阅读说明：本技术 心肌肌钙蛋白i特异性核酸适配体及其应用 (Cardiac troponin I specific nucleic acid aptamer and application thereof ) 是由 罗昭锋 方晓娜 何军林 王进军 张立云 于 2019-03-05 设计创作，主要内容包括：本发明提供一种特异性结合心肌肌钙蛋白I(以下简称cTnI蛋白)的单链DNA核酸适配体,其包含SEQ ID Nos.1-3中任一个所示的核苷酸序列。本发明的核酸适配体还可以是各种同源性较高的类似序列或由本发明序列得到的衍生物。本发明的核酸适配体与心肌肌钙蛋白I的结合能力更强,且序列更短,还具有分子量小、易于合成、生产时间短、合成的成本更低等优点。本发明还提供所述单链DNA核酸适配体的应用,它们可以单独地或组合地用于cTnI蛋白纯化或cTnI蛋白检测。本发明还提供一种用于检测cTnI蛋白的试剂盒,其包含本发明的单链DNA核酸适配体。(The invention provides a single-stranded DNA aptamer specifically binding cardiac troponin I (cTnI protein for short), which comprises a nucleotide sequence shown in any one of SEQ ID Nos. 1-3. The nucleic acid aptamers of the present invention may also be various analogous sequences having high homology or derivatives derived from the sequences of the present invention. The aptamer of the invention has stronger binding capacity with cardiac troponin I, has shorter sequence, and also has the advantages of small molecular weight, easy synthesis, short production time, lower synthesis cost and the like. The invention also provides the use of said single-stranded DNA aptamers, which can be used alone or in combination for cTnI protein purification or cTnI protein detection. The present invention also provides a kit for detecting a cTnI protein, which comprises the single-stranded DNA aptamer of the present invention.)

1. An aptamer that specifically binds cardiac troponin I, wherein the aptamer comprises or consists of: a nucleotide sequence shown in any one of SEQ ID nos.1 to 3, or a nucleotide sequence having high homology with the nucleotide sequence shown in any one of SEQ ID nos.1 to 3 and capable of specifically binding to cardiac troponin I, or a nucleotide sequence derived from the nucleotide sequence shown in any one of SEQ ID nos.1 to 3 and capable of specifically binding to cardiac troponin I.

2. The aptamer according to claim 1, wherein the nucleotide sequence of the aptamer of cardiac troponin I is modified, said modification being selected from phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopolation.

3. The aptamer according to claim 1, wherein the nucleotide sequence of the aptamer is linked to a fluorescent label, a radioactive substance, a therapeutic substance, biotin, digoxigenin, a nano-luminescent material, a small peptide, siRNA or an enzyme label.

4. The aptamer according to claim 1, which consists of a nucleotide sequence as set forth in any one of SEQ ID Nos.1 to 3.

5. An aptamer that specifically binds to cardiac troponin I, wherein the aptamer comprises or consists of any one of the following three sequences:

(1) a nucleotide sequence having a homology of 60% or more with the nucleotide sequence of the aptamer according to claim 1;

(2) a nucleotide sequence capable of hybridizing with the nucleotide sequence of the aptamer according to claim 1 under stringent conditions; or

(3) An RNA sequence transcribed from the nucleotide sequence of the aptamer according to claim 1.

6. A nucleic acid aptamer derivative, wherein the derivative is a phosphorothioate backbone sequence derived from the backbone of the nucleotide sequence of the aptamer of any one of claims 1 to 5, or a peptide nucleic acid modified from the aptamer of any one of claims 1 to 5.

7. Use of the aptamer according to any one of claims 1 to 5 or the aptamer derivative according to claim 6 for the preparation of a kit for purifying cardiac troponin I or for detecting cardiac troponin I.

8. A kit, wherein the kit comprises one or more of the aptamer of any one of claims 1 to 5 and the aptamer derivative of claim 6.

9. The kit of claim 8, wherein the kit is for detecting cardiac troponin I levels in serum of a subject.

10. The kit of claim 8, wherein the kit is for diagnosing whether a subject has myocardial cell injury.

Technical Field

The invention relates to the fields of biology and medicine, in particular to a nucleic acid aptamer capable of being used for binding cardiac troponin I (cTnI) and application thereof.

