with formula (1) to obtain KD.

[0058]

[0059] Where Bmax is the maximum fluorescence signal value obtained by fitting, and KD is the dissociation constant.

[0060] 3. Identification of specificity and affinity of nucleic acid aptamers

[0061] 1) Screening and identification of DNA aptamers for D-serine

[0062] 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.

[0063] 2. Experimental Results

[0064] 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 aptamer 6.2 (sequence shown in Table 1) incubated with D-serine was higher during qPCR, while the control sequence did not change, indicating that aptamer 6.2 can specifically bind to D-serine ( Figure 1 ).

[0065] Table 1. Aptamer 6.2 sequence

[0066]

[0067] 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 6.2 electrode for 5 minutes. After washing three times with binding buffer solution, the electrical impedance test was performed. The results showed that aptamer 6.2 can specifically recognize D-serine, but not L-serine ( Figure 2 ).

[0068] The evanescent wave sensor based on D-serine fixation was used for measurement: The test results 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 using formula (1) to obtain the KD value. The results showed that the dissociation constant KD value of aptamer 6.2 and D-serine was about 85nM ( Figure 3 ).

[0069] 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. A nucleic acid aptamer that specifically binds to D-serine, characterized in that: The nucleotide sequence of the nucleic acid aptamer has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2; Preferably, the nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO:

2.

2. Use of the nucleic acid aptamer according to claim 1 in detecting D-serine.

3. The use according to claim 2, characterized in that: The nucleic acid aptamer is connected to a functional group.

4. The use according to claim 3, characterized in that: The functional group includes a detectable agent.

5. The use according to claim 4, characterized in that: The detectable agents include fluorescent labels, radioisotopes, chemiluminescent molecules, paramagnetic ions or spin-trapping agents.

6. Use of the nucleic acid aptamer according to claim 1 in preparing a product for detecting D-serine.

7. The use according to claim 6, characterized in that: The products include test kits and test strips.

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

9. Use of the nucleic acid aptamer according to claim 1 in inhibiting D-serine.

10. Use of the nucleic acid aptamer according to claim 1 in preparing a product that inhibits D-serine.

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