Nucleic acid aptamer recognizing d-serine and use thereof

Abstract

The application discloses a nucleic acid aptamer for recognizing D-serine and application thereof. The nucleic acid aptamer for specifically combining 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 the D-serine is shortened, the rapid detection effect can be achieved, and the nucleic acid aptamer has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to nucleic acid aptamers that recognize D-serine and their applications. Background Technology

[0002] D-serine is an endogenous brain signaling molecule that can be used to prevent or treat brain injury caused by ischemia or hypoxia. D-serine is also an important intermediate for the pharmaceutical intermediates D-cycloserine and lacosamide. Cycloserine is a broad-spectrum antibiotic with some efficacy against tuberculosis, non-tuberculous respiratory infections, otitis media, and diarrhea. Clinically, it is mainly used to treat Mycobacterium tuberculosis, especially for multidrug-resistant tuberculosis, where market demand is significant. In addition, cycloserine also has a large market potential abroad as a psychotropic drug. Currently, there is no effective detection method for D-serine.

[0003] Nucleic acid aptamers are oligonucleotide fragments obtained from in vitro screening using exponential enrichment ligand evolution technology. They possess biological characteristics such as low cost and modifiability, and can rapidly and specifically bind closely to their corresponding targets, specifically recognizing various targets. However, there are currently no reports on nucleic acid aptamer sequences targeting D-serine and their applications. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a nucleic acid aptamer that recognizes D-serine and its application.

[0005] To achieve the above objectives, 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 the application of the nucleic acid aptamer described in the first aspect of the present invention in the detection of D-serine.

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

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

[0011] Furthermore, the detectable reagents include fluorescent labels, radioactive isotopes, chemiluminescent molecules, paramagnetic ions, or spin-trapping reagents.

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

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

[0014] Furthermore, the products include reagent kits and test strips.

[0015] A 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 described is not for diagnostic purposes.

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

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

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

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

[0021] This application is the first to discover a nucleic acid aptamer that specifically binds to D-serine. This nucleic acid aptamer can specifically recognize D-serine but not L-serine, thus shortening the detection time of D-serine and achieving rapid detection, which has broad application prospects. Attached Figure Description

[0022] Figure 1 This is a qPCR diagram illustrating the specific binding of candidate aptamer 6.2 to D-serine.

[0023] Figure 2 This is a diagram showing the specific binding of the aptamer 6.2 sequence to D-serine using electrochemical (electrical impedance) methods.

[0024] Figure 3 This is a KD diagram showing the binding constant of aptamer 6.2 sequence to D-serine determined by evanescent wave method. Detailed Implementation

[0025] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, 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 pertains.

[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, the terms "nucleic acid aptamer" and "aptamer" are used interchangeably, referring to nucleic acid molecules capable of specifically binding to target molecules. In the embodiments of this application, as long as the nucleic acid aptamer has the function of specifically binding to D-serine, there is no upper limit to its length. As long as it does not impair the function of the nucleic acid aptamer, the same sequence can be linked with different base sequences or nucleic acid aptamers.

[0028] In some embodiments, specificity refers to the ability of the nucleic acid aptamer involved in this application to bind to D-serine without binding to other serines.

[0029] In some embodiments, a nucleotide refers to a glycoside comprising a sugar moiety, a base moiety, and a covalently linked group (linking group), such as a phosphate ester or thiophosphate nucleotide linking group, and includes naturally occurring nucleotides such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and / or base moieties.

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

[0031] This invention provides the application of the above-mentioned nucleic acid aptamer in the detection of D-serine.

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

[0033] In some embodiments, a functional group refers to a compound suited to perform at least one function. The function, without limitation, includes the ability to specifically bind D-serine or other receptor D-serine, the ability to inhibit D-serine or other receptor D-serine, and direct and indirect detectability. Those skilled in the art will understand that a functional group can be associated with one or more functions. Non-limiting examples of functional groups include detectable reagents.

[0034] The detectable reagents include fluorescent labels, radioactive isotopes, chemiluminescent molecules, paramagnetic ions, or spin-trapping reagents.

[0035] The fluorescent markers include, but are not limited to, one or more of the following: 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, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, or Texas Red.

[0036] Radioactive isotopes, including but 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 them.

[0037] Paramagnetic ions include, but are not limited to, ions of chromium (III), manganese (II), iron (III), 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] This invention provides the application of nucleic acid aptamers in the preparation of products for detecting D-serine.

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

[0040] In some embodiments, the kit includes one or more reagents for various assays, including, for example, immunoassays such as ELISA (sandwich or competitive forms). Kit components may be pre-attached to a solid support, or applied to the surface of a solid support when the kit is used. In some embodiments, the signal generation means may be pre-bound to the nucleic acid aptamers of this application, or may need to be combined with one or more components such as buffers, antibody-enzyme conjugates, enzyme substrates, etc., before use. The kit may also include additional reagents, such as blocking reagents, washing reagents, enzyme substrates, etc., to reduce non-specific binding to the solid surface. The solid surface may be in the form of tubes, beads, microtiter plates, microspheres, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In certain aspects, enzymes that catalyze the formation of chemiluminescent or chromogenic products or the reduction of chemiluminescent or chromogenic substrates are components of the signal generation means. Such enzymes are well known in the art. The kit may include any of the capture and detection reagents described in this application. Optionally, the kit may also include instructions for carrying out the methods of the present invention.

