A molecular beacon-peptide probe and its application

Abstract

The present invention discloses a molecular beacon-peptide probe and its application, the molecular beacon-peptide probe comprises a loop part complementary to the target miRNA, a stem part comprising a double-stranded section and a single-stranded section; the 3'-terminal A base of the double-stranded section is connected with biotin, and the T base complementary thereto is connected with a reporter polypeptide; the single-stranded section extends along a polynucleotide on one side of the double-stranded section connected to the reporter polypeptide, which contains a nickase recognition site and a base modified with a thiophosphate bond at the end. After the molecular beacon-peptide probe loop part binds to the target miRNA, a DNA-RNA hybrid double-strand is formed, and a new DNA is synthesized under the action of a DNA polymerase to replace the miRNA, forming a DNA-DNA hybrid ring, which is cut off with a base modified with a thiophosphate bond under the action of a nickase, exposing a nuclease recognition site, and the nuclease cuts one side of the single-stranded section into fragments and frees the DNA single-stranded section complementary to the loop part and the reporter polypeptide, and the DNA single-stranded section enters the next cycle, and after the cycle ends, the reporter polypeptide is quantified by mass spectrometry SRM.

Description

Technical Field

[0001] The present invention relates to the technical field of multi-component miRNAs quantitative detection, and in particular to a molecular beacon-polypeptide probe and application thereof. Background Art

[0002] Currently, most technical means are used to study the changes in the content of these miRNAs in a qualitative or semi-quantitative manner. In addition, the data between laboratories are not standardized and cannot provide accurate quantitative data, resulting in the inability to perform comparative analysis of data from different laboratories.

[0003] Secondly, most methods can only detect one miRNA at a time, and the detection efficiency of multi-component miRNAs cannot meet the technical accuracy requirements. Currently, the main detection methods for miRNA include Northern blotting, polymerase chain reaction (PCR), rolling circle amplification (RCA), etc. These methods can only measure a specific miRNA and lack multi-channel detection capabilities.

[0004] At present, the commonly used multi-channel detection methods mainly include microarray based assays and large-scale sequencing methods. Due to the existence of cross-reactions, the microarray method has a high "false positive" rate and cannot quantify the miRNA content. Its sensitivity and specificity are difficult to meet the needs of clinical detection. Sequencing is only suitable for large-scale miRNA detection and is not suitable as a routine clinical diagnostic tool. New methods developed in recent years, such as DQAMmiR, use locked nucleic acid-DNA probes (LNA-DNA probes) and electrophoresis technology, but the separation ability of electrophoresis technology has certain limitations on the application of multi-channels. Other methods that rely on spectral separation often require a certain difference in absorbance, fluorescence, and refractive index. The signal overlap phenomenon greatly limits the multi-channel capabilities of these technologies. For example, the signal band generated by a single fluorescent group is very wide, usually covering about 100nm in a wavelength window of about 600nm. In addition, the fluorescence signal is easily affected by temperature and pH.

[0005] In view of this, overcoming the defects of the prior art is an urgent problem to be solved in this technical field. Summary of the invention

[0006] In view of the technical difficulties in sensitivity and analytical throughput of current multi-component miRNAs quantitative methods, the present invention designs a molecular beacon-peptide probe, and utilizes it to establish a high-sensitivity, high-throughput, and high-specificity multi-component miRNAs quantitative method.

[0007] The technical solution provided by the present invention is specifically as follows:

[0008] In a first aspect, the present invention provides a molecular beacon-polypeptide probe, comprising:

[0009] a loop portion, which is complementary to the target miRNA;

[0010] The stem portion comprises a double-stranded section and a single-stranded section; the 3'-terminal A base of the double-stranded section is connected to biotin, and the complementary T base is connected to a reporter polypeptide suitable for mass spectrometry multiple reaction monitoring; the single-stranded section extends along the side of the double-stranded section connected to the reporter polypeptide, and its end contains a base modified by a thiophosphate bond, and the polynucleotide chain between the thiophosphate bond modified base and the reporter polypeptide contains a nicking enzyme recognition site.

[0011] In some embodiments provided by the present invention, the sequence of the reporter polypeptide is one or more of AVQLGVDPFR (Seq_1), AVLGDPFR (Seq_2), AVDLGVDPFR (Seq_3), AVLGVDPFR (Seq_4), AVVGVDPFR (Seq_5), and ADVGVDPFR (Seq_6).

[0012] In some embodiments provided by the present invention, the front end of the reporter polypeptide is provided with an amino acid sequence GDR-, and the terminal G amino acid is connected to the T base complementary to the A base at the 3' end of the double strand through an azide-alkyne click reaction.

[0013] In a second aspect, the present invention provides a composition suitable for quantitatively detecting multi-component miRNAs using mass spectrometry multiple reaction monitoring technology, the composition comprising:

[0014] A variety of molecular beacon-peptide probes, which can bind to a variety of target miRNAs to form a variety of DNA-RNA hybrid double strands;

[0015] DNA polymerase, which is used to synthesize a single-stranded DNA complementary to the loop portion using the DNA-RNA hybrid double strand as a template, thereby forming a DNA-DNA hybrid loop to replace the target miRNA;

[0016] A nickase, which is used to cut off the nickase recognition site after forming a DNA-DNA hybridization loop, remove the 5' front end base modified with a phosphorothioate bond, thereby exposing the recognition site of Lambda exonuclease;

[0017] Lambda exonuclease is used to cut a single strand of a DNA-DNA hybridization loop exposing a lambda exonuclease recognition site into fragments, so that the DNA single strand complementary to the loop portion is completely retained.