Background

Cardiovascular diseases are one of the important diseases affecting human health in today's society, and acute myocardial infarction is an important cause of death of patients among cardiovascular diseases. Since the abnormality of the myocardial damage marker is one of the main bases for diagnosing myocardial infarction, many diagnostic reagents for serum markers of early myocardial damage exist at the present stage. Cardiac troponin I (cTnI) is a specific protein of cardiac muscle, has special clinical value for detecting tiny myocardial injury, normally, the level of cTnI in circulation is very low, when cardiac muscle cells are injured, the cTnI rapidly enters blood before other biochemical indexes, rises within 3-5 hours, and with the aggravation of injury, the concentration of the cTnI in the blood continuously rises, reaches a peak within 12-36 hours, and forms a longer time window. Numerous studies have shown that cTnI has proven to be one of the most specific and most sensitive serum markers of myocardial cell injury. Therefore, rapid, agile and accurate detection of cTnI has important clinical significance.

The aptamer (aptamer) refers to a DNA or RNA molecule obtained by screening and separating by an exponential enrichment ligand system evolution technology (SELEX), and can be combined with other targets such as proteins, metal ions, small molecules, polypeptides and even whole cells with high affinity and specificity, so that the aptamer has a wide prospect in the aspects of biochemical analysis, environmental monitoring, basic medicine, new drug synthesis and the like. Compared with an antibody, the aptamer has the advantages of small molecular weight, better stability, easy modification, no immunogenicity, short preparation period, artificial synthesis and the like, and a series of processes of animal immunization, feeding, protein extraction and purification and the like are omitted. The resulting aptamer sequence is usually subjected to related modification to obtain better performing aptamers, wherein sequence truncation is an important means, mainly by removing parts that are not essential for target interaction. Several researchers have published the aptamer sequences of some of the cTnI proteins they obtained. However, due to the existing means of aptamer screening, the obtained aptamer is not the simplest sequence to bind to the target, and the affinity and stability are not optimal. In practical application, the cost is increased, the stability is low, and therefore, the cTnI aptamer with shorter sequence, higher affinity and higher stability is required.

Disclosure of Invention

In order to solve the above problems, the present invention provides three cardiac troponin I aptamers and uses thereof. The present inventors have identified a sequence cTnI-14 that specifically binds cTnI based on the sequence cTnI-14 obtained by the SELEX method in their previous applications: 5 'TTCAGCACTCCACGCATAGCTACGGCGGCTACAATGCAGTGGGGAGGGACTTGTTGTAACCCTATGCGTGCTACCGTGAA 3' (80nt) was modified to simplify the sequence by analyzing the critical regions of binding and to remove the unrelated sequences of binding, to obtain the three aptamers to cardiac troponin of the invention and to test their binding ability to the cTnI protein. Compared with the original sequence, the obtained three new sequences have stronger binding capacity, shorter sequence, easy synthesis and lower cost. On this basis, the present inventors have completed the present invention.

In a first aspect, the present invention provides an aptamer that specifically binds to a cTnI protein, comprising the nucleotide sequence set forth in any one of SEQ ID nos.1 to 3, or a nucleotide sequence having high homology to the nucleotide sequence set forth in any one of SEQ ID nos.1 to 3 and capable of specifically binding to a cTnI protein, or a nucleotide sequence derived from the nucleotide sequence set forth in any one of SEQ ID nos.1 to 3 and capable of specifically binding to a cTnI protein. Wherein said high homology may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology to the nucleotide sequence as set forth in any one of SEQ ID Nos. 1-3. The binding constants of the aptamer (SEQ ID Nos.1-3) and cTnI detected by a surface plasmon resonance method are in a pM level, which shows that the aptamer provided by the invention has high affinity with a target protein.

Preferably, the aptamer of the present invention that specifically binds to a cTnI protein consists of a nucleotide sequence shown in any one of SEQ ID nos.1 to 3.

