CN113151283B - ssDNA aptamer for specifically recognizing N-acetylneuraminic acid and application thereof - Google Patents
ssDNA aptamer for specifically recognizing N-acetylneuraminic acid and application thereof Download PDFInfo
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Abstract
The invention relates to a ssDNA aptamer for specifically recognizing N-acetylneuraminic acid and application thereof, wherein 4 ssDNA aptamer sequences capable of being highly specifically combined with Neu5Ac are obtained by screening through a magnetic bead-SELEX technology, and ap1 has the highest affinity and specificity and a stable structure. Truncating the sequence on the basis of the ap1 sequence according to a secondary structure and a molecular simulation docking result to obtain 1 aptamer ap2 with the sequence length of 21nt, wherein the affinity is improved compared with that before truncation. The ssDNA aptamer of the invention has high specificity and affinity for binding with Neu5Ac, and has high sensitivity when being applied to Neu5Ac detection.
Description
Technical Field
The invention relates to the fields of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry, in particular to ssDNA aptamers for specifically recognizing N-acetylneuraminic acid and applications thereof.
Background
N-acetylneuraminic acid (Neu 5 Ac) is the most studied and most profound sialic acid, a precursor for the synthesis of other sialic acid species. Sialic acid is an important component of glycoconjugates, and negative charges carried by sialic acid can promote mutual repulsion between cells, so that the sialic acid plays roles in stabilizing cell membranes, increasing the viscosity of coupled glycoproteins and the like. Sialic acid is also an important biological information transfer molecule, such as participating in recognition of host-pathogen and cell-cell reactions, phagocytosis of senescent erythrocytes by macrophages, and the like. It has been previously demonstrated that, on the one hand, aberrant expression of Neu5Ac in glycoproteins is strongly associated with various cancers; on the other hand, the number of free sialic acid and lipid and protein bound sialic acid in the plasma of cancer patients is higher than in healthy individuals. Sialic acid has also been found to be an important component of brain gangliosides, which play a critical role in the transmission and storage of information in the brain, and Neu5Ac plays a very important role in brain development and in the normal function of cell membranes, membrane receptors and subcellular organelles. Therefore, how to accurately identify Neu5Ac and further carry out tracking detection on Neu5Ac monomers and biomacromolecules modified by the Neu5Ac monomers is greatly helpful for the diagnosis and treatment of diseases.
Currently, the detection methods reported at home and abroad for Neu5Ac mainly comprise high performance liquid chromatography, polarography, spectrophotometry, thin layer chromatography, chemiluminescence, immunoassay, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, voltammetry and the like. The instrument analysis method has high sensitivity and good accuracy, but the equipment is expensive and difficult to popularize, the requirement on experimenters is high, the pretreatment on samples is complex, the analysis is time-consuming, and the portable and efficient field detection is difficult to meet. The main method for detecting Neu5Ac by an immunoassay method is an enzyme-linked immunosorbent assay. The enzyme linked immunosorbent assay is a detection and analysis technology which detects Neu5Ac by using combined antibody-antigen immunoreaction and enzyme-catalyzed reaction, forms an enzyme-labeled complex by combining an antibody (antigen) and enzyme, catalyzes the reaction of the enzyme-labeled complex in a corresponding substrate, and achieves the detection purpose by electric signals, optical signals or naked eye identification. At present, some ELISA kit products for detecting Neu5Ac residues exist in the market, but the price is high, the types of enzyme-labeled antibodies are limited, and Neu5Ac is a small molecular compound, so that the immunogenicity is not possessed, and the production and the application are limited. These have all limited the use of immunoassay methods for Neu5Ac detection to a large extent.
Disclosure of Invention
In order to solve the technical problems, the invention provides the ssDNA aptamer for specifically recognizing the N-acetylneuraminic acid, which has strong stability, high specific binding with Neu5Ac and high affinity.
The ssDNA aptamer specifically recognizing the N-acetylneuraminic acid has one of sequences (ap 1-5) shown in SEQ ID NO. 1-5.
Of the above ap1 and ap3-5 sequences, the ap1 sequence has the highest affinity. According to the secondary structure and the result of molecular simulation docking, the binding sites of the ap1 sequence and Neu5Ac are all concentrated in the random sequence N35 region, the length of the stem region of the secondary structure is shortened, a ssDNA aptamer with the length of 21nt, namely an ap2 sequence, is obtained, and the affinity is improved. The aptamer sequence is truncated and optimized, the binding site of the aptamer sequence and the target molecule is reserved, a large number of redundant sequences are deleted, and the synthesis cost can be greatly reduced.