[0018] In some embodiments provided by the present invention, the above composition further comprises dNTP and a primer, and the 5' front end of the primer contains a base modified with a phosphorothioate bond.

[0019] In some embodiments provided by the present invention, the above composition further comprises:

[0020] Streptavidin beads, which are used to bind and remove biotin-containing components; and

[0021] Trypsin, which is used to release the reporter polypeptide.

[0022] In a third aspect, the present invention provides an application of a composition suitable for quantitative detection of multi-component miRNAs by mass spectrometry multi-reaction, the application comprising:

[0023] A) Non-disease therapeutic and / or diagnostic applications in the quantitative detection of multi-component miRNAs; or

[0024] B) Application in the preparation of multi-component miRNAs therapeutic and / or diagnostic products.

[0025] In a fourth aspect, the present invention provides a kit suitable for quantitative detection of multi-component miRNAs, wherein the kit comprises the above-mentioned composition suitable for quantitative detection of multi-component miRNAs by mass spectrometry multiple reaction.

[0026] In a fifth aspect, the present invention provides a multi-component miRNAs quantitative detection method for non-disease diagnosis purposes, comprising:

[0027] Obtaining samples from subjects;

[0028] Using the above composition to perform double cycle amplification of exponential amplification and linear amplification on the sample;

[0029] Streptavidin beads were used to bind and remove biotin-containing components, and trypsin was used to release the reporter peptide;

[0030] The content of reporter peptide was detected by mass spectrometry multiple reaction detection method.

[0031] In a sixth aspect, the present invention provides a system for quantitatively detecting multi-component miRNAs, comprising:

[0032] (1) Collection module: obtaining samples from subjects;

[0033] (2) Amplification module: using the above composition to perform exponential amplification and linear amplification on the sample;

[0034] (3) Separation module: using streptavidin beads to bind and remove biotin-containing components and using trypsin to release the reporter peptide;

[0035] (4) Detection module: Use mass spectrometry multi-channel monitoring reaction to detect the content of reporter peptide.

[0036] The various molecular beacon-peptide probes designed in the present invention can bind to multi-component miRNAs and perform a double cycle of exponential amplification and linear amplification. The reporter polypeptides of the molecular beacon-peptide probes can combine the powerful multi-channel, high-resolution, and high-accuracy detection capabilities of mass spectrometry with the double cycle mediated by the molecular beacon-peptide probe to achieve quantitative detection of multi-component miRNAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the multi-component miRNAs quantitative detection method provided by the present invention;

[0038] Figure 2 It is a schematic diagram of the design of the molecular beacon-peptide probe provided by the present invention, and the molecular beacon-peptide probe is divided into a stem portion and a loop portion;

[0039] Figure 3 This is the daughter ion map of the six reporter peptides screened by the present invention, and this figure is the daughter ion map of the synthesized pure peptide;

[0040] Figure 4 It is the SRM channel chromatogram of six reporter peptides provided by the present invention, and three parent ion-daughter ion channels are selected for each peptide segment;

[0041] Figure 5 This is an example of a molecular beacon and a chromatogram after the molecular beacon is combined with a polypeptide provided in an embodiment of the present invention. The chromatographic conditions are as follows: chromatographic column: ThermoBiobasic-C8 (150 mm×4.6 mm, 5 μm, 300A); mobile phase: A: 50 mM triethylamine B: acetonitrile; gradient: B phase 5 (0 min)-16 (16 min)-20 (20 min)-5 (25 min); wavelength: λ=260 nm;

[0042] Figure 6 : is a curve diagram of the effect of different enzymes on the cycle provided by the embodiment of the present invention; wherein, MB: molecular beacon, NE: Nb·BbvCI nickase, LE: λ exonuclease, BP: BstDNA polymerase;

[0043] Figure 7 is a standard curve obtained by measuring samples of different concentrations using miR-21 as an example provided in an embodiment of the present invention;

[0044] Figure 8 It is a standard curve of various miRNAs provided in the embodiments of the present invention; it includes 1 fM to 5 nM, and the relative peak area ratio of the reporter polypeptide and the corresponding stable isotope-labeled internal standard is plotted against the concentration. DETAILED DESCRIPTION

[0045] Mass spectrometry has rapidly become a very important tool in the field of analysis in recent years due to its advantages such as high accuracy, good repeatability, fast analysis, and low sample requirement. Among them, the selected or multiple reaction monitoring (SRM / MRM) technology has shown the quantitative advantages of high accuracy and high throughput when analyzing complex biological samples. This technology can not only remove the interference of a large number of impurity ion signals, but also detect multiple detection substances at the same time (>1000 SRM channels / hour). For example, Bisson N et al. used this technology to study 90 proteins at the same time. SRM technology has been widely used in protein quantification. Its main principle is to use SRM to monitor the fragment ion signals of characteristic peptides produced after target proteolysis, so as to quantify the target protein at the peptide level.