Wherein the nucleotide sequences shown in SEQ ID Nos.1 to 3 are respectively shown as follows:

SEQ ID No.1(cTnI-14-7)：

5’-CCAATGCAGTGGGGAGGGACTGCGTTGG-3’

SEQ ID No.2(cTnI-14-3)：

5’-CCAATGCAGTGGGGAGGGACTTGTTGG-3’

SEQ ID No.3(cTnI-14-16)：

5’-CCAATGCAGTGGGGGGACTTGTTGG-3’

in addition, it will be appreciated by those skilled in the art that modifications may be made to the nucleic acid aptamers described above at a position in their nucleotide sequences, for example, phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotyping, provided that the aptamer sequences so modified have desirable properties, for example, may have an affinity for binding to cTnI proteins equal to or greater than the parent aptamer sequence prior to modification, or may have greater stability, although not significantly increased, or may have a specific luminophore to facilitate visualization.

It will be appreciated by those skilled in the art that as an improvement to the above-described embodiments, a fluorescent substance, a radioactive substance, a therapeutic substance, biotin, digoxigenin, a nano-luminescent material, a small peptide, an siRNA or an enzyme label, etc., may be attached to the nucleotide sequence of the aptamer, provided that the aptamer sequence thus modified has desirable properties, e.g., may have an affinity for binding to cTnI protein equal to or higher than the parent aptamer sequence before modification, or may have higher stability although the affinity is not significantly improved.

In other words, the above nucleic acid aptamer sequences, whether partially substituted or modified, have substantially the same or similar molecular structure, physicochemical properties and functions as the original nucleic acid aptamer, and are all applicable to binding to cTnI proteins.

As a general technical concept, the nucleic acid aptamer of the present invention may also comprise any one of the following three sequences:

(1) a nucleotide sequence having a homology of 60% or more with the nucleotide sequence of the aptamer in all the aforementioned embodiments (for example, a nucleotide partially complementary to the aforementioned aptamer sequence may be deleted or added), preferably, the homology may be 70% or more, 80% or more, 90% or more, or 99% or more; preferably a nucleotide sequence having a homology of 60% or more with the nucleotide sequence shown in any one of SEQ ID Nos.1 to 3, and preferably, the homology with the nucleotide sequence shown in any one of SEQ ID Nos.1 to 3 may be 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more;

(2) a nucleotide sequence capable of hybridizing with the nucleotide sequence of the aptamer under stringent conditions in all the aforementioned technical schemes; or

(3) RNA sequences transcribed from the nucleotide sequences of the aptamers described in all of the preceding claims;

wherein, the nucleotide sequences in the (1) to (3) can be specifically combined with cTnI protein.

The term "homology" refers to two or more sequences having a specified percentage of nucleotides that are identical over a specified region when compared and aligned for maximum identity over a comparison window using methods known in the art (such as sequence comparison algorithms). The percent "homology" between two sequences can be determined using the BLASTP algorithm version 2.2.2(Altschul, stephenf., Thomas l.madden, Alejandro a.Jinghui Zhang, Zheng Zhang, webb miller, and David j.lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database searches", Nucleic Acids Res.) were determined using default parameters.

The term "stringent conditions" refers to conditions that are generally understood in the art as stringent. Examples of stringent conditions for hybridization are 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68 ℃ or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at about 42 ℃ (see Sambrook et al, Molecular Cloning: analytical Manual,2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

Furthermore, as a general technical concept, the present invention also provides aptamer derivatives, which are phosphorothioate backbones derived from the backbone of the nucleotide sequence of the aptamer in all the aforementioned technical embodiments, or corresponding peptide nucleic acids modified from the aptamer in all the aforementioned technical embodiments.

The above derived aptamers or other derivatives derived therefrom have substantially the same or similar molecular structure, physicochemical properties and functions as the original aptamers.

