Method for detecting small molecules based on aptamer enzyme trans-cleavage fluorescent RNA aptamer

Abstract

The invention provides a method for detecting small molecules based on an aptamer enzyme trans-cleavage fluorescent RNA (Ribonucleic Acid) aptamer. The conformation change of the aptamer is caused by the combination of small molecules and the aptamer, and the activity of the aptamer trans-cleavage luminous RNA aptamer is activated, so that the SAM and theophylline can be quantitatively and highly specifically detected. The aptamer enzyme is constructed by fusing an SAM or theophylline aptamer with hammerhead ribozyme and locking a binding arm of the hammerhead ribozyme, the substrate probe is designed by adopting a Mango fluorescent RNA aptamer as signal output, and compared with other fluorescent aptamers, the aptamer has the advantages of shorter base length and easiness in design and coding modification. The combination of SAM or theophylline and the aptamer opens the combination arm of the aptamer enzyme, so that the trans-cleavage activity of the aptamer enzyme is activated, and a specially designed substrate probe is trans-cleaved. The aptamer enzyme designed in the invention has high specificity, and even if other molecules with similar structures exist, the aptamer enzyme can also detect SAM or theophylline with high specificity. The method for detecting small molecules based on the aptamer enzyme trans-cleavage fluorescent RNA aptamer has the advantages of being simple, easy to operate, capable of effectively working in complex environmental samples and the like, and can become an ideal choice for detecting small molecules in biological samples.

Description

Technical Field

[0001] The invention belongs to the technical field of bioanalysis, and in particular relates to a method for detecting small molecules based on aptamer enzyme trans-cutting fluorescent RNA aptamer. Background Art

[0002] The detection of small molecules in biological systems is crucial for the study of physiological functions, clinical diagnosis, drug development and other fields. Therefore, the development of effective small molecule detection technology is of great significance for both scientific research and practical applications. Although traditional techniques such as spectroscopy, chromatography and enzyme-linked immunosorbent assay (ELISA) have been widely used in the detection of small molecule compounds, these methods usually require complex sample pretreatment steps, which are not only time-consuming and labor-intensive, but also costly, limiting their application in field detection and general applicability. In recent years, biomacromolecule-based detection technologies, such as electrochemical sensors and nucleic acid biosensors, have attracted widespread attention due to their advantages such as simple operation, low cost and fast response. In particular, nucleic acid biosensors, such as aptamers and ribozymes, have become ideal choices for small molecule detection due to their programmability, high affinity and high specificity. Nucleic acid biosensors can be designed into sensors that can specifically recognize and bind to target small molecules through molecular engineering methods, thereby achieving sensitive detection of small molecules.

[0003] Hammerhead ribozyme (HHR) is the most distinctive and smallest ribozyme. Due to its highly flexible structure and highly programmable allosteric properties, it has become the most promising tool for designing aptazyme biosensors. At present, strategies to precisely regulate the structure and function of aptazymes by controlling the activity of aptazymes through exogenous conditions such as small molecules, nucleic acids and proteins have been developed and widely used in biosensing systems. For example, the P1 stem of the TPP aptamer is connected to the stem II of the hammerhead ribozyme to make a TPP-responsive aptazyme for rapid detection of TPP in whole blood; however, the destruction of the stem II domain of the hammerhead ribozyme reduces the cleavage efficiency of the hammerhead ribozyme, limiting the feasibility of large-scale application. Although the construction of most biosensors relies on the self-cleavage activity of the hammerhead ribozyme, the trans-cleavage activity of the hammerhead ribozyme also provides new ideas for the design of biosensors. For example, by designing a biosensor circuit, small molecules are induced to specifically bind to proteins, and then the hammerhead ribozyme is transcribed to trans-cleave specific substrate probes to achieve sensitive detection of small molecules; this method involves the use of proteins and the design of circuits, which is costly and slow. Therefore, it is necessary to develop small molecule detection biosensors with sensitive response and high specificity by regulating the trans-cleavage activity of aptazymes through aptamer-mediated conformational changes.