[0046] There are only four kinds of nucleotides that make up nucleic acids, which leads to many different miRNAs having the same ion pair signals, and their specificity is far lower than that of proteins composed of 20 amino acids. Therefore, it is impossible to directly quantify miRNA using SRM technology. The applicant introduced a DNA-peptide probe, and through the hybridization of miRNA and DNA-peptide probe, the miRNA signal was converted into the mass spectrometry signal of the reporter peptide, and the high selectivity, high sensitivity and quantitative ability of mass spectrometry SRM technology were successfully grafted into the analysis and research of miRNA. Among them, the DNA and miRNA sequences are completely complementary, and the reporter peptide needs to meet the following requirements: ① It has sequence uniqueness and is not the same as a known protein or polypeptide sequence; ② It has a high mass spectrometry response and can reach the pM level (~10 -10 mol / L); ③ can be completely enzymatically released from the substrate peptide; ④ can generate high-quality SRM. However, the detection limit of DNA-peptide probes is limited (~100pM), and it is unable to detect samples with lower concentrations.

[0047] Molecular beacon (MB) is a hairpin molecule formed by oligonucleotides. This stem-loop probe can undergo conformational changes after hybridization with the nucleic acid target molecule. This structural change brings a lot of convenience to the design, especially the molecular beacon-mediated isothermal nucleic acid amplification, which has the characteristics of no need for reversal, easy control of conditions, strong intrinsic fixation, strong specificity, and economic suitability compared to RT-PCR amplification. The molecular beacon stem-loop-mediated double-cycle amplification method can achieve an amplification efficiency of 2.5×10 10 times, and its amplification efficiency is much higher than that of RT-PCR, DSNSA, and Nanopore. However, as mentioned above, the signal overlap caused by the use of fluorescent probes in this method severely limits the multi-channel detection capability of this technology.

[0048] The present invention combines the powerful multi-channel, high-resolution, and high-accuracy detection capabilities of mass spectrometry with probe-mediated double-cycle amplification through the design of multiple molecular beacon-peptide probes, thereby realizing the quantitative detection of multi-component miRNAs. Specifically: the molecular beacon-peptide probe provided by the present invention includes a loop portion complementary to the target miRNA and a stem portion comprising a double-stranded section and a single-stranded section, the 3'-terminal A base of the double-stranded section is connected to biotin, and the complementary T base is connected to a reporter polypeptide suitable for mass spectrometry multi-reaction monitoring; the single-stranded section extends along one side of the double-stranded section connected to the reporter polypeptide, and its end contains a base modified by a phosphorothioate bond, and the polynucleotide chain between the base modified by the phosphorothioate bond and the reporter polypeptide contains a recognition site for a nicking enzyme. Specifically, the double-stranded stem of the molecular beacon-peptide probe has 8-10 base pairs, is not complementary to the target miRNA, does not interfere with the nickase cleavage, and can be pulled apart after the loop binds to the target miRNA. The base modified by the thiophosphate bond refers to the base in which the oxygen in the phosphate is replaced by sulfur. In the present invention, the molecular beacon-peptide probe contains 2 such bases at the end to avoid nonspecific cleavage by nuclease exonucleases. In the present invention, the reporter polypeptide suitable for mass spectrometry multiple reaction monitoring should meet the following conditions: the polypeptide length is 6-16 amino acids; it is unique; it does not contain cysteine ​​or methionine; it does not contain RK, RR, KR series; it contains at least 40% branched-chain amino acids. For example, one or more of AVQLGVDPFR, AVLGDPFRR, AVDLGVDPFR, AVLGVDPFR, AVVGVDPFR, and ADVGVDPFR, these reporter polypeptides do not exist in all proteins in the human body.

[0049] To improve the enzymatic hydrolysis efficiency, in some embodiments provided by the present invention, the amino acid sequence GDR- is connected to the front end of the reporter polypeptide, which we call the substrate polypeptide, to form polypeptide sequences containing the reporter polypeptide, such as GDRAVQLGVDPFR (Seq_7), GDRAVLGDPFR (Seq_8), GDRAVDLGVDPFR (Seq_9), GDRAVLGVDPFR (Seq_10), GDRAVVGVDPFR (Seq_11), and GDRADVGVDPFR (Seq_12).

[0050] Based on the above molecular beacon-peptide probes, the present invention provides a composition suitable for quantitative detection of multi-component miRNAs by mass spectrometry multiple reaction monitoring technology, the composition comprising a plurality of molecular beacon-peptide probes, DNA polymerase, nickase and lambda exonuclease; the plurality of molecular beacon-peptide probes bind to a plurality of target miRNAs to form a plurality of DNA-RNA hybrid double strands; under the action of DNA polymerase and primers, the DNA-RNA hybrid double strands are used as templates to synthesize a DNA single strand complementary to the loop, thereby forming a DNA-DNA hybrid loop to replace the target miRNA, and the replaced miRNA and the new molecular beacon- The polypeptide probes recognize and bind to each other, and enter the next round to form a DNA-RNA hybrid double strand. This cycle now exhibits linear amplification. After the DNA-DNA hybrid ring is formed, the nickase cuts off the base modified by the thiophosphate bond at the end of the single strand to expose the recognition site of the Lambda exonuclease. Then, the Lambda exonuclease cuts one strand of the DNA-DNA hybrid ring that exposes the recognition site of the Lambda exonuclease into fragments, so that the DNA single strand complementary to the ring can be completely retained and enter the next round, forming a DNA-probe hybrid ring with a new molecular beacon-polypeptide probe, generating a new round of hybridization, and now exhibiting exponential amplification.