In a second aspect, the present invention also provides a use of the aforementioned aptamer or aptamer derivative. For example, the aptamer of the present invention or a derivative thereof can be used for the purification or detection of cTnI protein, and the aptamer of the present invention or a derivative thereof can be used to detect the concentration of cTnI protein in a sample, thereby determining whether the subject has myocardial cell damage. Preferably, the cTnI protein purification or detection can be performed using any one or more of the aptamers shown in SEQ ID Nos. 1-3. The concentration of the cTnI protein in the sample can be detected by using any one or more aptamers shown in SEQ ID Nos.1-3, so as to judge whether the subject has myocardial cell damage. And further, whether the subject has a disease associated with myocardial cell injury, such as, but not limited to, acute myocardial infarction, may be diagnosed based on the above information.

In a third aspect, the present invention provides a kit for purifying a cTnI protein, the kit comprising an aptamer according to the first aspect of the present invention. Preferably, the kit comprises any one or more of the aptamers shown in SEQ ID nos.1 to 3 or derivatives thereof. More preferably, the kit comprises any one or more of the aptamers shown in SEQ ID Nos.1 to 3.

Since the aptamer of the present invention can specifically bind to cTnI protein, the aptamer of the present invention can be used to purify cTnI protein in a sample. For example, when purifying a cTnI protein, the specific procedure may be: incubating any one or more aptamers or modified sequences shown in SEQ ID NOS.1-3 with a sample solution containing cTnI protein, wherein the aptamers can specifically bind to the cTnI protein, recovering a complex, eluting the bound cTnI protein by high salt or other methods, and purifying to obtain the cTnI protein; or any one or more aptamers or modified sequences shown in SEQ ID Nos.1 to 3 are firstly fixed on a solid phase matrix, a sample liquid containing the cTnI protein is slowly flowed through the solid phase matrix, the aptamers can be specifically combined with the cTnI protein but not with other unrelated proteins, then the solid phase matrix is washed by a buffer solution, the unbound unrelated proteins are removed, and the combination of the aptamers and the cTnI protein is destroyed by high salt or other methods, so that the cTnI protein is specifically eluted and collected. The person skilled in the art will be able to select an appropriate purification method depending on the actual requirements.

The result of surface plasmon resonance (SPR for short) experiments shows that the cTnI aptamer provided by the invention has obvious combination phenomenon with cTnI protein and has no combination phenomenon with interference protein.

In a fourth aspect, the present invention provides a kit for detecting a cTnI protein, the kit comprising an aptamer according to the first aspect of the present invention. Preferably, the kit comprises any one or more of the aptamers shown in SEQ ID nos.1 to 3 or derivatives thereof. More preferably, the kit comprises any one or more of the aptamers shown in SEQ ID Nos.1 to 3. The kit can accurately determine the concentration of the cTnI protein in a sample.

Still further, the present invention provides a kit for detecting the concentration of a cTnI protein in a sample, the kit comprising an aptamer according to the first aspect of the present invention. Preferably, the kit comprises any one or more of the aptamers shown in SEQ ID nos.1 to 3 or derivatives thereof. More preferably, the kit comprises any one or more of the aptamers shown in SEQ ID Nos.1 to 3. The kit can rapidly, quickly and accurately detect the concentration of the cTnI protein in the sample, and can further judge whether the subject has myocardial cell damage according to the detected concentration of the cTnI protein in the sample. In other words, using the concentration of cTnI protein in a sample to determine whether the subject has myocardial cell damage is a rapid and accurate diagnostic method. The aptamer provided by the invention is inspected by spot hybridization, and the result shows that the depth of the spots after incubation with cTnI proteins with different concentrations has larger gradient difference, so that the aptamer provided by the invention is proved to have better binding with the cTnI, and has no or little binding with control proteins BSA and TNF alpha.

The present invention provides a kit for diagnosing whether a subject has myocardial cell injury, the kit comprising an aptamer according to the first aspect of the invention. Preferably, the kit comprises any one or more of the aptamers shown in SEQ ID nos.1 to 3 or derivatives thereof. More preferably, the kit comprises any one or more of the aptamers shown in SEQ ID Nos.1 to 3. More preferably, the kit comprises the aptamer shown in SEQ ID No. 1. The kit can rapidly, quickly and accurately detect the concentration of the cTnI protein in the sample, and can further judge whether the subject has myocardial cell damage according to the detected concentration of the cTnI protein in the sample.