[0004] Mango RNA aptamer is a high-affinity ribonucleotide aptamer that binds to a thiazole orange fluorophore derivative (TO1-Biotin) with nanomolar affinity and increases the fluorescence intensity of the fluorophore several times. Compared with other fluorescent aptamers, Mango RNA aptamer has a shorter base length and is easier to design and encode. It is used to combine with the target dye (TO1-Biotin) to produce a bright fluorescent signal, which is used as the signal characterization system in this study. Summary of the invention

[0005] The present invention aims to solve the problems of complicated steps, high cost, insufficient specificity and sensitivity in the existing SAM and theophylline detection methods. The present invention provides a method for detecting SAM and theophylline based on aptamer trans-cutting fluorescent RNA aptamer. The detection method consists of two modules: constructing aptamer and constructing substrate probe. First, through the closure of the 5' end binding arm of the hammerhead ribozyme and the SAM aptamer, it is designed as a domain that can partially complement the base of the SAM aptamer. The binding arm of the 5' end of the hammerhead ribozyme is locked, and the binding on the 3' end alone cannot recognize and trans-cut the substrate probe, and the hammerhead ribozyme is converted into a SAM-responsive aptamer (SAM-HHR). The fluorescent RNA aptamer "Mango" is selected as the signal input module. The fluorescent RNA aptamer "Mango" is designed as a double-stem hairpin structure (Anti-Mango) to destroy the secondary structure so that it cannot bind to the dye. At the same time, the hairpin loop is designed as a sequence that can be recognized and bound by the hammerhead ribozyme. The double-stem hairpin structure can firmly lock "Mango", greatly reducing the background signal. The binding of SAM and aptamer opens the binding arm of the 5' end of the aptamer, exposing the complete binding arms on both sides, identifying and cutting the substrate probe, releasing the "Mango" fluorescent RNA aptamer, "Mango" restores the correct secondary structure, binds to the dye TO1-Biotin, and restores the fluorescent signal to achieve the detection purpose. Second, the 3' end binding arm of the hammerhead ribozyme is connected in series with the theophylline aptamer. Through the domain of partial base complementarity, the theophylline aptamer locks the 3' end binding arm of the hammerhead ribozyme. Only relying on the binding on the 5' end, the aptamer cannot trans-cleave the substrate probe, and the hammerhead ribozyme loses its trans-cleavage activity and is converted into a theophylline-responsive aptamer (Theo-HHR). The fluorescent RNA aptamer "Mango" is selected as the signal output module, and a double "Mango" substrate probe (dMango) with a special structure is designed. The dMango substrate probe is composed of two complete "Mango" fluorescent RNA aptamers fused through a symmetrical structure. It can bind to two TO1-Biotin dyes at the same time, greatly enhancing the fluorescent signal. In addition, the symmetrical structure connection part of the dMango substrate probe has a site for hammerhead ribozyme recognition and cleavage. The binding of theophylline to the aptamer opens the binding arm of the 3' end of the aptamer, exposing the complete binding arms on both sides, recognizing and cleaving the substrate probe, destroying the correct secondary structure of the "Mango" fluorescent RNA aptamer on the double hairpin structure, making it unable to bind to the dye TO1-Biotin, reducing the fluorescent signal, and achieving the purpose of detection.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a biosensor for detecting small molecules based on aptamer enzyme trans-cleavage of fluorescent RNA aptamers, the sensor comprising SAM-HHR, Theo-HHR, Anti-Mango and dMango; wherein the SAM-HHR is an aptamer enzyme that specifically binds to SAM; Theo-HHR is an aptamer enzyme that specifically binds to theophylline; Anti-Mango and dMango are substrate probes with enzyme cleavage sites.

[0008] According to a preferred technical solution, the aptamers SAM-HHR and Theo-HHR contain aptamers that can specifically bind to SAM and theophylline.

