Methylation-driven construction of dual-color luminescent RNA nanosensors for label-free and ultrasensitive detection of multiple DNA methyltransferases

Abstract

The present invention belongs to the field of biological analysis, and provides a methylation-driven dual-color luminescent RNA nanosensor for label-free and ultra-sensitive detection of multiple DNA methyltransferases. The dual-color luminescent RNA nanosensor is constructed by integrating cascade nucleic acid amplification and dual-color luminescent RNA aptamers, thereby realizing label-free and ultra-sensitive detection of multiple DNA methyltransferases. The ultra-high specificity of MDREs for methylated DNA, efficient double-loop cascade amplification, and high signal-to-noise ratio of luminescent RNA aptamer-fluorophore pairs are utilized. Compared with the experiments reported for DNA MTase, this nanosensor has obvious advantages: (1) ensuring accurate quantification of low-abundance DNA MTases; (2) significantly improving amplification efficiency; (3) realizing label-free and simultaneous detection of multiple DNA MTases at a high signal-to-noise ratio; and detecting in a label-free manner, which is convenient to operate and reduces costs.

Description

Technical Field

[0001] The invention belongs to the field of biological analysis, and particularly relates to a methylation-driven dual-color luminescent RNA nanosensor for label-free and ultrasensitive detection of multiple DNA methyltransferases. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] DNA methylation is a well-known epigenetic modification that usually occurs at the C-5 position of cytosine (C) or the N-6 position of adenine (A) in genomic DNA and plays an important role in various biological processes in both prokaryotes and eukaryotes. DNA methyltransferases (MTases) are an important superfamily of methylases that can catalyze DNA methylation by transferring methyl groups from S-adenosyl-L-methionine (SAM) to specific bases to balance the cellular methylation pattern. Abnormal DNA methylation may alter DNA structure, lead to genomic instability, inactivate tumor suppressor genes, cause transcriptional silencing, and is closely related to many types of human diseases (such as neurodegenerative diseases and immune system disorders) and cancers (such as breast cancer, lung cancer, thyroid cancer, colon cancer, liver cancer, kidney cancer, and cervical cancer). Due to their important functions in establishing and maintaining methylation patterns, DNA MTases have been widely considered as potential biomarkers for disease occurrence and therapeutic targets for cancer treatment. Therefore, accurate and sensitive detection of multiple DNA MTases is of great significance for biochemical research and clinical diagnosis of human diseases related to DNA methylation.

[0004] So far, various methods have been developed to measure the activity of DNA MTases. Traditional methods are usually based on isotope-labeled gel electrophoresis, immunoblot analysis with 5-mC monoclonal antibodies, and high-performance liquid chromatography (HPLC) using a methanol-water mobile phase. However, they usually involve irradiated hazardous materials, labor-intensive procedures, and delicate equipment. In addition, some new methods have been developed, including colorimetry, surface-enhanced Raman spectroscopy (SERS), electrochemistry, and fluorescence analysis. Colorimetry is a visualization detection of the activity of DNA cytosine-5 methyltransferase (Dnmt 1) and Dam MTase using enzyme-responsive DNA-AuNP nanoassembly and end-protection-induced DNA-gold nanoparticle (AuNP) diffusion. SERS detection uses multiplexed signal amplification triggered by methylation cleavage to detect Dam MTase activity. Electrochemical analysis combines methylation-sensitive restriction endonucleases (MSREs) with various nanomaterials, such as AuNPs, silver nanoparticles (AgNPs), and graphene oxide (GO), to detect the activities of Dnmt 1, M.SsssI, and Dam MTases. Fluorescence analysis uses fluorescence resonance energy transfer (FRET) based on GO and quantum dots (QD) to quantify the activities of Dam and HaeIII MTases. However, these methods lack sensitivity, require delicate operations, and require complex nanomaterial synthesis and cumbersome electrode modification, which limits their practical applications, especially in detecting low-abundance targets. To improve sensitivity, a variety of nucleic acid isothermal amplification methods have been introduced in the detection of DNA MTases, including rolling circle amplification (RCA), isothermal exponential amplification reaction (EXPAR), enzyme repair-based amplification (ERA), and exonuclease / endonuclease-assisted signal amplification (EASA). Although the sensitivity is improved, their applications are usually monitored by fluorescent dyes, such as SYBR Green I, TaqMan, and molecular beacons. SYBR Green I can be inserted into any double-stranded DNA (dsDNA) fragment, has a tendency to false positives, and is not suitable for multiplex detection. TaqMan and molecular beacons can be used for homogeneous analysis, but the dual modification of fluorophore and quencher increases the experimental cost. It is worth noting that due to the diversity of catalytic substrates and the rarity of methylation-dependent restriction endonucleases (MDREs), previously reported methods can only detect one type of DNA MTase. Therefore, it is very necessary to develop a new method that is more convenient and economical to simultaneously detect multiple DNA MTases.

[0005] Recently, luminescent RNA aptamers that bind to cognate fluorophores were discovered through an in vitro selection technique called systematic evolution of ligands by exponential enrichment (SELEX). As RNA mimics of enhanced green fluorescent proteins (eGFPs), Mango and Spinach aptamers can bind and activate the fluorescence of the corresponding fluorophores TO1-3PEG-biotin (TO1-biotin) and 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), respectively. Due to their high signal-to-noise ratio, excellent resistance to photobleaching, rapid fluorescence activation, and broad applicability to target molecules, the Mango / Spinach fluorophore pair has become a candidate for the development of luminescent biosensing platforms for the detection of various molecular biomarkers. Despite the successful application of Mango / Spinach-based biosensors, several challenges remain. The selection of appropriate sites for recognition element insertion is a key issue, and the binding affinity of the redesigned Mango and Spinach aptamers to the target molecules is reduced due to the increased complexity of the sequences. To date, Mango / Spinach-based biosensors have rarely utilized signal amplification strategies, and their sensitivity is usually limited for the detection of low-abundance targets. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for constructing a methylation-driven dual-color luminescent RNA nanosensor for label-free and ultrasensitive detection of multiple DNA methyltransferases.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a method for constructing a methylation-driven dual-color luminescent RNA nanosensor for label-free and ultrasensitive detection of multiple DNA methyltransferases, comprising:

[0009] In the presence of CpG methyltransferase M.SssI and adenine methyltransferase Dam MTase, dumbbell probe 1, DP1, was methylated at cytosine C-5 and adenine N-6, respectively, forming the catalytic substrates of GlaI and DpnI;

[0010] It is cleaved simultaneously by GlaI and DpnI to produce two cleavage products, CP1 and CP2;

[0011] The cleavage products CP1 and CP2 hybridize with the 3' end sequence of the middle loop of dumbbell probe 2-DP2, respectively, and then act as primers to initiate a bidirectional SDA reaction in the presence of Bst DNA polymerase, Nt.BspQI and dNTPs to generate triggers 1 and 2;

[0012] The triggers 1 and 2 hybridize with the 5' end sequence of the stem of DP2 and then serve as transcription templates to activate dual transcription amplification in the presence of T7 RNA polymerase and NTPs to produce mango and spinach aptamers;

[0013] The mango and spinach aptamers are combined with TO1-biotin and DFHBI to generate enhanced dual-color fluorescence signals, and the activities of M.SssI and Dam MTases are detected simultaneously.

[0014] T7-based transcriptional amplification is a new T7 promoter-specific RNA amplification technology. It transcribes a large number of ssRNA sequences from the downstream DNA sequence of the T7 promoter through repeated polymerization of ribonucleoside triphosphates by T7 RNA polymerase. T7-based transcriptional amplification has the advantages of high sequence design flexibility and good compatibility with target-dependent signal amplification, and has been applied to the detection of nucleic acids and DNA-binding enzymes. Here, inspired by the unique characteristics of T7-based transcriptional amplification, the present invention demonstrates for the first time the construction of a dual-color luminescent RNA nanosensor by integrating cascade nucleic acid amplification and methylation-driven dual-color luminescent RNA aptamers, achieving label-free and ultrasensitive detection of multiple DNA methyltransferases. Utilizing the ultrahigh specificity of MDREs for methylated DNA, efficient dual-loop cascade amplification, and high signal-to-noise ratio of the luminescent RNA aptamer-fluorophore pair, the nanosensor has good specificity and high sensitivity, with a detection limit of 3.77×10 for M.SssI MTase. -3 U / mL, and the detection limit for Dam MTase was 7.21×10 -5 In addition, it can be used to screen potential inhibitors and quantify M.SssI and Dam MTases from E. coli cells and human serum samples, providing a general and powerful platform for the detection of multiple DNA MTases.

[0015] The second aspect of the present invention provides a methylation-driven dual-color luminescent RNA nanosensor, comprising:

[0016] Two dumbbell probes: DP1 and DP2;

[0017] Among them, the stem of DP1 contains the dinucleotide sequences 5′-ACGT-3′ / 3′-TGCA-5′ and 5′-GATC-3′ / 3′-CTAG-5′;

[0018] The stem of DP2 consists of a double-stranded region and a single-stranded region. The double-stranded region contains part of the recognition sequence of the cutting enzyme, and the single-stranded region contains the other part of the recognition sequence of the cutting enzyme, the T7 promoter sequence, and the DNA sequences of mango and spinach adapters.

[0019] TO1-biotin and DFHBI;

[0020] Bst DNA polymerase, Nt.BspQI, and dNTPs;

[0021] T7 RNA polymerase and NTPs.

[0022] Beneficial effects of the present invention

[0023] (1) Good specificity and high sensitivity

[0024] Due to the good specificity of MDREs for methylated DNA, efficient double-loop cascade amplification, and high signal-to-noise ratio of the luminescent RNA aptamer-fluorophore pair, the sensor has good specificity and ultrahigh sensitivity, with a detection limit of 3.77×10 -3 U / mL, and the detection limit for Dam MTase was 7.21×10 -5 U / mL, this nanosensor has significant advantages such as low cost, simple operation, good specificity, high sensitivity, high reliability and wide universality. It provides a new paradigm for in vitro luminescent RNA aptamer engineering and a universal platform for the detection of multiple DNA MTases, and has broad application prospects in biomedical research, clinical diagnosis and drug resistance discovery.

[0025] (2) Label-free rapid detection

[0026] The method can be completed uniformly in one tube within 2 hours and is detected in a label-free manner, without involving any methylation-independent cleavage, complicated manipulation, and fluorescent labeling process in conventional methyltransferase assays.

[0027] (3) Practicality and versatility

[0028] In this study, the nanosensor can be used to distinguish M.SssI and Dam MTases from interferases, screen potential inhibitors, and quantify the activities of M.SssI and Dam MTases in Escherichia coli cells and human serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings in the specification, which constitute a part of the present application, are used to provide further understanding of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0030] Figure 1Schematic diagram of the engineered dual-color luminescent RNA nanosensor driven by methylation for label-free and ultrasensitive detection of multiple DNA MTases. The strategy consists of three steps: (1) cleavage of dumbbell substrates catalyzed by DNA MTases, (2) methylation-cleavage-guided dual-loop cascade amplification induced production of two luminescent RNA aptamers, and (3) simultaneous detection of dual-color fluorescence signals of TO1-biotin and DFHBI.