It will be appreciated by those skilled in the art that the kit for diagnosing whether a subject has myocardial cell injury of the present invention may also be used for diagnosing a disease associated with myocardial cell injury, for example, acute myocardial infarction.

In a fifth aspect, the present invention also provides a method for detecting the concentration of a cTnI protein in a sample, the method being carried out using the aptamer according to the first aspect of the present invention. Preferably, the method is performed using any one or more of the aptamers shown in SEQ ID Nos.1 to 3 or derivatives thereof. More preferably, the method is performed using any one or more of the aptamers shown in SEQ ID Nos.1 to 3. More preferably, the method is performed using an aptamer as shown in SEQ ID No. 1.

The detection of the concentration of cTnI protein in a sample using the aptamer of the present invention can be performed according to a method in the art using a conventional aptamer for detection of a target, for example, the concentration of cTnI protein in a sample of a patient with acute myocardial infarction can be detected using the aptamer of the present invention by a dot hybridization assay.

The present invention also provides a method of diagnosing whether a subject has myocardial cell injury, the method being carried out using an aptamer according to the first aspect of the invention. Preferably, the method is performed using any one or more of the aptamers shown in SEQ ID Nos.1 to 3 or derivatives thereof. More preferably, the method is performed using any one or more of the aptamers shown in SEQ ID Nos.1 to 3.

In various aspects of the invention, the sample may be serum of the subject, or a dilution or treatment of the serum.

The invention has the advantages that: compared with some aptamers of cTnI protein which are published at present, the aptamer obtained by the invention has higher affinity to the cTnI protein, shorter sequence, easy synthesis and lower cost, and can be used for quickly, quickly and accurately detecting the cTnI protein.

Drawings

The above features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows data (SPR data) of affinity detection of cTnI-14-7(SEQ ID No.1) and cTnI protein obtained in example 1 of the present invention. The cTnI-14-7 is detected to be combined with the cTnI protein by an SPR instrument, and the KD value is 695 pM.

FIG. 2 shows data (SPR data) of affinity assay of cTnI-14-3(SEQ ID No.2) and cTnI protein obtained in example 1 of the present invention. The cTnI-14-3 was detected by SPR to bind to cTnI protein with a KD of 138 pM.

FIG. 3 shows data (SPR data) of affinity assay of cTnI-14-16(SEQ ID No.3) and cTnI protein obtained in example 1 of the present invention. The cTnI-14-16 was detected by SPR instrument to bind to cTnI protein with a KD value of 86 pM.

FIG. 4 shows affinity assay data for cTnI-14.

FIG. 5 shows data of affinity detection (SPR data) of three aptamers to a target protein and an interfering protein, as described in example 1 of the present invention. FIG. 5A shows that the affinity of three aptamers to the target protein cTnI described in example 1 of the present invention is high. FIG. 5B shows that the three aptamers described in example 1 of the present invention have no or very low affinity to TNF α protein. FIG. 5C shows that the three aptamers described in example 1 of the present invention have no or very weak affinity to bovine serum albumin (BSA for short). FIG. 5D shows that the three aptamers described in example 1 of the present invention have no or very weak affinity to streptavidin (SA protein for short),

FIG. 6 shows the application of the aptamer screened by the method of dot hybridization in the detection of cTnI protein. FIG. 6A shows the results of a dot hybridization experiment of three aptamers (SEQ ID Nos.1-3) according to example 1 of the present invention to a cTnI protein, wherein the three aptamers bind strongly to the target cTnI protein and do not bind to the control BSA protein. FIG. 6B shows the results of experiments on the detection of the membrane-hybridized cTnI protein by three aptamers (SEQ ID Nos.1-3), and it can be seen that the color of the developed spots becomes darker with the increase of the concentration of the cTnI protein, which indicates that all three aptamers can be used for the detection of the membrane-hybridized cTnI protein and have higher sensitivity.

Detailed Description

The present invention is further described below with reference to specific examples, but it will be understood by those skilled in the art that the following examples are for better understanding of the present invention and the present invention is not limited to these specific examples.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all conventional biochemical reagents, and are commercially available, unless otherwise specified.