[0009] In a preferred technical solution, the substrate probes Anti-Mango and dMango contain a hammerhead ribozyme trans-cleavage site NUX (N is any base, and X is A, C or U).

[0010] The second aspect of the present invention provides a method for detecting SAM and theophylline based on aptamer trans-cleavage fluorescent RNA aptamer sensor as described in the first aspect, comprising the following steps:

[0011] 1. A fluorescent RNA aptamer sensor based on aptamer enzyme trans-cleavage to detect SAM.

[0012] S1, mixing the aptamer enzyme SAM-HHR and the SAM solution and then incubating them, so that SAM binds to the aptamer, the 5′ binding arm of the SAM-HHR is opened, and the cleavage activity of the aptamer is restored;

[0013] S2, mixing the SAM-HHR with trans-cleavage activity in step S1 and the substrate probe Anti-Mango, allowing the aptamer enzyme to recognize and trans-cleave the substrate probe to release the "Mango" fluorescent RNA aptamer, and "Mango" recovers its correct secondary structure;

[0014] S3, mix the "Mango" fluorescent RNA aptamer with the correct secondary structure with TO1-Biotin and incubate;

[0015] S4. The product after incubation in step S3 is detected for fluorescence signal by F7000 fluorescence spectrophotometer.

[0016] In the preferred technical solution, in step S1, the incubation reaction buffer comprises 10 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 100 mM KCl; reaction temperature is 37°C; time is 15 min.

[0017] According to a preferred technical solution, in step S2, the aptamer enzyme trans-cleavage reaction temperature is 25° C. and the reaction time is 25 min.

[0018] In a preferred technical solution, in step S2, the trans-cleavage mixture includes 1× lysis buffer, pH 8.0, containing 8 mM KCl, 5 mM MgCl 2 , 50 mM Tris-HCl, and 100 mM NaCl.

[0019] In the preferred technical solution, in step S3, the incubation reaction buffer contains 40 mM HEPES (pH 7.6), 10 mM MgCl 2 , 100mM KCl; reaction temperature is room temperature; time is 10min.

[0020] According to a preferred technical solution, in step S4, the fluorescence detection setting parameters are: excitation wavelength 495 nm, emission wavelength 515 nm, and slit width 5 nm.

[0021] 2. A fluorescent RNA aptamer sensor based on aptamer trans-cleavage to detect theophylline.

[0022] S1, same as S1 in step 1, except that SAM is replaced with theophylline;

[0023] S2, mix the substrate probe dual "Mango" fluorescent RNA aptamer and TO1-Biotin and incubate;

[0024] S3, mixing the Theo-HHR with trans-cleavage activity in step S1 and the double "Mango" fluorescent RNA aptamer incubated in step S2 to perform a trans-cleavage reaction;

[0025] S4: Same as S4 in step 1.

[0026] A preferred technical solution is that in step S1, the incubation reaction buffer is the same as step S1 in step 1;

[0027] The preferred technical solution is that in step S2, the incubation reaction buffer is the same as step S3 in step 1;

[0028] In a preferred technical solution, in step S3, the trans-cleavage lysis buffer is the same as that in step S2 in step 1;

[0029] According to a preferred technical solution, in step S4, the fluorescence detection setting parameters are the same as those in step 1 S4.

[0030] Compared with the prior art, a method for detecting SAM and theophylline based on aptamer trans-cleavage of fluorescent RNA aptamers has the following beneficial effects:

[0031] (1) The method for detecting SAM and theophylline based on aptamer trans-cleavage fluorescent RNA aptamer sensor described in the present invention has strong specificity, high sensitivity and no need for labeling, and the detection limits are as low as 0.72nM and 0.16nM.

[0032] (2) The method for detecting SAM and theophylline based on aptamer trans-cleavage fluorescent RNA aptamer sensor described in the present invention has the ability to detect real samples.

[0033] (3) The method for detecting SAM and theophylline based on aptamer trans-cleavage fluorescent RNA aptamer sensor described in the present invention is universal and can be used to detect proteins, nucleic acids, etc., providing a new idea for small molecule detection.