[0031] Figure 2 .(A) Non-denaturing PAGE analysis of the products of the 5-mC cleavage-directed double-loop cascade amplification. Lane M, DNA ladder marker; Lane 1, synthetic DP1; Lane 2, synthetic DP2-1; Lane 3, in the presence of GlaI+DP1; Lane 4, synthetic CP1; Lane 5, in the presence of M.SssI+GlaI+DP1; Lane 6, synthetic trigger 1; Lane 7, in the presence of M.SssI+GlaI+DP1+DP2-1+Bst DNA polymerase+Nt.BspQI; Lane 8, synthetic transcript-1; Lane 9, in the presence of M.SssI+GlaI+DP1+DP2-1+Bst DNA polymerase+Nt.BspQI+T7 RNA polymerase. (B) Non-denaturing PAGE analysis of the products of the 6-mA cleavage-directed double-loop cascade amplification. Lane M, DNA ladder marker; Lane 1, synthetic DP1; Lane 2, synthetic DP2-2; Lane 3, in the presence of DpnI+DP1; Lane 4, synthetic CP2; Lane 5, in the presence of Dam+DpnI+DP1; Lane 6, synthetic trigger 2; Lane 7, in the presence of Dam+DpnI+DP1+DP2-2+Bst DNA polymerase+Nt.BspQI; Lane 8, synthetic transcript-2; Lane 9, in the presence of Dam+DpnI+DP1+DP2-2+Bst DNA polymerase+Nt.BspQI+T7 RNA polymerase. (C) Fluorescence measurement of the engineered luminescent RNA nanosensor driven by 5-cytosine methylation in the absence (black line) and presence (red line) of M.SssI. The inset shows the fluorescence intensity of TO1-biotin in the absence (black bars) and presence (red bars) of M.SssI. (D) Fluorescence measurements of the engineered luminescent RNA nanosensor driven by 6-adenine methylation in the absence (grey line) and presence (green line) of Dam. The inset shows the fluorescence intensity of DFHBI in the absence (grey bars) and presence (green bars) of Dam.

[0032] Figure 3(A) Fluorescence emission spectra of TO1-biotin in response to different concentrations of M.SssI MTase. (B) Changes in fluorescence intensity of TO1-biotin corresponding to different concentrations of M.SssI MTase. The inset shows the linear relationship between fluorescence intensity and the logarithm of M.SssI MTase concentration. (C) Fluorescence emission spectra of DFHBI in response to different concentrations of Dam MTase. (D) Changes in fluorescence intensity of DFHBI corresponding to different concentrations of Dam MTase. The inset shows the linear relationship between fluorescence intensity and the logarithm of Dam MTase concentration. Error bars represent the standard deviation of three independent experiments.

[0033] Figure 4 .Measurement of fluorescence intensity of TO1-biotin and DFHBI in response to 100U / mL M.SssI MTase+100U / mL Dam MTase, 100U / mL M.SssIMTase, 100U / mL Dam MTase, 100U / mL MspI MTase, 100U / mL M.CviPI MTase, 0.1g / L BSA and a control with only reaction buffer. Error bars represent standard deviations of three independent experiments.

[0034] Figure 5 (A) Changes in relative activity of M.SssI MTase in response to different concentrations of decitabine. (B) Changes in relative activity of Dam MTase in response to different concentrations of gentamicin. Experiments were performed using 100 U / mL of M.SssI MTase and 100 U / mL of Dam MTase. Error bars represent standard deviations of three independent experiments.

[0035] Figure 6 .Activity detection graphs of Dam MTase in GW5100 (Dam positive) and JM110 (Dam negative) E. coli cells, including (A) DFHBI fluorescence signals in GW5100 E. coli, JM110 E. coli, and lysate, (B) fluorescence intensity of DFHBI in response to different concentrations of GW5100 E. coli total protein, and the linear relationship between the fluorescence intensity of DFHBI and the logarithm of the total protein concentration (C) (inset), (C) fluorescence signal of TO1-biotin in response to different concentrations of M.SssI MTase in GW5100 E. coli cell extracts, and the linear relationship between the fluorescence intensity of TO1-biotin (F) and the logarithm of the M.SssI MTase concentration (C) (inset). DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0037] As described in the background art, DNA methylation is one of the most important epigenetic modifications and is involved in regulating a variety of cellular processes in prokaryotes and eukaryotes. DNA methyltransferases (MTases) can maintain the methylation pattern of genomic DNA, and their abnormal activity may lead to various diseases including cancer. However, due to the diversity of catalytic substrates and the scarcity of methylation-dependent restriction endonucleases, accurate and sensitive detection of multiple DNA MTases remains challenging. Here, inspired by the intrinsic advantages of luminescent RNA aptamers and the unique characteristics of T7 transcription amplification, the present invention demonstrates for the first time a methylation-driven construction of a dual-color luminescent RNA nanosensor for label-free and ultrasensitive detection of multiple DNA MTases. In the presence of CpG (M.SssI) and adenine methyltransferase (Dam), dumbbell probe 1 (DP1) is methylated at cytosine (C-5) and adenine (N-6), respectively, to form catalytic substrates of GlaI and DpnI, which are then cleaved simultaneously by GlaI and DpnI to produce two cleavage products (CP1 and 2). CP1 and 2 can hybridize with the 3' end sequence of the loop in DP2 and act as primers to initiate a bidirectional SDA reaction to produce triggers 1 and 2. Triggers 1 and 2 can hybridize with the 5' end sequence of the stem in DP2 and act as transcription templates to activate dual transcription amplification in the presence of T7 RNA polymerase, producing two luminescent RNA transcripts (i.e., mango and spinach aptamers). TO1-biotin and DFHBI can specifically bind to mango and spinach aptamers to produce enhanced dual-color fluorescence signals and simultaneously detect the activities of M.SssI and Dam MTases. The nanosensor has good specificity and high sensitivity, with a detection limit of 3.77×10 for M.SssI MTase. -3 U / mL, and the detection limit for Dam MTase was 7.21×10 -5 U / mL. In addition, it can be used to screen potential inhibitors and quantify the activities of M.SssI and Dam MTases in E. coli cells and human serum samples. Importantly, this nanosensor can be completed uniformly in one tube within 2 hours and detected in a label-free manner without any methylation-independent cleavage, complicated operation and fluorescent labeling, providing a convenient, novel and powerful platform for detecting multiple DNA MTases in biomedical research and clinical treatment.