[0051] In some embodiments provided by the present invention, the above composition further comprises dNTP and primers, and the 5' front end of the primer contains a base modified with a phosphorothioate bond to prevent non-specific cleavage of the primer by exonuclease.

[0052] In some embodiments provided by the present invention, the above-mentioned composition also includes streptavidin beads and trypsin. After the exponential amplification is completed, the remaining molecular beacon-peptide probe and biotin-containing fragments are bound to the streptavidin beads and removed by centrifugation. The supernatant solution is enzymatically digested by trypsin to release the reporter polypeptide, which is then detected and quantified by mass spectrometry SRM.

[0053] Based on the above composition, the present invention provides a multi-component miRNAs quantitative detection method for non-disease diagnosis purposes, comprising: obtaining a sample from a subject; using the above composition to perform double-cycle amplification of exponential amplification and linear amplification on the sample; using streptavidin beads to bind and remove biotin-containing components, and using trypsin to release the reporter polypeptide; and using a mass spectrometry multiple reaction detection method to detect the content of the reporter polypeptide.

[0054] The present invention also provides a method for preparing a molecular beacon-peptide probe, comprising connecting an alkyne-modified molecular beacon with an azide-modified substrate polypeptide through a click chemistry reaction, and chromatographically purifying to obtain the molecular beacon-peptide probe. Specifically, the alkyne is connected to the base T complementary to the A base at the 3' end of the double-stranded chain, and the azide is connected to the G amino acid at the front end of the reporter polypeptide.

[0055] Preferably, for the reporter polypeptides screened out to meet the above requirements, the synthesized pure reporter polypeptides are verified by mass spectrometry SRM technology, 3-5 detection channels are established for each reporter polypeptide, and the mass spectrometry response intensity and enzymatic efficiency are verified to meet the preset requirements; wherein the preset requirements include that the mass spectrometry response must be less than 100pM and the enzymatic efficiency must be higher than 90%. Preferably, the target miRNA includes one or more of miR-21, miR-141, miR-let7a, miR-200c, miR-125a, and miR-15b.

[0056] In the present invention, the substrate polypeptide includes a reporter polypeptide, and the reporter polypeptide is formed after enzyme cleavage.

[0057] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0058] Embodiment 1:

[0059] This example takes the molecular beacon-peptide probe designed for the target miRNA miR-145 (sequence UCCTAAGGACCCTTTTACCTG (Seq_13)) as an example to illustrate the molecular beacon-peptide probe provided by the present invention in detail. Figure 2The molecular beacon-peptide probe is designed for miR-145. The loop of the molecular beacon-peptide probe is the recognition region, and its sequence is complementary to the miR-145 to be detected, and the sequence is 5'AGGGATTCCTGGGAAAATGGAC-3' (Seq_14), referred to as DNA1. The double-stranded stem of the molecular beacon-peptide probe is complementary to TGTCCAAGA-3' (Seq_15) and ACAGGTTCT (Seq_16). The 3' end of TGTCCAAGA-3' is connected to biotin, the terminal T base of ACAGGTTCT is connected to the reporter polypeptide, and the outer side of the terminal T base is connected to the single-stranded 5'AAGCTGAGG- (Seq_17). The O on the phosphate of the terminal AA is replaced by S, and the 5'GCTGAGG on the single-stranded 5'AAGCTGAGG is the recognition site of the nickase. The nickase cuts between CT and removes AA. When designing molecular beacon-peptide probes based on other target miRNAs, it is only necessary to replace the DNA1 sequence with the complementary sequence of the target miRNA.

[0060] The synthesis and characterization of molecular beacon-peptide probes specifically include: first, adding excess substrate peptides to the molecular beacon and substrate peptides for click chemistry reaction, reacting for 4 hours at 37°C under shaking conditions, and then purifying using preparative chromatography. The molecular beacon-peptide product can be separated from unreacted DNA and peptides by HPLC and quantified using the external calibration peak area method. To further characterize the newly synthesized molecular beacon-peptide probe, the product can be digested with trypsin. The digested DNA and peptide fragments are verified by HPLC respectively.

[0061] The following uses miR-145 in multi-component miRNAs as an example to elaborate on the quantitative detection method of multi-component miRNAs:

[0062] Step 1: Figure 2 As shown, the loop DNA1 of the molecular beacon-peptide probe binds to miR-145 to form a DNA1-miR-145 hybrid double strand;

[0063] Step 2: Under the action of DNA polymerase and primer T*C*TTGGAC (* represents that the oxygen on the phosphate of the base is replaced by sulfur, and the base is called a thiophosphate-modified base), a new DNA single strand (referred to as DNA2) is synthesized to replace the miR-145 on the DNA1-miR-145 hybrid double strand. The replaced miR-145 enters the next cycle, recognizes and binds to the new molecular beacon-peptide probe, and forms a DNA-RNA hybrid double strand. This cycle manifests as linear amplification.