[0034] (4) The method for detecting SAM and theophylline based on aptamer trans-cleavage fluorescent RNA aptamer sensor described in the present invention does not require expensive enzymes and amplification in the entire detection process, is easy to operate, and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to further enhance the understanding of the present invention, the following is a brief introduction to the drawings required for the specific implementation method. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention. In the drawings:

[0036] Figure 1 The schematic diagram of the principle of detecting SAM and theophylline based on the aptamer trans-cleavage fluorescent RNA aptamer sensor of the present invention is shown in FIG. A. Schematic diagram of the principle of detecting SAM; B. Schematic diagram of the principle of detecting theophylline.

[0037] Figure 2 This is a diagram of the optimized fluorescence signal intensity of the Anti-Mango hairpin substrate in the present invention.

[0038] Figure 3 The diagrams are the results of the experiment on the optimization of the blocking length of the binding arms of the aptamers of the present invention, wherein: A. The results of the experiment on the optimization of the blocking length of the binding arms of the SAM-responsive aptamers; B. The results of the experiment on the optimization of the blocking length of the binding arms of the theophylline-responsive aptamers.

[0039] Figure 4 The non-denaturing polyacrylamide gel electrophoresis diagrams of the feasibility of the verification method in the present invention are shown in Figure 1. A. The feasibility gel electrophoresis diagram of SAM detection; B. The feasibility gel electrophoresis diagram of theophylline detection

[0040] Figure 5 This is a diagram showing the influence of potassium ion concentration and magnesium ion concentration on the detection of theophylline by the sensor in the present invention.

[0041] Figure 6This is a diagram showing the effects of aptamer enzyme cleavage reaction time and temperature on the detection of theophylline by the sensor in the present invention.

[0042] Figure 7 The results of the sensitivity test of the target method of the present invention are shown in Figure 1. A. Sensitivity test of SAM at different concentrations; B. Sensitivity test of theophylline at different concentrations.

[0043] Figure 8 The linear relationship diagrams of the present invention are fitted according to the fluorescence signals of targets of different concentrations. A. The linear relationship diagram of the fluorescence signals of SAM of different concentrations; B. The linear relationship diagram of the fluorescence signals of theophylline of different concentrations.

[0044] Fig. 9 The results of the verification method specificity in the present invention are shown in Figures A. Result of SAM-responsive aptazyme specificity; B. Result of theophylline-responsive aptazyme specificity.

[0045] Fig.10 The results of the physical sample detection capability of the verification method in the present invention are shown in FIG. A. Comparison between the detection of SAM concentrations in different cell lysates by the present invention and that by HPLC; B. Comparison between the detection of different theophylline concentrations in the same cell lysate by the present invention and that by HPLC. DETAILED DESCRIPTION

[0046] Sequence Listing

[0047]

[0048]

[0049] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0051] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0052] The experimental methods in the following examples are all conventional methods unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0053] Example 1

[0054] A method for detecting SAM and theophylline based on aptamer trans-cleavage fluorescent RNA aptamer sensor, the specific synthesis steps are as follows:

[0055] 1. Synthesis steps of a fluorescent RNA aptamer sensor for detecting SAM based on aptamer enzyme trans-cleavage:

[0056] S1. Binding reaction of aptamer enzyme SAM-HHR and SAM: 1.2 μL 200 μM SAM-HHR was mixed with 1 μL SAM solution of different concentrations, and 1.9 μL 10 mM Tris-HCl (pH 7.6), 1.9 μL 10 mM MgCl 2 , 1.9μL 100mM KCl and 11.1μL RNase-free ddH 2 O, incubate the mixed system at 37°C for 15 min, and obtain SAM-HHR with trans-cleavage activity through the binding of SAM and aptamer;