[0038] The present invention provides a method for constructing a methylation-driven dual-color luminescent RNA nanosensor for label-free and ultrasensitive detection of multiple DNA methyltransferases, comprising:

[0039] In the presence of CpG (M.SssI) and adenine methyltransferase (Dam), dumbbell probe 1 (DP1) is methylated at cytosine (C-5) and adenine (N-6), respectively, forming catalytic substrates for GlaI and DpnI, and then cleaved by GlaI and DpnI simultaneously to produce two cleavage products (CP1 and 2). CP1 and 2 can hybridize with the 3' end sequence of the loop in DP2 and act as primers to initiate the bidirectional SDA reaction to produce triggers 1 and 2. Triggers 1 and 2 can hybridize with the 5' end sequence of the stem in DP2 and act as transcription templates to activate dual transcription amplification in the presence of T7 RNA polymerase, producing two luminescent RNA transcripts (i.e., mango and spinach aptamers). TO1-biotin and DFHBI can specifically bind to mango and spinach aptamers to produce enhanced dual-color fluorescence signals, and simultaneously detect the activities of M.SssI and Dam MTases. Compared with the reported experiments on DNA MTase, this nanosensor has obvious advantages: (1) MDREs (i.e., GlaI and DpnI) specifically cleave 5-mC and 6-mA, ensuring accurate quantification of low-abundance DNA MTases; (2) The introduction of double-loop cascade amplification (i.e., SDA and transcription amplification) significantly improves the amplification efficiency; (3) The luminescent RNA aptamer-fluorophore pair can generate a dual-color fluorescent signal, achieving label-free and simultaneous detection of multiple DNA MTases at a high signal-to-noise ratio; (4) This experiment can be completed uniformly within 2 h, does not involve any methylation-independent cleavage, complex operations, and fluorescent labeling, and performs detection in a label-free manner, which is easy to operate and reduces costs.

[0040] In some embodiments, the stem of DP2 consists of a double-stranded region and a single-stranded region. The double-stranded region contains a partial recognition sequence of the cutting enzyme, and the single-stranded region contains other partial recognition sequences of the cutting enzyme, a T7 promoter sequence, and DNA sequences of mango and spinach adapters.

[0041] In some embodiments, the cleavage product of DP1 can mediate bidirectional SDAs after hybridization with DP2, thereby inducing dual transcription amplification to produce two luminescent RNA aptamers;

[0042] In some embodiments, an NH 2 and three unpaired bases.

[0043] In some embodiments, the stem in the DP1 comprises the dinucleotide sequences 5'-ACGT-3' / 3'-TGCA-5' and 5'-GATC-3' / 3'-CTAG-5'.

[0044] In some embodiments, the two loop sequences of DP1 are respectively complementary to partial sequences of the loop of DP2.

[0045] In some embodiments, the catalytic substrate of GlaI is 5'-A-mC-GT-3'.

[0046] In some embodiments, the catalytic substrate of DpnI is 5'-G-mA-TC-3'.

[0047] In some embodiments, if M.SssI and Dam MTases are lacking in the sample, no fluorescent signals of TO1-biotin and DFHBI are detected; otherwise, M.SssI and Dam MTases are present.

[0048] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0049] In the following examples, in dumbbell probe 1 (DP1), the underlined regions represent the recognition sites of M.SssI and Dam MTases, respectively. In dumbbell probes 2-1 and 2-2, the underlined portions represent the recognition sites of Nt.BtpQI, the italic regions represent the T7 promoter sequences, and the bold portions represent the DNA sequences of mango and spinach aptamers, respectively.

[0050]

[0051]

[0052] Among them, Dumbbell probe 1 (SEQ ID NO.1 in sequence listing), Dumbbell probe 2-1 (SEQ ID NO.2 in sequence listing), Dumbbell probe 2-2 (SEQ ID NO.3 in sequence listing), Cleavage product 1 (SEQ ID NO.4 in sequence listing), Cleavage product 2 (SEQ ID NO.5 in sequence listing), Trigger 1 (SEQ ID NO.6 in sequence listing), Trigger 2 (SEQ ID NO.7 in sequence listing), Transcript-1 (SEQ ID NO.8 in sequence listing), and Transcript-2 (SEQ ID NO.9 in sequence listing).

[0053] Example 1

[0054] Methylation-driven construction of dual-color luminescent RNA nanosensors for label-free detection of multiple DNA methyltransferases. All oligonucleotides were dissolved in 1× Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 7.4) to prepare stock solutions. Dumbbell probe 1 (DP1), dumbbell probe 2-1 (DP2-1), and dumbbell probe 2-2 (DP2-2) were hybridized in 1× hybridization buffer (1.5 mM MgCl 2 , 10mM Tris-HCl, pH 8.0) was diluted to 10μM, incubated at 95℃ for 5min, cooled to room temperature and folded into a stable hairpin structure and double-stranded DNA duplex. Subsequently, 1μL of synthetic DP1 and DP2 was added to 10μL of reaction solution containing different concentrations of M.SssI and DamMTases, 320μM SAM, 10U GlaI, 6U DpnI and 1μL 10×SEBuffer Y, and incubated at 37℃ for 35min. After the cleavage reaction, the product was added to 20μL of reaction solution containing 1U Bst DNA polymerase, 5U Nt.BspQI, 0.25mM dNTPs, 20U RNase inhibitor, 1μL 10×ThermoPol reaction buffer, 2μL 10×NEBuffer3.1, and incubated at 55℃ for 30min. The obtained reaction product was added to 30 μL of a reaction solution containing 30 U T7 RNA polymerase, 0.5 mM NTPs, 100 nM TO1-biotin, 10 μM DFHBI and 3 μL 10× RNAPol reaction buffer, and reacted at 37° C. for 45 min.

[0055] Electrophoresis analysis and steady-state fluorescence measurement. To analyze the cleavage, polymerization, and transcription products, 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) was performed in 1×TBE buffer (9mMTris-HCl, 9mM boric acid, 0.2mM EDTA, pH 7.9) at room temperature for 50 min at a constant voltage of 110V. After electrophoresis, the gel was stained with SYBR gold and then visualized with a ChemiDoc MP imaging system (Hercules, CA). For fluorescence measurement, 30μL of the reaction product was diluted to a final volume of 60μL with ultrapure water. The fluorescence spectrum was measured by a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). At a scan rate of 20nm / s, the excitation wavelengths were 400nm and 495nm, respectively, and the fluorescence intensity of DFHBI and TO1-biotin was recorded at emission wavelengths of 505nm and 532nm for further data analysis.