[0064] Step 3: After DNA2 replaces miR-145 on the DNA1-miR-145 hybrid double strand, a DNA1-DNA2 hybrid ring is formed on the molecular beacon-peptide probe; under the action of the nickase, the base (AA) modified with a thiophosphate bond at the 5' front end of the molecular beacon-peptide probe is cut off, exposing the recognition site of the Lambda exonuclease, and then the Lambda exonuclease cuts DNA1 into fragments, freeing the substrate peptide segment, while retaining the intact DNA2 to enter the next round of hybridization, combining with the new molecular beacon-peptide probe or the DNA1-miR-145 hybrid double strand to form a DNA1-DNA2 hybrid ring, which shows exponential amplification.

[0065] Step 4: After exponential amplification is completed, the remaining molecular beacon-peptide probes and 3'-end biotin-modified fragments are separated and removed by binding to streptavidin beads. The supernatant solution is digested with trypsin to release the reporter peptide fragments, which are then detected and quantified by mass spectrometry SRM.

[0066] When multi-component miRNAs are detected simultaneously, multiple molecular beacon-peptides can be added to the system. This method combines the powerful multi-channel, high-resolution, and high-accuracy detection capabilities of mass spectrometry with probe-mediated double-cycle amplification to achieve quantitative detection of multi-component miRNAs.

[0067] In the multi-component miRNAs quantitative detection method of the present invention, some pre-optimization processing is usually required, including: determining 3-5 SRM channels for each reporter polypeptide; using a triple quadrupole liquid-mass spectrometer to detect fragment ion signals from the polypeptide; selecting parent ion-daughter ion pairs according to the mass number and abundance of the fragment ions; optimizing the SRM channels by adjusting the sample preparation process and chromatographic separation and mass spectrometry conditions, including improving enzymatic efficiency, optimizing parent ion-daughter ion pairs, improving chromatographic peak shape, and improving mass spectrometry signal-to-noise ratio. In order to correct the errors caused by the instrument and operation, an internal standard is required for correction.

[0068] Embodiment 2:

[0069] Based on Example 1 of the present invention, this embodiment also proposes a specific implementation method details of a multi-component miRNAs quantitative detection method, including:

[0070] The amplification reaction solution system is 200 μL, and 110 μL of DEPC water, 20 μL of 10×NEB buffer2, 8 μL of 10 mM triphosphate deoxynucleotide, 10 μL of 10 μM molecular beacon-peptide probe, 2 μL of 20 U RNase inhibitor (TAKARA), 2 μL of 10 μg / μL bovine serum albumin, 8 μL of 16 U Bst DNA polymerase, 6 μL of 15 U Nb·BbvCI nickase, 10 μL of 12.5 U λ exonuclease, 8 μL of 100 μM primer are added in sequence, and 16 μL of extracted RNA solution (total RNA of the sample to be tested extracted by Trizol) or standard solution is added, and incubated at 37°C for 2 h. Then heat to 100℃ for 10min to inactivate the enzyme, add streptavidin beads, centrifuge at 3000g for 5min, take the supernatant, add sequencing grade trypsin for enzymatic digestion at 37℃ for 6h, add 0.1% trifluoroacetic acid to inactivate the trypsin, add internal standard solution, filter with 0.22μm filter membrane, and retain the sample for final injection and detection.

[0071] Optimization of temperature, time, solution and investigation of amplification efficiency of dual-cycle amplification The amount of enzyme, temperature, time and solution selection of dual-cycle amplification will affect the amplification efficiency. First, the relationship between the amount of different enzymes and the amplification efficiency was investigated to determine the amount of enzymes that affect dual-cycle amplification. The amplification efficiency of each miRNA was investigated in the designed temperature range of 4°C to 52°C gradient and the amplification time of 0.5h to 6h gradient. The content of the reporter polypeptide was detected by LC-MS / MS each time to calculate the amplification efficiency.

[0072] Due to the different physical and chemical properties of different miRNAs, the amplification efficiency is different for the same amplification conditions. For multiple miRNAs to be detected, the amplification multiple is related to the enzyme concentration, temperature, and amplification time. In addition, the interference between different miRNAs needs to be investigated and these parameters need to be quantified.

[0073] Embodiment 3:

[0074] This example mainly introduces two aspects: screening and verification of reporter peptides and synthesis of molecular beacon-peptide probes.

[0075] 1. According to the conditions for screening reporter peptides, six high-response reporter peptides AVQLGVDPFR (peptide A), AVLGDPFR (peptide B), AVDLGVDPFR (peptide C), AVLGVDPFR (peptide D), AVVGVDPFR (peptide E), ADVGVDPFR (peptide F) and their corresponding mass spectrometry ion scans were screened out, among which, Figure 4The daughter ion diagram of the six selected reporter peptides is the daughter ion spectrum of the synthesized pure peptide. We selected the common b1, b2 ions and y1, y2, y3, yn ions. These ion spectra show that the six selected peptides have specific ions.