[0057] S2. Aptamer SAM-HHR trans-cleaves Anti-Mango to detect SAM: 5.5 μL SAM-HHR with trans-cleavage activity in step S1 and 1 μL 200 μM Anti-Mango were mixed, and 1 μL 50 mM Tris-HCl (pH 7.6) and 1.1 μL 5 mM MgCl were added to the mixture. 2 , 1μL 8mM KCl, 2μL 10mM Nacl and 8.4μL RNase-free ddH 2 O, incubate at 25°C for 25 min;

[0058] S3, mix 2 μL of the mixed solution after trans-cleavage in step S3 and 0.5 μL of 1 mM TO1-Biotin solution, add 4 μL of 40 mM HEPES (pH 7.4), 1 μL of 10 mM MgCl 2 , 10μL 100mM KCl and 2.5μL RNase-free ddH 2 O; reaction temperature is room temperature; time is 10min;

[0059] S4. Collecting fluorescence signals: Collect the fluorescence spectrum of the mixture on a F7000 spectrophotometer. The excitation wavelength is 495nm, the emission wavelength is 515nm to 615nm, and the slit width is 5nm. The data analysis uses the "Mango" fluorescence intensity at 535nm.

[0060] 2. Synthesis steps of a fluorescent RNA aptamer sensor for detecting theophylline based on aptamer enzyme trans-cleavage:

[0061] S1, same as S1 of step 1 in Example 1, except that SAM-HHR is replaced by Theo-HHR, and SAM is replaced by theophylline;

[0062] S2. Obtaining double-hairpin dMango with high fluorescence value: Mix 2.5 μL 200 μM dMango substrate probe with 0.5 μL 1 mM TO1-Biotin solution, add 4 μL 40 mM HEPES (pH 7.4), 1 μL 10 mM MgCl 2 , 10μL 100mM KCl and 2μL RNase-free ddH 2 O; reaction temperature is room temperature; time is 10min;

[0063] S3. Aptazyme Theo-HHR trans-cleaves dMango to detect theophylline: 5.5 μL of Theo-HHR with trans-cleavage activity in step S1 and 2.5 μL of double-hairpin dMango with high fluorescence value in step S2 were mixed, and 1 μL of 50 mM Tris-HCl (pH 7.6) and 1.1 μL of 5 mM MgCl were added to the mixture. 2 , 1μL 8mM KCl, 2μL 10mM Nacl and 8.8μL RNase-free ddH 2 O. Incubate at 25°C for 25 min;

[0064] S4, collecting fluorescence signals: the same as S4 of step 1 in Example 1.

[0065] Example 2

[0066] Design and screening of a fluorescent RNA aptamer sensor based on aptamer trans-cleavage for detecting SAM and theophylline.

[0067] (1) Optimization of the Anti-Mango Hairpin Substrate Probe

[0068] The signal suppression and effective recovery of the fluorescent RNA aptamer "Mango" affect the performance of the detection method. In order to successfully suppress the "Mango" signal, a sequence was extended at the 3′ end of "Mango". This sequence is complementary to some of the "Mango" bases. On this basis, three different hairpin structures were designed to suppress the "Mango" fluorescent signal. The substrate was cut using a hammerhead ribozyme (HHR) without a tandem aptamer to test the signal recovery efficiency of the substrate. The double-stem hairpin structure of M-3 can effectively suppress the fluorescent signal and release the "Mango" fluorescent RNA aptamer by cutting the hairpin structure in the loop region of the stem. The results are shown in Figure 2. Figure 2 shown.

[0069] (2) Structural optimization of aptamer enzyme SAM-HHR.

[0070] The length of the 5′-end binding arm of the aptamer SAM-HHR is crucial to prevent the substrate probe from being trans-cleaved. To this end, we explored the effects of different lengths of the blocking binding arm on the cleavage efficiency of the hammerhead ribozyme. Four aptamers with different lengths of blocking binding arms (SAM-HHR-1, SAM-HHR-2, SAM-HHR-3, SAM-HHR-4) were designed to cleave the double-stem hairpin substrate probe M-3, among which the hammerhead ribozyme SAM-HHR-4 (blocked 8 bases) completely lost its cleavage efficiency. The results are shown in Figure 3 As shown in A.