[0056] Inhibitor experiment. For the inhibition test of M.SssI MTase, different concentrations of decitabine were added to the reaction mixture (1μM DP1, 1μM DP2-1, 100U / mL M.SssI, 10U GlaI, 320μM SAM and 1μL 10×SEBufferY) and incubated at 37°C for 35min. For the inhibition test of Dam MTase, different concentrations of gentamicin were pre-incubated with the reaction solution (1μM DP1, 1μL 10×SEBuffer Y) at 37°C for 15min. Then 1μM DP2-2, 100U / mL Dam MTase, 6U DpnI, 320μM SAM were added to the above reaction mixture and incubated at 37°C for 35min. The reaction products were used for the activity detection of M.SssI and Dam MTases according to the above steps. The relative activity (RA) of M.SssI MTase was calculated based on the equation: RA(%) = C i / C t ×100%=10 (Fi–Ft) / 446.38 ×100%, where F t represents the fluorescence intensity of TO1-biotin in the presence of 100 U / mL M.SssI MTase; F i It shows the fluorescence intensity of TO1-biotin in the presence of M.SssI MTase and decitabine. i and C t according to Figure 3 The linear correlation equation in B is obtained. According to the equation RA (%) = C i / C t ×100%=10 (Fi–Ft) / 269.14 ×100% to calculate the relative activity (RA) of Dam MTase, where F t is the DFHBI fluorescence intensity in the presence of 100 U / mL Dam MTase; F i Represents the fluorescence intensity of DFHBI in the presence of Dam MTase and gentamicin. i and C t according to Figure 3 The linear correlation equation of D is obtained.

[0057] Cell culture and cell extract preparation. Colonies of GW5100 and JM110 Escherichia coli were placed in 3 mL of liquid culture medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride) and cultured in a rotary shaker at 37°C and 250 rpm for 12 h. Then 100 μL of bacteria were added to 3 mL of liquid culture medium and incubated under the same conditions for 2.5 h. In the exponential growth phase, E. coli cells were collected and centrifuged at 8000 rpm for 10 min to obtain cell pellets. The cells were washed with 10× phosphate buffer (PBS, 1.6 M sodium chloride, 10.6 mM potassium dihydrogen phosphate (KH 2 PO 4 ), 29.7 mM sodium phosphate dihydrate (Na 2 HPO 4 .7H 2 After washing three times with 4% paraformaldehyde (pH 7.4), the cells were lysed with cell lysis buffer (P0013, Beyotime Biotech, Shanghai, China), and the protein concentration was quantified using the BCA protein assay kit (P0010S, Beyotime Biotech, Shanghai, China).

[0058] Detection of DNA MTase activity in human serum. For the recovery experiment, different concentrations of M.SssI MTase were added to the reaction solution (10% human serum, 1μM DP1, 1μM DP2-1, 320μM SAM, 10U GlaI, 1μL 10×SEBuffer Y) and incubated at 37°C for 35min. Similarly, different concentrations of Dam MTase were added to the reaction mixture (10% human serum, 1μM DP1, 1μM DP2-2, 320μM SAM, 6U DpnI, 1μL 10×SEBuffer Y) and incubated at 37°C for 35min. The above method was used to determine and according to the fitting formula ( Figure 3 (B and D, and related illustrations) quantification of M.SssI and Dam MTases according to the formula R (%) = C m / C 0 ×100% to calculate the recovery rate (R), where C m are the concentrations of M.SssI and Dam MTases in the presence of human serum, C 0 are the concentrations of M.SssI and Dam MTases in the absence of human serum, respectively.

[0059] Principle of multiple methyltransferase assay. The present invention demonstrates the construction of a dual-color luminescent RNA nanosensor driven by methylation, using CpG methyltransferase (M.SssI) and adenine methyltransferase (Dam MTase) as target models to perform label-free and ultrasensitive detection of multiple DNA methyltransferases. In higher eukaryotes, M.SssI MTase can methylate the C-5 position of cytosine in the palindromic sequence 5'-CG-3', while Dam MTase can methylate the N-6 position of adenine in the prokaryotic palindromic sequence 5'-GATC-3'. The principle of the methylation-driven dual-color luminescent RNA nanosensor is as follows Figure 1 As shown. In this strategy, the present invention designs two dumbbell probes (DP1 and DP2), each of which has two loops and a stem. The stem in DP1 contains dinucleotide sequences 5'-ACGT-3' / 3'-TGCA-5' and 5'-GATC-3' / 3'-CTAG-5', which serve as catalytic substrates for DNA methyltransferases (i.e., M.SssI and Dam), and become recognition sequences for DNA endonucleases (i.e., GlaI and DpnI) after methylation. The loop sequence of DP1 ( Figure 1 Red and cyan) are respectively related to the loop sequence of DP2 ( Figure 1 The loop in DP2 is complementary to the loop in DP1 that is cut by MDREs, triggering a bidirectional strand displacement amplification (SDA) reaction and producing two different transcription templates. The stem of DP2 consists of a double-stranded region and a single-stranded region. The double-stranded region contains a partial recognition sequence for the cutting enzyme, and the single-stranded region contains the other partial recognition sequence for the cutting enzyme, the T7 promoter sequence, and the DNA sequences of mango and spinach aptamers. After the cleavage product is complementary to DP2, it can mediate bidirectional SDAs, thereby inducing dual transcription amplification and producing two luminescent RNA aptamers. In addition, an NH 2-terminus is modified at the 3' end of DP1, DP2-1, and DP2-2. 2and three unpaired bases to prevent nonspecific aggregation. The nanosensor includes three consecutive steps: (1) cleavage of the dumbbell substrate caused by methylation catalyzed by DNA MTases; (2) two luminescent RNA aptamers induced by dual-loop cascade amplification directed by methylation cleavage; (3) simultaneous detection of the fluorescence signals of dual-color TO1-biotin and DFHBI. In the presence of M.SssI MTase, the methyl group of SAM is transferred to the 5-cytosine of the palindromic sequence 5'-ACGT-3' of DP1, forming the catalytic substrate 5'-A-mC-GT-3' of GlaI. Subsequently, the methylated DP1 is cleaved at the 5-mC site to produce a 25nt cleavage product 1 (CP1). Similarly, in the presence of Dam MTase, DP1 is methylated to form the catalytic substrate 5'-G-mA-TC-3' of DpnI, and the methylated DP1 is cleaved at the 6-mA site to generate a 23nt cleavage product 2 (CP2). After adding DP2 to the reaction system, the generated CP1 and CP2 hybridize with the 3' end sequence of the loop in DP2, and then act as primers to initiate a bidirectional SDA reaction in the presence of Bst DNA polymerase, Nt.BspQI, and dNTPs to generate triggers 1 and 2. Importantly, triggers 1 and 2 can hybridize with the 5' end sequence of the stem of DP2, and then act as transcription templates to activate dual transcription amplification in the presence of T7 RNA polymerase and NTPs, generating two RNA transcripts (i.e., mango and spinach aptamers) ( Figure 1 , yellow and pink). After adding TO1-biotin and DFHBI fluorophores, they specifically bind to the mango and spinach aptamers, respectively, and produce significantly enhanced dual-color fluorescence signals. In contrast, in the absence of M.SssI and DamMTases, DP1 cannot be methylated and cleaved by endonucleases, and neither SDA nor transcription amplification can occur, so no luminescent RNA aptamers are produced. Therefore, no fluorescence signals of TO1-biotin and DFHBI are detected. Compared with the reported experiments on DNA MTase, this nanosensor has obvious advantages: (1) MDREs (i.e., GlaI and DpnI) specifically cleave 5-mC and 6-mA, ensuring accurate quantification of low-abundance DNA MTases; (2) The introduction of double-loop cascade amplification (i.e., SDA and transcription amplification) significantly improves the amplification efficiency; (3) The luminescent RNA aptamer-fluorophore pair can generate a dual-color fluorescent signal, achieving label-free and simultaneous detection of multiple DNA MTases at a high signal-to-noise ratio; (4) This experiment can be completed evenly within 2 h, does not involve any methylation-independent cleavage, complex operations, and fluorescent labeling, and is detected in a label-free manner, which is easy to operate and reduces costs.