[0076] 2. By optimizing the mass spectrometry conditions of six synthetic reporter peptides AVQLGVDPFR (peptide A), AVLGDPFR (peptide B), AVDLGVDPFR (peptide C), AVLGVDPFR (peptide D), AVVGVDPFR (peptide E), ADVGVDPFR (peptide F), AVDIGDPFR (peptide G), and AIVGPFR (peptide H), the corresponding SRM spectra were obtained, and the corresponding parent ion and daughter ion channels were marked in the figure. Among them, Figure 5 This is the SRM channel chromatogram of six reporter peptides. Three parent ion-daughter ion channels are selected for each peptide. Each channel established is unique. These peptide sequences do not exist in the body and are therefore unique.

[0077] 3. Molecular beacon and chromatogram after molecular beacon and reporter peptide binding. Taking miR-145 as an example,

[0078] GDRAVQLGVDPFR is a substrate polypeptide, and the sequence is 5′-AAGCTGAGGTCTTGGACAAGGGATTCCTGGGAAAATGGACTGTCCAAGA-biotin-3′ (Seq_18). Figure 6 Example of chromatograms of molecular beacons and molecular beacons combined with peptides. Chromatographic conditions: Column: Thermo

[0079] Biobasic-C8 (150 mm × 4.6 mm, 5 μm, 300A); mobile phase: A: 50 mM triethylamine; B: acetonitrile; gradient: B phase 5% (0 min) -16% (16 min) -20% (20 min) -5% (25 min); excitation wavelength: λ = 260 nm. Figure 6 It can be seen that the spectra before and after synthesis can be clearly separated, indicating that the molecular beacon-peptide probe was successfully synthesized.

[0080] Embodiment 4:

[0081] Design of the method in the embodiment of the present invention and feasibility of double circulation:

[0082] In this design, the characteristics of three enzymes and molecular beacon-peptide probes are fully utilized. First, DNA polymerase is used. This enzyme can use DNA as a template, but cannot synthesize from scratch. Under the action of primers, DNA complementary to the molecular beacon-peptide probe can be synthesized to replace the target miRNA. The second enzyme is a nicking enzyme. The nicking enzyme has a specific recognition site and only cuts the sequence with this site, but it only works when the target is double-stranded, so the probe will not be cut when the double strand is not formed. Once the double strand is formed, the site will be cut, thereby breaking the terminal base. The third enzyme is Lambda exonuclease, which is a highly progressive 5'→3' exodeoxyribonuclease. It selectively cuts the 5'-phosphorylated chain of double-stranded DNA. The enzyme shows low activity against single-stranded DNA and non-phosphorylated DNA. The design that the 5' front end base of the primer and probe is modified by a phosphorothioate bond avoids its non-specific cutting. The design of the molecular beacon-peptide probe can cleverly combine the characteristics of the three enzymes, greatly increasing the efficiency of amplification, while the mass spectrometry signal conversion of the peptide makes multi-channel detection possible.

[0083] In order to illustrate the feasibility of double cycle, 5'-FAM fluorescence and 3'-quencher group DABCYL were used to replace biotin and reporter peptide on the probe in the embodiment of the present invention, and the probe modified with the 5'-FAM fluorescence and 3'-quencher group DABCYL was verified by fluorescence spectrophotometer. Specifically, the amplification reaction solution system is 200 μL, and DEPC water 110 μL, 10×NEB buffer2 20 μL, 10 mM dNTPs 8 μL, 10 μM molecular beacon 10 μL, 20 U RNase inhibitor 2 μL, 10 μg / μL bovine serum albumin 2 μL, 16 U Bst DNA polymerase 8 μL, 15 U Nb·BbvCI nickase 6 μL, 12.5 U λ exonuclease 10 μL, 100 μM primer 8 μL, 16 μL extracted RNA solution or standard solution was added, and incubated at 37 ° C for 2 hours. Then quickly stored at -20 ° C. The control variable method was used to determine the effects of different enzymes on the cycle, such as Figure 6 As shown in the figure, when only Nb·BbvCI nickase or λ exonuclease is added, there is no amplification. When only Bst DNA polymerase is added, obvious amplification signals can be seen, indicating that Bst DNA polymerase works first and triggers the cycle on the right. When all three enzymes are present, the signal is the highest, indicating that a double cycle is formed.

[0084] 2. Selection and investigation of reporter peptides

[0085] To detect multiple miRNAs simultaneously, different miRNA-probe hybrids must be technically separated. So far, spatial and spectral separations have been mainly used for this purpose. For spatial separations such as electrophoresis and chromatography, the maximum number of miRNAs that can be analyzed (called peak capacity) depends on the average peak width and the analysis time. There is evidence that the optimized peak capacity of electrophoretic separation is unlikely to exceed 30-40 peaks. On the other hand, traditional spectral separation, which usually requires pre-processing of miRNAs for differences in absorbance, fluorescence, refractive index or reflectivity, may be more difficult. For example, the signal generated by fluorophores is very broad, usually covering about 100nm in a wavelength window of about 600nm. Therefore, the multi-channel potential of current technologies is highly limited due to signal overlap. In contrast, the signal from mass spectrometry covers an area of ​​6Da in a mass window of about 1000Da considering isotopes, which will facilitate the simultaneous analysis of many mass tags. In addition, the two filtering conditions (i.e., parent ion and daughter ion) can achieve quantitative detection with specificity. Previous studies have shown that 120 polypeptides can be processed simultaneously by predetermined SRM. This high selectivity provides the possibility of monitoring multiple reporter polypeptides.