[0071] (3) Structural optimization of the aptamer enzyme Theo-HHR.

[0072] The length of the 3′-end binding arm of the aptamer Theo-HHR is crucial to prevent the substrate probe from being trans-cleaved. To this end, we explored the effect of different lengths of the blocking binding arm on the cleavage efficiency of the hammerhead ribozyme. Four aptamers with different lengths of blocking binding arms (Theo-HHR-1, Theo-HHR-2, Theo-HHR-3, Theo-HHR-4) were designed to cleave the double-hairpin substrate probe dMango, among which the hammerhead ribozyme Theo-HHR-4 (blocked 12 bases) completely lost its cleavage efficiency. The results are shown in Figure 3 As shown in B.

[0073] Example 3

[0074] A feasibility analysis of aptamer-based trans-cleavage fluorescent RNA aptamer sensor for detecting SAM and theophylline.

[0075] In order to investigate the feasibility of the method of the present invention for detecting SAM and theophylline, the results of polyacrylamide gel electrophoresis were analyzed. Figure 4 .

[0076] The feasibility of the method of the present invention for SAM detection is shown in Figure 4 A, with lane 1 (Anti-Mango), lane 2 (HHR) and lane 3 (SAM-HHR) as references, the electrophoresis band after the substrate probe Anti-Mango was trans-cleaved by HHR can be clearly observed in lane 4, and no cleavage band can be observed in lane 5 without the target molecule SAM. After adding SAM to lane 6, the cleavage band can be clearly observed, and the position is the same as that in lane 4, which proves that SAM-HHR can recognize SAM and be activated for trans-cleavage activity, thereby trans-cleaving the substrate probe.

[0077] The feasibility of the method of the present invention for theophylline detection is shown in Figure 4 B. After theophylline was incubated with Theo-HHR, it was reacted with the double-hairpin substrate probe dMango in the cleavage reaction buffer and analyzed by gel electrophoresis. A clear band with a faster electrophoretic migration speed (lane6) was obtained. This band corresponds to the cleavage product (lane4) of the double-hairpin substrate probe dMango catalyzed by the hammerhead ribozyme. This proves that theophylline can bind to the aptamer, restore the correct secondary structure of the aptamer, and the aptamer restores the trans-cleavage activity. The double-hairpin substrate probe dMango is trans-cleaved by the aptamer.

[0078] Example 4

[0079] The effects of different reaction conditions on the detection of SAM and theophylline by aptamer-based trans-cleavage fluorescent RNA aptamer sensor were tested.

[0080] (1) Optimization of potassium ion concentration

[0081] The preparation method of the sensor is the same as that of Example 1, except that the concentration of potassium ions in the buffer solution is changed to 1mM, 10mM, 50mM, 100mM, 150mM, and 200mM. Figure 5 A.

[0082] from Figure 5 As can be seen in A, the fluorescence signal is highest when the potassium ion concentration is 50 mM.

[0083] (2) Optimization of magnesium ion concentration

[0084] The preparation method of the sensor is the same as that of Example 1, except that the concentration of magnesium ions in the buffer solution is changed to 0mM, 1mM, 2mM, 5mM, 10mM, and 20mM. Figure 5 B.

[0085] from Figure 5 As can be seen in B, the fluorescence signal is highest when the magnesium ion concentration is 2 mM.

[0086] (3) Optimization of Theo-HHR cleavage dMango reaction time

[0087] The preparation method of the sensor was the same as that of Example 1, except that the reaction time of Theo-HHR cleavage dMango was changed to 10 min, 20 min, 30 min, 40 min, and 50 min. Figure 6 A.

[0088] from Figure 6 As shown in A, the signal-to-noise ratio was the highest and the fluorescence signal was the best when the reaction time of Theo-HHR cleavage dMango was 40 min.