[0060] (1) Feasibility experiment:

[0061] The detection of multiple DNA MTases relies on the methylation of dumbbell substrates and subsequent double-loop cascade amplification to produce two luminescent RNA aptamers. In order to study the feasibility of the nanosensor, the present invention performed non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis and fluorescence detection. When GlaI+DP1 was present, only a 90nt band was observed ( Figure 2 A, lane 3), which is consistent with the size of synthetic DP1 (90 nt) ( Figure 2 A, lane 1), indicating that DP1 was neither methylated nor cleaved. When M.SssI+GlaI+DP1 was present, two different bands of 65nt and 25nt were detected ( Figure 2 A, lane 5), the size is consistent with the cleavage product ( Figure 2 A, lane 4), longer (65nt) and shorter (25nt), indicating that M.SssIMTase can methylate the 5-cytosine in the palindromic sequence 5'-ACGT-3', inducing GlaI to cut DP1 to produce CP1 (25nt). With the addition of DP2-1+DNA polymerase+Nt.BspQI, a characteristic band of 72nt can be observed ( Figure 2 A, lane 7), which is the length of the SDA product ( Figure 2 A, lane 6), which means that the cleavage of 5-mC initiated SDA to produce trigger 1 (72nt). T7 RNA polymerase was further added and a characteristic band of 40nt was observed ( Figure 2 A, lane 9), which is the length of the transcript ( Figure 2 A, lane 8), indicating that transcriptional amplification was activated, producing transcript -1 (40 nt) (i.e., Mango aptamer). Similarly, when Dam+DpnI+DP1 was present, two distinct bands of 67 nt and 23 nt were observed ( Figure 2 B, lane 5), indicating that Dam MTase methylates the 6-adenine in the palindromic sequence 5'-GATC-3', inducing DpnI to cleave DP1 to produce CP2 (23 nt) ( Figure 2 B, lane 4), while when DpnI+DP1 was present, only the synthetic DP1 band (90 nt) was detected ( Figure 2 B, lane 3), which means that due to the absence of Dam MTase, DP1 was neither methylated nor cleaved. After adding DP2-2+DNA polymerase+Nt.BspQI+T7 RNA polymerase, characteristic bands of 87nt and 55nt were observed ( Figure 2 B, lanes 7 and 9), which correspond to the sizes of the SDA products ( Figure 2 B, lane 6) and the size of the transcript ( Figure 2 B, lane 8). This means that the cleavage of 6-mA initiated SDA and transcription amplification, generating trigger 2 (87nt) and transcript-2 (55nt) (i.e., spinach aptamer), respectively. In order to further verify the feasibility of the nanosensor, the present invention added TO1-biotin and DFHBI fluorescent molecules to the reaction solution, and performed fluorescence detection under different conditions ( Figure 2 In the absence of DNAMTases, no fluorescence signal of TO1-biotin was detected ( Figure 2 No fluorescence signal of DFHBI was detected (C, black curve) Figure 2 In the presence of only M.SssI or Dam MTase, there is an obvious fluorescence signal of TO1-biotin at the characteristic emission peak of 532 nm in response to M.SssI MTase ( Figure 2 C, red curve), or in response to Dam MTase, there is an obvious DFHBI fluorescence signal at the characteristic emission peak of 505 nm ( Figure 2 These results ( Figure 2 ) showed that the nanosensor was able to detect the activities of multiple DNAMTases simultaneously in a label-free manner with good specificity and high sensitivity.