[0086] The basic principle of selecting reporter peptides, based on previous experience, needs to meet the following conditions: the peptide length is 6-13 amino acids; it is unique; it does not contain cysteine ​​or methionine; it does not contain RK, RR, KR series; it contains at least 40% branched-chain amino acids. In addition, computer predictions can be made through various algorithms and computational tools to predict which peptides and product ions are most suitable for SRM. Finally, experimental data can confirm the reliability of the selection. In this way, we selected 6 peptide sequences, namely AVQLGVDPFR, AVLGDPFR, AVDLGVDPFR, AVLGVDPFR, AVVGVDPFR, and ADVGVDPFR, all of which have responses below 100pM.

[0087] After investigating the SRM of the peptide, the efficiency of enzymatic hydrolysis of the substrate peptide also affects the generation of the reporter peptide. In order to make the enzymatic hydrolysis more efficient, the present invention adds GD-amino acid in front of the R or K amino acid that can be cleaved by trypsin to play a role of steric hindrance, and the combination is GDR-reporter peptide. The enzymatic hydrolysis efficiencies of the six substrate peptides were calculated to be 99.95%, 99.36%, 99.78%, 99.26%, 99.43%, and 99.13% respectively using the same amount of substrate peptides for hydrolysis. It can be seen that the substrate peptides are basically converted into reporter peptides.

[0088] 3. Synthesis and verification of molecular beacon-peptide probes

[0089] For molecular beacons and peptide probes, the commonly used synthesis method in the laboratory is to perform Michael addition of thiol-modified DNA and maleimide. It is very difficult to modify the thiol directly on the T base, so the present invention chooses to modify the alkyne on the T base and the azide on the G amino acid of the front end GDR of the reporter peptide by click chemistry. In order to ensure that the molecular beacon can react as much as possible, an excess of peptide (1:20 times) is added during the synthesis process. Figure 5 As shown in the HPLC graph, a new product peak is generated after the reaction, which is the molecular beacon-peptide probe peak. After we prepared it by preparative chromatography, we further enzymatically hydrolyzed the product by mass spectrometry to obtain the reporter peptide SRM peak, proving that the synthesis was successful.

[0090] 4. Method feasibility verification

[0091] Previous studies have demonstrated the feasibility of the double cycle, so we conducted further verification on whether the molecular beacon-peptide probe is feasible. We used miR-21 as a template, the sequence is 5'-A*A*GCTGAGG / iDBCOdT / CTTGGACATCAACATCAGTCTGATAAGCTATGTCCAAGA-biotin-3' (Seq_36, * represents that the oxygen on the phosphate of the base is replaced by sulfur), and (6-Azido)-GDRAVQLGVDPFR as a substrate peptide to synthesize a molecular beacon-peptide probe. And amplified at different concentrations at 37°C for 4h, then separated and digested, and finally quantitatively studied by mass spectrometry. The results are as follows Figure 7 As shown, the minimum concentration of this method is 1fM and the detection range is 1fM-5nM.

[0092] Furthermore, multiple miRNAs were detected simultaneously with multiple molecular beacon-peptide probes. It is worth noting that cross-hybridization may also come from the interaction between the target miRNA and other molecular beacon-peptide probes in multiplex detection. To address this issue, we monitored the hybridization between each molecular beacon-peptide probe and different target miRNAs. A low level of cross-effect interference was observed. In order to be more accurate in sample quantification, we corrected for cross-reactions. This correction method uses a matrix that includes all possible cross-reaction interferences from each molecular beacon-peptide probe. The SRM intensity of the reporter peptides is then multiplied by the matrix to obtain their actual signals. After cross-reaction correction, the calibration curve contains all six molecular beacon-peptide probes, but only one target miRNA (increasing concentration), showing target-specific signal intensity.

[0093] 5. Specificity of the molecular beacon-peptide probe dual cycle method

[0094] Hybridization is a key factor affecting the specificity of multi-component detection methods, and examining the cross-hybridization between miRNAs and probes is essential for the accuracy of the method. For simple hybridization methods, a series of miRNAs that form single or double mismatches with molecular beacon-peptide probes are usually designed to evaluate the cross-hybridization effect from other miRNAs, especially their highly homologous family members. In the present invention, each molecular beacon-peptide probe is hybridized in parallel with the mismatched miRNA and the target miRNA, as shown in Table 1 below. The detected peak intensity decreases in the order of match > single mismatch > double mismatch, which provides evidence that the molecular beacon-peptide probe can distinguish between the target miRNA and the mismatched miRNA, as shown in Table 2.