[0089] (4) Optimization of reaction temperature for Theo-HHR cleavage of dMango

[0090] The preparation method of the sensor was the same as that of Example 1, except that the reaction temperature of Theo-HHR cleavage dMango was changed to 10°C, 20°C, 30°C, 40°C, and 50°C. Figure 6 B.

[0091] from Figure 6 As shown in B, the signal-to-noise ratio and the fluorescence signal were the best when the reaction temperature of Theo-HHR cleavage dMango was 30°C.

[0092] Example 5

[0093] Performance analysis experiment of aptamer trans-cleavage fluorescent RNA aptamer sensor for detecting SAM and theophylline.

[0094] (1) Sensitivity test

[0095] The preparation method of the fluorescent RNA aptamer sensor based on aptamer enzyme trans-cleavage is the same as that in Example 1, except that the concentration of SAM is changed to 0nM, 80nM, 300nM, 600nM, 1μM, 1.5μM, 2μM, 3μM, 8μM, 12μM, and 15μM. The results are shown in Figure 7 A.

[0096] from Figure 7 As shown in A, the fluorescence intensity gradually increases with the increase of SAM concentration.

[0097] The preparation method of the fluorescent RNA aptamer sensor based on aptamer enzyme trans-cleavage is the same as that in Example 1, except that the concentration of theophylline is changed to 0nM, 20nM, 50nM, 100nM, 200nM, 0.5μM, 1μM, 1.5μM, 2μM, 5μM, and 10μM. The results are shown in Figure 7 B.

[0098] from Figure 7 As can be seen in B, as the concentration of theophylline increases, the fluorescence intensity gradually decreases.

[0099] (2) Establishment of standard curve

[0100] use Figure 7 The fluorescence intensity corresponding to different SAM or theophylline concentrations was fitted nonlinearly and linearly. The results are shown in Figure 8 .

[0101] from Figure 8 As shown in A, there is a good linear relationship between fluorescence intensity and SAM concentration from 80 nM to 1.5 μM, and the regression equation is F = 199.74X + 129.68 (R 2 =0.9921), and the detection limit was 0.72 nM.

[0102] from Figure 8 As shown in B, there is a good linear relationship between fluorescence intensity and theophylline concentration from 20 nM to 500 nM, and the regression equation is F = -1558.89X + 1540.43 (R 2 =0.9968), and the detection limit was 0.16 nM.

[0103] (3) Specificity detection

[0104] Specific detection of SAM

[0105] The preparation method of the fluorescent RNA aptamer sensor based on aptamer enzyme trans-cleavage is the same as that in Example 1, except that the target molecules are changed to theophylline, S-adenosyl-L-homocysteine, adenosine, methionine, and adenine. Fig. 9 A.

[0106] from Fig. 9 As can be seen in A, the fluorescence intensity increases only in the presence of SAM small molecules, indicating that SAM can be specifically detected.

[0107] Specific detection of theophylline

[0108] The preparation method of the fluorescent RNA aptamer sensor based on aptamer enzyme trans-cleavage is the same as that in Example 1, except that the target molecule is changed to S-adenosylmethionine, tetracycline, guanine, adenosine, and adenine. Fig. 9 B.

[0109] from Fig. 9 As can be seen in B, theophylline small molecules can induce the most obvious change in fluorescence intensity, indicating that theophylline can be specifically detected.

[0110] Example 6

[0111] Physical sample testing of theophylline and SAM based on aptamer trans-cleavage fluorescent RNA aptamer sensor

[0112] The physical sample detection of SAM was performed using human cell lysates of HEK-293T (human embryonic kidney cells), HeLa (cervical cancer cells), HepG2 (liver cancer cells) and MCF-7 (breast cancer cells). 500nM SAM was added to the culture medium of each of the above cell lines. After transfection for 5 hours, the culture medium was replaced with fresh culture medium without SAM. The culture medium was replaced again one day after transfection, and then resuspended in PBS buffer, treated with 40μL of 400g / L TCA, stirred the mixture, incubated on ice for 30 minutes, centrifuged at 13000r / min for 15 minutes at 4°C using a high-speed centrifuge, and the supernatant was collected. SAM-HHR was added to the supernatant of different cell lysates, and the preparation method and the buffer used were the same as in Example 1. After fluorescence detection, it was found that there were strong fluorescence signals in HeLa cell lysate and HepG2 cell lysate, and the fluorescence values ​​in HEK-293T cell lysate and MCF-7 cell lysate were lower. The concentrations in different cell lysates were detected by high performance liquid chromatography and were found to be comparable to the concentrations detected in the present invention. Fig.10 As shown in A, the application of the present invention in detecting SAM in real samples is confirmed.