Further, the 3 'end or 5' end of the ssDNA aptamer is modified with a functional group or molecule.
Further, the functional group or molecule is a fluorescent group, an isotope, an electrochemical label, an enzyme label, an affinity ligand or a thiol group. The functional groups or molecules are used to increase the stability of the aptamer, provide a detectable signal, or to link the aptamer with other substances to form a composition.
The invention also claims the application of the ssDNA aptamer in Neu5Ac detection.
A composition for detecting Neu5Ac, comprising one or more of the ssDNA aptamers described above.
A kit for detecting Neu5Ac, comprising one or more of the ssDNA aptamers described above.
A test strip for detecting Neu5Ac, comprising one or more of the ssDNA aptamers described above.
A chip for detecting Neu5Ac, comprising one or more of the ssDNA aptamers described above.
A fluorescent biosensor for detecting Neu5Ac comprising one or more of the ssDNA aptamers described above; also included are fluorophores and fluorescence quenchers.
Further, the fluorescence quencher is graphene oxide.
The method comprises the steps of constructing a fluorescence biosensor by using ssDNA aptamers and Graphene Oxide (GO) to detect Neu5Ac, incubating the ssDNA aptamers modified with fluorophores with the GO together, adsorbing the ssDNA aptamers and quenching the fluorophores by the GO, and competing the ssDNA aptamers marked by the fluorophores from the surface of the GO when the target molecule Neu5Ac exists, so as to recover fluorescence, and realizing determination of the Neu5Ac by using the quantitative relation between the fluorescence intensity and the concentration of the Neu5 Ac.
By the scheme, the invention at least has the following advantages:
(1) The invention provides a high specificity aptamer sequence with good stability, high affinity, easy preparation, easy modification and marking for Neu5Ac detection.
(2) The detection method established by the invention has high sensitivity, strong specificity and low cost.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a schematic diagram of magnetic bead-SELEX screening of Neu5 Ac-specific ssDNA aptamers;
FIG. 2 shows K for ssDNA aptamer sequences ap1, ap3, ap4 and ap5 d A fitted graph of values;
FIG. 3 is a schematic diagram of a fluorescence biosensor constructed in example 4;
FIG. 4 shows the results of example 3 for fluorescence detection of the specificity of ssDNA aptamer sequence ap 1;
FIG. 5 is a standard graph of a fluorescent biosensor constructed in example 4 using the ssDNA aptamer sequence ap2.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1
(1) Construction of random ssDNA libraries and their primers:
(a) Construction of a random ssDNA library of 79 bases in length
5'-TAGGGAATTCGTCGACGGATCC-N35-CTGCAGGTCGACGCATGCGCCG-3', wherein N represents any one of bases A, T, C and G.
(b) Synthesis of the Forward primer
Forward primer 1:5 'TAGGGAATTCGTCGACGGAT-3';
forward primer 2:5'-FAM-TAGGGAATTCGTCGACGGAT-3';
(c) Synthesis of reverse primer
Reverse primer 1:5 'CGGCGCATGCGTCGACCTG-3';
reverse primer 2:5'-biotin-CGGCGCATGCGTCGACCTG-3'.
(2) In vitro screening of aptamers:
in order to screen ssDNA aptamers with high affinity and high specificity to Neu5Ac, 10 rounds of aptamer screening were performed.
(a) The PCR amplification system for 25. Mu.L is shown in Table 1.
TABLE 1 substances and amounts of 25. Mu.L PCR amplification System
| Starting materials | Concentration of | Volume of |
| |
10μmol·L -1 | 1μL |
| Reverse primer 2 | 10μmol·L -1 | 1μL |
| Template DNA | 1μmol·L -1 | 1μL |
| 2×PrimeSTARMax Premix | / | 12.5μL |
| ddH 2 O | / | Make up to 25 μ L system |
Amplification conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s; annealing at 55 ℃ for 30s; extension at 72 ℃ for 15s; extending for 5min at 72 ℃;30 cycles.
(b) Major steps of in vitro screening
Washing streptavidin coupled magnetic beads with PBS buffer for 5 times, dissolving 200 μ L of initial library PCR product in 200 μ L of binding buffer solution, adding streptavidin coupled magnetic beads, shaking gently at room temperature for 2h, placing on a magnetic separator, removing supernatant, washing magnetic beads with binding buffer solution for 5 times, adding 100 mmol. L -1 The Neu5Ac solution was gently shaken at room temperature for 2h and then placed on a magnetic separator for 3min, and the supernatant was collected and put into the next round of screening.