[0062] (2) Sensitivity test

[0063] Under optimal experimental conditions, the sensitivity of the proposed sensor was studied by measuring the fluorescence signal in response to different concentrations of DNA MTases. When the concentration of M.SssI MTase increased from 0.005 to 100 U / mL, the fluorescence signal of TO1-biotin increased in a concentration-dependent manner ( Figure 3 (A). When the concentration of M.SssI MTase increased from 0.005 to 100U / mL, the fluorescence intensity increased significantly. In the range of 0.005 to 100U / mL, there was a linear relationship between the fluorescence intensity (F) and the logarithm of the M.SssI concentration (C), and the regression equation was F = 446.38log 10 C+1216.35, the correlation coefficient is 0.9972( Figure 3 (B) The detection limit was calculated to be 3.77 × 10 by evaluating the negative control signal without M.SssI MTase plus 3 times the standard deviation. -3The sensitivity is 87.5 times higher than that of the electrochemical analysis method based on photoelectrochemical (PEC) immunosensor (0.33U / mL), and is comparable to the fluorescence method based on hybridization chain reaction (IR-HCR) (0.024U / mL) and the nanosensor based on AuNP (0.002U / mL). Figure 3 As shown in Figure C, when the concentration of Dam MTase increased from 0.0001 to 100 U / mL, the fluorescence signal of DFHBI increased in a concentration-dependent manner. In the range of 0.0001 to 100 U / mL, there was a linear relationship between the fluorescence intensity (F) and the logarithm of the Dam MTase concentration (C). The regression equation was F = 269.14log 10 C+1219.84, the correlation coefficient is 0.9995( Figure 3 The detection limit was calculated to be 7.21×10 -5 U / mL. Compared with the AuNP-based colorimetric method (1.0 U / mL), it is improved by 4 orders of magnitude. Compared with the AuNP- and Ru(III)-mediated dual amplification electrochemical method (0.3 U / mL), it is improved by 3 orders of magnitude. Compared with the enzyme-assisted amplification fluorescence method (0.06 U / mL), it is improved by 2 orders of magnitude. It is 27.7 times that of the QD-based FRET method (0.002 U / mL). The improved sensitivity of the nanosensor can be attributed to: (1) the high specificity of MDREs (i.e., GlaI and DpnI) for methylated DNA (i.e., 5-mC and 6-mA); (2) efficient dual-loop cascade amplification (i.e., bidirectional SDAs and dual transcription amplification); and (3) the inherent high sensitivity of the luminescent RNA nucleic acid aptamer-fluorophore pair.

[0064] (3) Specificity detection

[0065] DNA MTase is an enzyme superfamily composed of a large number of members, and distinguishing one DNA MTase from other members has always been a challenge. The present invention uses MspI MTase, M.CviPI MTase and bovine serum albumin (BSA) as negative controls to investigate the specificity of the method. MspI and M.CviPI MTase are both members of the DNA MTase superfamily, and they can methylate the C-5 position of cytosine in 5'-CCGG-3' and 5'-GC-3' palindromic sequences, respectively. BSA is not a member of the DNA MTase superfamily. Figure 4 As shown, when MspI MTase, M.CviPI MTase and BSA were present, neither the fluorescence signal of TO1-biotin nor the fluorescence signal of DFHBI was observed, which was the same as the fluorescence value obtained by the control with only reaction buffer ( Figure 4). In contrast, when M.SssI MTase was present, an obvious fluorescence signal of TO1-biotin was detected, but no fluorescence signal of DFHBI was detected. When Dam MTase was present, an obvious fluorescence signal of DFHBI was detected, but no fluorescence signal of TO1-biotin was detected. In addition, when M.SssI and Dam MTases were present at the same time, enhanced fluorescence signals of TO1-biotin and DFHBI could be detected simultaneously ( Figure 4 The good selectivity can be attributed to: (1) GlaI and DpnI have good resolution for 5-mC and 6-mA; (2) dumbbell probe end modification can effectively inhibit nonspecific aggregation amplification; (3) high signal-to-noise ratio of mango-TO1-biotin and spinach-DFHBI.

[0066] (4) Inhibitor analysis

[0067] DNA MTase is considered to be a biomarker and drug target for predicting cancer, and the screening of inhibitors is the key to early diagnosis and clinical treatment. In order to study the feasibility of the proposed nanosensor for inhibitor experiments, the present invention uses decitabine and gentamicin as inhibitors. Decitabine is a 2'-deoxycytidine analog (i.e., 5-aza-2'-deoxycytidine), which can be phosphorylated to form aza-dCTP. Aza-dCTP inhibits DNA methylation by replacing cytosine in CpG sequences by forming a covalent bond with DNA MTase. Gentamycin is a broad-spectrum aminoglycoside antibiotic that is widely used as a methyltransferase inhibitor to control infections caused by Gram-negative bacteria. When the concentration of decitabine increased from 0.2μM to 8μM, the relative activity of M.SssI MTase decreased in a concentration-dependent manner ( Figure 5 (A) The IC was calculated based on the fitted calibration curve. 50 The inhibitor concentration required to reduce enzyme activity by 50% was 0.65 μM ( Figure 5 The value obtained by the fluorescence method based on SDA and DNAzyme amplification (0.50 μM) and the fluorescence method based on the combination of graphene oxide and restriction endonuclease site-specific cleavage (0.63 μM) are consistent. Similarly, when the concentration of gentamicin increased from 6 μM to 50 μM, the relative activity of DamMTase decreased accordingly ( Figure 5 In B), IC was calculated based on the fitted calibration curve. 50 The value is 12.41μM ( Figure 5The results clearly show that this strategy can be used to screen inhibitors of multiple DNA MTases simultaneously and has great potential in the development of anticancer drugs.

[0068] (5) Actual sample analysis

[0069] Accurate quantification of multiple DNA MTases in real samples is of great significance for clinical diagnosis and tumor treatment. In order to study the potential of this strategy in real sample analysis, the present invention measured the activity of Dam MTase in GW5100 (Dam positive) and JM110 (Dam negative) Escherichia coli cells. A high DFHBI fluorescence signal ( Figure 6 In contrast, only a low DFHBI fluorescence signal was detected in JM110 E. coli ( Figure 6 A, green column) (t test, P < 0.001), which is consistent with the value obtained by the control with only lysate ( Figure 6 These results indicate that Dam MTase is highly expressed in GW5100 E. coli cells. In addition, the fluorescence signal of DFHBI increases with the increase of the total protein concentration extracted from GW5100 E. coli cells ( Figure 6 In B). -5 to 1.5×10 -2 In the range of mg / mL, there is a good linear relationship between the logarithm of the fluorescence intensity (F) and the total protein concentration (C) ( Figure 6 B, and related illustrations). The relevant equation is F = 282.16log 10 The correlation coefficient of C+1513.99 is 0.9959, and the detection limit is calculated to be 1.36×10 -5 The sensitivity of this method is higher than that of the fluorescence method based on target circulation (4.5×10 -5 In addition, the present invention measured the concentration of 1.5×10 -5 mg / mL) in response to M.SssI MTase Figure 6 In E. coli cell extracts, the fluorescence signal of TO1-biotin increased with the increase of M.SssI MTase concentration, and there was a linear relationship between the fluorescence intensity (F) and the logarithm of M.SssI MTase concentration (C) in the range of 0.005 to 100 U / mL ( Figure 6(C, inset). The regression equation is F = 428.77 log 10 C+1219.51, the correlation coefficient is 0.9997, and the detection limit is calculated to be 2.95×10 -3 These results indicate that the nanosensor can accurately detect the activities of multiple DNA MTases in complex environments.