[0113] Physical sample testing of theophylline detection based on aptamer trans-cleavage fluorescent RNA aptamer sensor

[0114] Theo-HHR and dMango were added to the supernatant of cell lysates with different theophylline concentrations, and the preparation method and the buffer used were the same as those in Example 1. The theophylline concentration in cell lysates with different theophylline concentrations was detected by high performance liquid chromatography (HPLC). The theophylline concentration in cell lysates with different theophylline concentrations was detected by high performance liquid chromatography (HPLC), and it was found to be comparable to the concentration detected by the present invention. Fig.10 As shown in B, the application of the present invention in detecting theophylline in physical samples is confirmed.

Claims

1. A sensor for small molecule detection based on aptamer enzyme trans-cleavage of fluorescent RNA aptamer, characterized in that: The steps include: (1) Modification of small molecule-responsive aptamers: SAM aptamers or theophylline aptamers are linked in series through hammerhead ribozyme binding arms, and the hammerhead ribozyme binding arms with trans-cleavage activity are locked using the principle of base complementary pairing, so that the hammerhead ribozyme loses its trans-cleavage activity. The binding of SAM or theophylline to the aptamer will restore the aptamer to the correct conformation, and the trans-cleavage activity of the hammerhead ribozyme will be activated, thereby trans-cleaving the substrate probe; (2) inserting the aptamer sequence with T7 promoter and the specially designed substrate probe sequence into the restriction endonuclease site of the pUC57 plasmid vector, and linearizing the plasmid with the restriction endonuclease BamHI to obtain the full-length DNA products of the aptamer and substrate probe; (3) transcribing the DNA product obtained in step (2) into RNA; (4) reacting the aptamer RNA obtained in step (3) with SAM or theophylline in an incubation buffer in the presence of K ions, and then mixing with a lysis buffer loaded with a substrate probe, followed by sequential PAGE gel analysis and "Mango" fluorescence signal detection.

2. according to the method described in claim 1, there are restriction endonuclease sites and T7 promoter on the wherein said pUC57 plasmid vector, the plasmid vector can be linearized by restriction endonuclease BamHI, and then can transcribe aptamer RNA and substrate probe under the effect of T7 RNA polymerase.

3. The sensor according to claim 1, characterized in that The substrate probe contains the aptamer trans-cleavage site NUX (N is any base, X is A, C or U).

4. According to the method described in claims 1-3, the transcription product aptamer can react with SAM or theophylline to activate the trans-cleavage activity of the hammerhead ribozyme, thereby recognizing and cleaving the substrate probe.

5. The detection method according to claim 4, characterized in that: PAGE gel analysis after aptamer trans-cleavage of the substrate probe was performed using 10% non-denaturing polyacrylamide gel.

6. The detection method according to claim 1, characterized in that: The pH of the incubation buffer used in the incubation process in step (4) is 8.0, and it contains 50 mM Tris-HCl and 5 mM magnesium chloride.

7. The detection method according to claim 1, characterized in that: The pH of the 1× lysis buffer used for loading in step (4) is 8.0, and it contains 8 mM potassium chloride, 5 mM magnesium chloride, 50 mM Tris-HCl and 100 mM sodium chloride.

8. The method according to claims 1, 6 and 7, characterized in that: The fluorescence detection setting parameters after trans-cleavage of the substrate probe were: excitation wavelength 495 nm, emission wavelength 515 nm, and slit width 5 nm.

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