(c) Determination of screening frequency
After each round of screening, 100. Mu.L of the supernatant was collected, 100. Mu.L of the binding buffer was mixed, and the fluorescence (emission wavelength 520nm, excitation wavelength 494 nm) was measured using a multifunction plate reader. The difference between the fluorescence of the experimental and control groups is the fluorescence of the ssDNA sequence bound to Neu5 Ac. The screening process is stopped until there is no further tendency for the fluorescence to increase.
(d) The next round of screening was repeated 10 times according to the above screening method, and the fluorescence intensity tended to be stable after the 4 th round of screening.
(3) Screening the obtained ssDNA clones, sequencing:
and (3) performing PCR amplification on ssDNA obtained by final round screening by using a forward primer 1 and a reverse primer 1, loading the whole amplification product onto 3% agarose, and recovering the PCR product. The purified PCR product was ligated with pMD19-TVector vector with reference to the T vector instructions, and after ligation overnight at 16 ℃, it was transformed into Escherichia coli JM109 and cultured overnight. And verifying a correct transformant through colony PCR and agarose gel, selecting a plurality of positive clones, extracting plasmids of the positive clones for sequence determination, and sequencing to obtain a plurality of aptamers with different sequences ap1 and ap3-ap 5. A schematic diagram of the magnetic bead-SELEX technology for screening Neu5 Ac-specific ssDNA aptamers is shown in FIG. 1.
Example 2
Fluorometric determination of the dissociation constant K of aptamer sequences d Value of
The stability and secondary structure of the multiple aptamer sequences obtained in example 1 were analyzed using Mfold online software. Aptamer sequences with different concentrations and modified by fluorescent group 6-carboxyfluorescein (FAM) are added into a binding buffer solution, the volume of the aptamer sequences is supplemented to 200 mu L by the binding buffer solution, the aptamer sequences are denatured at 90 ℃ for 10min, the aptamer sequences are quickly subjected to ice bath for 10min, and the aptamer sequences are placed at normal temperature for 10min. Adding 10 mu mol Neu5Ac into the solution, slightly shaking, reacting at room temperature for 2h, adding graphene oxide, incubating at room temperature for 2h, and subjecting the mixture to 13000 r/min -1 Centrifuging for 5min, and collecting supernatant. Finally, the whole supernatant is added into a 96-well plate, and the fluorescence intensity is measured by a microplate reader. The amount of aptamer sequence is proportional to the fluorescence intensity.
From the equation: y = Bmax × free ssDNA/(K) d + free ssDNA), K for each aptamer sequence d The values were analyzed. Y in the equation represents the proportion of aptamer-bound Neu5Ac to total Neu5Ac, i.e., saturation; bmax represents the number of maximum binding sites; free ssDNA indicates the concentration of free ssDNA not bound to Neu5 Ac.
The fitted curves are shown in FIG. 2, and K was measured for ap1, ap3, ap4 and ap5 d The values are respectively 87.34. + -. 15.19 nmol. L -1 、393.72±143.16nmol·L -1 、103.63±18.52nmol·L -1 、169.95±41.53nmol·L -1 All have higher affinity, wherein the affinity of ap1 and Neu5Ac is the highest.
Example 3
Specific result of detecting ssDNA aptamer sequence ap1 by fluorescence method
Adding 200 μ L binding buffer solution into aptamer ap1 with the same concentration modified by fluorophore 6-carboxyfluorescein (FAM), denaturing at 90 deg.C for 10min, rapidly ice-cooling for 10min, and standing at room temperature for 10min. Then adding 1 mu mol of Neu5Ac, N-glycolylneuraminic acid (Neu 5 Gc), especiallyAdding a solution of rosoninic acid (KDN), glucose, sucrose and maltose into the solution, slightly shaking, reacting at room temperature for 2h, adding graphene oxide, incubating at room temperature for 2h, and incubating the mixture at 13000 r/min -1 Centrifuging for 5min, and collecting supernatant. Finally, the whole supernatant is added into a 96-well plate, and the fluorescence intensity is measured by a microplate reader.
The specific detection result is shown in fig. 4, and the detected fluorescence intensity is obviously higher than that of other saccharides in the presence of Neu5Ac, thereby proving that ap1 has good specificity.