[0095] Table 1. Sequences of different target miRNAs and mismatched miRNAs

[0096]

[0097] Table 2. Specificity of multi-channel detection

[0098]

[0099] 6. Quantitative Analysis of Cellular miRNA

[0100] To comprehensively evaluate the developed molecular beacon-peptide probe / dual-cycle assay, we determined the concentrations of target miRNAs in 2 breast cell lines, as shown in Table 3. In addition, by comparing MCF-7 cells and MCF-7 / ADR cells, all investigated miRNAs were involved in the acquisition of drug resistance.

[0101] Table 3. Molecular beacon-peptide probe / dual cycle detection results of six miRNAs in two cell lines

[0102]

[0103] In the above embodiments of the present invention, a multi-channel method for simultaneously measuring six miRNAs was established by combining the innovative molecular beacon-peptide probe and double cycle amplification. Figure 8 As shown in the figure, after 4 hours of amplification at 37°C, the six miRNAs can reach a minimum detection concentration of 1 fM. Because mass spectrometry has a powerful multi-channel capability, if necessary, channels can be further increased. The concentrations of the six miRNAs in the two cell lines were also measured. Among them, the specificity of multi-channel detection is shown in Table 2.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A molecular beacon-peptide probe, characterized in that: include: a loop portion, which is complementary to the target miRNA; The stem comprises a double-stranded section and a single-stranded section; the A base at the 3' end of the double-stranded section is connected to biotin, and the T base complementary thereto is connected to a reporter polypeptide suitable for mass spectrometry multiple reaction monitoring; the single-stranded section extends along the side of the double-stranded section connected to the reporter polypeptide, and the end thereof contains a base modified by a phosphorothioate bond, and the polynucleotide chain between the base modified by the phosphorothioate bond and the reporter polypeptide contains a nickase recognition site; The reporter polypeptide is a polypeptide sequence with high mass spectrometry response, and the polypeptide sequence is one or more of AVQLGVDPFR, AVLGDPFR, AVDLGVDPFR, AVLGVDPFR, AVVGVDPFR, and ADVGVDPFR; The front end of the reporter peptide is connected to the amino acid sequence GDR-, and the G amino acid at the N-terminus is connected to the T base complementary to the A base at the 3' end of the double-stranded chain through an azide-alkyne click reaction.

2. A composition suitable for quantitative detection of multi-component miRNAs by mass spectrometry multiple reaction monitoring technology, characterized in that: The composition comprises: A plurality of molecular beacon-peptide probes according to claim 1, which can bind to a plurality of target miRNAs to form a plurality of DNA-RNA hybrid double strands; DNA polymerase, which is used to synthesize a single-stranded DNA complementary to the loop portion using the DNA-RNA hybrid double strand as a template, thereby forming a DNA-DNA hybrid loop to replace the target miRNA; A nickase, which is used to cut off the nickase recognition site after forming a DNA-DNA hybridization loop, remove the 5' front end base modified with a phosphorothioate bond, thereby exposing the lambda exonuclease recognition site; Lambda exonuclease is used to cut a single strand of a DNA-DNA hybridization loop exposing a lambda exonuclease recognition site into fragments, so that the DNA single strand complementary to the loop portion is completely retained.

3. The composition suitable for mass spectrometry multi-reaction quantitative detection of multi-component miRNAs according to claim 2, characterized in that: The composition further comprises dNTP and a primer, wherein the 5' front end of the primer contains a base modified with a phosphorothioate bond.

4. The composition suitable for mass spectrometry multi-reaction quantitative detection of multi-component miRNAs according to claim 2 or 3, characterized in that: The composition further comprises: Streptavidin beads, which are used to bind and remove biotin-containing components; and Trypsin, which is used to release the reporter polypeptide.

5. Use of the composition suitable for mass spectrometry multi-reaction quantitative detection of multi-component miRNAs according to any one of claims 2 to 4, characterized in that: The applications include: A) Quantitative detection of multiple miRNAs for non-disease therapeutic and / or diagnostic applications; or B) Application in the preparation of multi-component miRNAs therapeutic and / or diagnostic products.

6. A kit suitable for quantitative detection of multi-component miRNAs, characterized in that: The invention comprises the composition suitable for the mass spectrometry multi-reaction quantitative detection of multi-component miRNAs as described in claim 2 or 3.

7. A multi-component miRNAs quantitative detection method for non-disease diagnosis purposes, characterized in that: include: Obtaining samples from subjects; Using the composition according to any one of claims 2 to 4 to perform double cycle amplification of exponential amplification and linear amplification on the sample; Streptavidin beads were used to bind and remove biotin-containing components, and trypsin was used to release the reporter peptide; The content of reporter peptide was detected by mass spectrometry multiple reaction detection method.

8. A system for quantitatively detecting multi-component miRNAs, characterized in that: include: (1) Collection module: obtaining samples from subjects; (2) Amplification module: using the composition according to any one of claims 2 to 4 to perform exponential amplification and linear amplification on the sample; (3) Separation module: using streptavidin beads to bind and remove biotin-containing components, and using trypsin to release the reporter peptide; (4) Detection module: Use mass spectrometry multi-channel monitoring reaction to detect the content of reporter peptides.

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