[0070] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Shandong Normal University <120> Methylation-driven construction of dual-color luminescent RNA nanosensors for label-free and ultrasensitive detection of multiple DNA methylation molecules GTPase <130> 2022 <160> 9 <170> PatentIn version 3.3 <210> 1 <211> 92 <212> DNA <213> Artificial sequence <220> <221> misc_feature <222> (91)..(91) <223> n is a, c, g, t or u <400> 1 ggcttacacg tgttctcgac tccatgctga acacgtgtaa gccttttgtc tggttgatca 60 ataactcggcgatcaattgatcaaccagacnh 92 <210> 2 <211> 103 <212> DNA <213> Artificial sequence <400> 2 cagtagatgc tctggtaata cgactcacta taggggtacg aaggaaggtt tggtatgggg 60 tagttgtcgt actgaagagc tccatgtgtt cagcatggag att 103 <210> 3 <211> 119 <212> DNA <213> Artificial sequence <400> 3 ccagagcatc tactgtaata cgactcacta tagggcggga gaaggacggg tccagcgttc 60 gcgctgttga gtagagtgtg agctccctga agagctcggc gtcaattgat cgccgatat 119 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <400> 4 gtgttctcga ctccatgctg aacacch 27 <210> 5 <211> 25 <212> DNA <213> Artificial sequence <400> 5 tcaataactc ggcgatcaat tgach 25 <210> 6 <211> 72 <212> DNA <213> Artificial sequence <400> 6 gtacgacaac taccccatac caaaccttcc ttcgtacccc tatagtgagt cgtattacca 60 gagcatctac tg 72 <210> 7 <211> 87 <212> DNA <213> Artificial sequence <400> 7 gggagctcac actctactca acagcgcgaa cgctggaccc gtccttctcc cgccctatag 60 tgagtcgtat tacagtagat gctctgg 87 <210> 8 <211> 40 <212> DNA <213> Artificial sequence <400> 8 gggguacgaa ggaagguuug guauggggua guugucguac 40 <210> 9 <211> 55 <212> DNA <213> Artificial sequence <400> 9 gggcgggaga aggacggguc cagcguucgc gcuguugagu agagugugag cuccc 55

Claims

1. A method for constructing a methylation-driven dual-color luminescent RNA nanosensor for detecting multiple DNA methyltransferases, characterized in that: include: In the presence of CpG methyltransferase M.SssI and adenine methyltransferase Dam MTase, dumbbell probe DP1 was methylated at cytosine C-5 and adenine N-6, respectively, forming the catalytic substrates of GlaI and DpnI; Cleavage under the action of GlaI and DpnI produces two cleavage products, CP1 and CP2; The cleavage products CP1 and CP2 hybridize with the 3' end sequence of the loop in the dumbbell probe DP2, respectively, and then serve as primers to initiate a bidirectional SDA reaction in the presence of BstDNA polymerase, Nt.BspQI and dNTPs to produce triggers 1 and 2; The triggers 1 and 2 hybridize with the 5' end sequence of the DP2 stem and then serve as transcription templates to activate dual transcription amplification in the presence of T7 RNA polymerase and NTPs to produce mango and spinach aptamers; The mango and spinach aptamers are combined with TO1-biotin and DFHBI to generate enhanced dual-color fluorescence signals, that is, to simultaneously detect the activities of M.SssI and Dam MTases; The nucleotide sequence of the DP1 is as follows: GGC TTA CA CG TG TTC TCG ACT CCA TGC TGA ACA CG T GTA AGC CTT TTG TCT GGTT TC A ATA ACT CGG CGA TCA ATT GAT C AA CCA GAC-NH2, in which the 3' end of DP1 is modified with an NH2; The nucleotide sequence of DP2 is shown in SEQ ID NO.2 and SEQ ID NO.3; The method is not intended for the diagnosis and treatment of diseases.

2. The method for constructing a dual-color luminescent RNA nanosensor driven by methylation as claimed in claim 1 for detecting multiple DNA methyltransferases, characterized in that: The two loop sequences of DP1 are complementary to the partial sequences of the loop of DP2 respectively.

3. The method for constructing a dual-color luminescent RNA nanosensor driven by methylation as claimed in claim 1 for detecting multiple DNA methyltransferases, characterized in that: The catalytic substrate of GlaI is 5'-A-mC-GT-3'.

4. The method for constructing a dual-color luminescent RNA nanosensor driven by methylation as claimed in claim 1 for detecting multiple DNA methyltransferases, characterized in that: The catalytic substrate of DpnI is 5'-G-mA-TC-3'.

5. The method for constructing a dual-color luminescent RNA nanosensor driven by methylation as claimed in claim 1 for detecting multiple DNA methyltransferases, characterized in that: If the sample lacks M.SssI and Dam MTases, no fluorescence signals of TO1-biotin and DFHBI can be detected. Otherwise, M.SssI and Dam MTases exist.

6. A methylation-driven dual-color luminescent RNA nanosensor, characterized in that: include: Two dumbbell probes: DP1 and DP2; The nucleotide sequence of the DP1 is as follows: GGC TTA CA CG TG TTC TCG ACT CCA TGC TGA ACA CG T GTA AGC CTT TTG TCT GGTT TC A ATA ACT CGG CGA TCA ATT GAT C AA CCA GAC-NH2, in which the 3' end of DP1 is modified with an NH2; The nucleotide sequence of DP2 is shown in SEQ ID NO.2 and SEQ ID NO.3; TO1-biotin and DFHBI; Bst DNA polymerase, Nt.BspQI, and dNTPs; T7 RNA polymerase and NTPs; GlaI and DpnI.

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