[0041] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0042] Example

[0043] I. Experimental Methods

[0044] 1. SELEX Filtering

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

[0046] 2) Specific nucleic acid aptamers are screened through steps such as incubation, washing, elution, and amplification. As the number of screening rounds increases, the screening pressure is gradually increased by reducing the amount of ssDNA input and appropriately increasing the reverse screening step, thereby obtaining nucleic acid aptamers that recognize D-serine.

[0047] a. Incubate the D-serine and ssDNA library at room temperature for 1 hour, then capture the D-serine and its bound ssDNA with activated carboxyl magnetic beads, wash with buffer 4 times, and finally elute the oligonucleotide ligands that specifically bind to the target protein. Prepare a secondary library by PCR amplification for the next round of screening.

[0048] 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 and then sequenced.

[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 the specificity and affinity of nucleic acid aptamers

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

[0052] Add 20 pmol of the identified nucleic acid aptamer and 60 pmol of D-serine 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 use only the supernatant as a qPCR template. Record the CT value to determine whether the nucleic acid aptamer binds to D-serine.

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

[0054] When using a biosensor based on nucleic acid aptamer immobilization for D-type serine detection, the binding of the nucleic acid aptamer immobilized on the electrode surface to D-type serine affects the secondary structure of the nucleic acid aptamer, thereby affecting the electrode impedance. The detection of D-type serine can be achieved by observing the change in electrode impedance.

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

[0056] 3) Streaming method for identifying the specificity of nucleic acid aptamer binding to D-serine.

[0057] Based on the evanescent wave sensor with nucleic acid aptamer immobilization, this study utilizes the in-situ enrichment of D-serine on the fiber surface and the in-situ purification of target D-serine by the nucleic acid aptamer modified on the fiber to achieve the detection of target D-serine by the aptamer. 500 nM of D-serine was immobilized on the fiber, and Cy5.5-labeled nucleic acid aptamer 6.2 with a concentration of 0-160 nM was prepared, with a sample volume of 300 μL. The fluorescently modified nucleic acid aptamer in the sample specifically binds to the D-serine immobilized on the fiber surface. After the fluorescent group enters the evanescent wave field, it is excited by the evanescent wave to generate fluorescence. As the concentration of nucleic acid aptamer in the sample increases, the number of nucleic acid aptamers binding to Hcy on the fiber increases, and the fluorescence signal is enhanced. A curve of fluorescence intensity changing with the concentration of nucleic acid aptamer was plotted and fitted with formula (1) to obtain KD.

[0058]

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

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

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

[0062] Using SELEX technology with D-serine as the target, specific screening was performed. After six rounds of screening, ssDNA libraries specifically binding to D-serine were obtained. Subsequent rounds 4 and 6 of enrichment library sequencing were then performed using high-throughput sequencing. The primary and secondary structures of the sequences were analyzed using RNA structure software, and seven candidate sequences were selected. Further affinity and specificity assessments were conducted on the candidate sequences.

[0063] II. Experimental Results

[0064] Using irrelevant sequences as control sequences, magnetic bead adsorption subtraction experiments revealed that, compared to the control sequence, aptamer 6.2 (sequence shown in Table 1) showed a higher CT value in the supernatant after incubation with D-serine during qPCR, while the control sequence showed no change. This indicates 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 corresponding concentrations of 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM using binding buffer, and L-serine was diluted to corresponding concentrations of 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM. These were then incubated with the blocked aptamer 6.2 electrode for 5 min. After washing three times with binding buffer, electrical impedance testing was performed. The results showed that aptamer 6.2 specifically recognized D-serine but not L-serine. Figure 2 ).

[0068] The fluorescence signal was determined using an evanescent wave sensor based on D-serine immobilization. Test results showed that as the concentration of the specific aptamer in the sample increased, the number of nucleic acid aptamers binding to D-serine on the optical fiber increased, leading to enhanced fluorescence signal. A curve of fluorescence intensity versus nucleic acid aptamer concentration was plotted and fitted using formula (1) to obtain the KD value. The results showed that the dissociation constant KD value between nucleic acid aptamer 6.2 and D-serine was approximately 85 nM. Figure 3 ).

[0069] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The application of nucleic acid aptamers in the detection of D-serine for non-diagnostic purposes, wherein the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The nucleic acid aptamer is linked to a functional group.

3. The application according to claim 2, characterized in that, The functional groups include detectable reagents.

4. The application according to claim 3, characterized in that, The detectable reagents include fluorescent labels, radioactive isotopes, chemiluminescent molecules, paramagnetic ions, or spin-trapping reagents.

5. Application of nucleic acid aptamers in the preparation of products for detecting D-serine, wherein the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO:

2.

6. The application according to claim 5, characterized in that, The products include reagent kits and test strips.

7. A method for detecting D-serine for non-diagnostic purposes, characterized in that, The method includes contacting a sample with a nucleic acid aptamer to detect D-serine in the sample, wherein the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO:2.

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