Example 4
ap1 truncation and detection applications
The results of molecular simulation docking show that the binding sites of ap1 and Neu5Ac are all concentrated in a random sequence region, and ap2 is obtained by performing sequence truncation on ap1 according to a secondary structure. Graphene oxide is used as a fluorescence quencher, a fluorescent group 6-FAM is modified at the 5' end of ap2, and a fluorescence biosensor is constructed, and the principle is shown in FIG. 3. First, 6. Mu.L of 6-FAM labeled aptamer (10. Mu. Mol. L) is taken -1 ) And mixing the solution and a combined buffer solution uniformly to obtain 200 mu L of an aptamer solution, performing thermal denaturation at 95 ℃ for 5min, cooling at 4 ℃ for 10min, mixing the solution and 20 mu L of graphene oxide solution uniformly, and performing shake incubation at room temperature for 30min. Then, the mixture is respectively treated at 12000 r.min -1 Centrifuging for 10min, collecting supernatant, adding into 96-well plate, respectively, and measuring fluorescence intensity with multifunctional microplate reader to obtain standard curve (FIG. 5) with excitation wavelength of 494nm and emission wavelength of 520 nm.
The actual sample was tested using milk, the binding buffer was replaced with milk, and the test method and amount were the same as above.
The ap2 sequence obtained in this example was determined to have K d The value was 55.71. + -. 12.29 nmol.L -1 The affinity is further improved. As can be seen from fig. 5, the fluorescence intensity strongly linearly correlated with Neu5Ac, and therefore, the determination of Neu5Ac was achieved using a quantitative relationship between the fluorescence intensity and the concentration of Neu5 Ac. The recovery rate of ap2 reaches over 90 percent by using milk to test actual samples.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Various other modifications and alterations will occur to those skilled in the art upon reading the foregoing description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Sequence listing
<110> university of south of the Yangtze river
<120> ssDNA aptamer capable of specifically recognizing N-acetylneuraminic acid and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 79
<212> DNA
<213> (Artificial sequence)
<400> 1
tagggaattc gtcgacggat cccgtggcgt ctgcaacgga aaagaattta tcttgtcctg 60
caggtcgacg catgcgccg 79
<210> 2
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 2
ggaaaagaat ttatcttgtc c 21
<210> 3
<211> 79
<212> DNA
<213> (Artificial sequence)
<400> 3
tagggaattc gtcgacggat ccgaatacac tatgactgtc ggaggtccga gtgcgggctg 60
caggtcgacg catgcgccg 79
<210> 4
<211> 79
<212> DNA
<213> (Artificial sequence)
<400> 4
tagggaattc gtcgacggat ccgcaggtat ttcgaggacg cgtttgtaac ggagctcctg 60
caggtcgacg catgcgccg 79
<210> 5
<211> 79
<212> DNA
<213> (Artificial sequence)
<400> 5
tagggaattc gtcgacggat ccgaatacac tatgactgtc ggatgtccga gtgcgggctg 60
caggtcgacg catgcgccg 79
Claims (10)
1. The ssDNA aptamer capable of specifically recognizing the N-acetylneuraminic acid is characterized in that the nucleotide sequence of the ssDNA aptamer is shown as SEQ ID No. 1.
2. The ssDNA aptamer of claim 1, wherein: the 3 'end or the 5' end of the ssDNA aptamer is modified with a functional group or molecule.
3. The ssDNA aptamer of claim 2, wherein: the functional group or molecule is a fluorescent group, an isotope, an electrochemical marker, an enzyme marker, an affinity ligand or a sulfhydryl group.
4. Use of the ssDNA aptamer of any of claims 1-3 in the preparation of an N-acetylneuraminic acid detection product.
5. A composition for detecting N-acetylneuraminic acid, which is characterized in that: comprising one or more of the ssDNA aptamers of any of claims 1-3.
6. A kit for detecting N-acetylneuraminic acid, characterized in that: comprising one or more of the ssDNA aptamers of any of claims 1-3.
7. A test paper for detecting N-acetylneuraminic acid is characterized in that: comprising one or more of the ssDNA aptamers of any of claims 1-3.
8. A chip for detecting N-acetylneuraminic acid, which is characterized in that: comprising one or more of the ssDNA aptamers of any of claims 1-3.
9. A fluorescent biosensor for detecting N-acetylneuraminic acid, characterized in that: comprising one or more of the ssDNA aptamers of any of claims 1-3; also included are fluorophores and fluorescence quenchers.
10. The fluorescence biosensor of claim 9, wherein: the fluorescence quencher is graphene oxide.
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