CN113088558B - Fluorescent biosensor for detecting DNA (deoxyribonucleic acid) methyltransferase as well as preparation and application thereof - Google Patents

Abstract

The invention discloses a fluorescent biosensor for detecting DNA (deoxyribonucleic acid) methyltransferase as well as preparation and application thereof, wherein the fluorescent biosensor comprises a symmetrical double-ring dumbbell and a 3D tetrahedral fluorescent bracket, wherein the symmetrical double-ring dumbbell contains DNA methylation sites, can form methylation double chains under the catalysis of the DNA methyltransferase, and then forms two single-chain dumbbell rings with consistent structures through enzyme digestion reaction; the 3D tetrahedron fluorescent scaffold has the same hairpin at each top, and is modified with fluorescent group and quenching group, the fluorescent group and the quenching group are close to each other to cause fluorescence quenching, and the sheared dumbbell ring can combine with the hairpin to generate fluorescence, and DNA methyltransferase catalytic reactions with different concentrations can generate fluorescence signals with different intensities. The DNA methylation transferase detection method established based on the sensor has good stability and specificity and high sensitivity, can be used for screening MTase inhibitors, and has important significance for early detection and diagnosis of cancers, drug research and the like in clinic.

Description

Fluorescent biosensor for detecting DNA (deoxyribonucleic acid) methyltransferase as well as preparation and application thereof

Technical Field

The invention relates to the technical field of fluorescent biosensors and biomarker detection, in particular to a fluorescent biosensor for detecting DNA (deoxyribonucleic acid) methyltransferase as well as preparation and application thereof.

Background

DNA methylation refers to the epigenetic modification of the 5' carbon atom of CpG dinucleotide cytosine in a genomic DNA nucleic acid sequence by a DNA methyltransferase with S-adenosylmethionine (SAM) as a methyl donor to covalently bond a methyl group. DNA methylation plays an important role in the genetic control aspects of body X chromosome inactivation, genome imprinting, gene expression change and the like. DNA methylation is a common form of covalent modification of bases in eukaryotes and is also the major epigenetic form of mammals, and plays a critical role in important genetic controls such as X-chromosome inactivation, genomic imprinting and gene expression, while DNA methyltransferases can establish and maintain the stability of the methylation state of genomic DNA sequences. DNA methyltransferase is an important biomarker for clinical detection, and related researches show that various genetic diseases, cancers and the like are closely related to the occurrence and the development of the genetic diseases.

The traditional detection method of the methyltransferase mainly comprises HPLC, methylation specific PCR, a radiolabeling method and the like, and the defects of complex experimental operation, high experimental hazard, expensive experimental equipment, complex sample preparation process, high false positive and the like are generally involved in the detection, so that more rapid and simple detection methods, such as a colorimetric method, an electrochemical detection method, an electrochemiluminescence detection method, a chemiluminescent detection method and the like, are focused on, the detection effect of the DNA methyltransferase is improved, but the detection accuracy is also influenced by the detection instability, the interference of surrounding environmental factors and the like.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a fluorescent biosensor for detecting DNA methyltransferase, and preparation and application thereof, which are used for solving the problems of poor stability and specificity of the detection method of DNA methyltransferase in the prior art.

To achieve the above and other related objects, a first aspect of the present invention provides a fluorescent biosensor for detecting DNA methyltransferase, comprising symmetrical double-ring dumbbell (SDRDs) and 3D tetrahedral fluorescent scaffold (DTFS) structures, wherein the symmetrical double-ring dumbbell is a symmetrical dumbbell structure containing 5'-GATC-3' DNA methylation sites, which sites can be specifically recognized by Dam methyltransferase, and the symmetrical double-ring dumbbell forms a methylated double chain under the catalysis of the DNA methyltransferase Dam methyltransferase, and then forms a single-chain dumbbell ring with completely identical two half structures through enzyme digestion reaction; the 3D tetrahedron fluorescent scaffold is synthesized by 4 long chains, the sequences of the 4 long chains are shown as SEQ ID NO.2-5, each top end of the tetrahedron contains the same hairpin structure, meanwhile, a fluorescent group and a fluorescence quenching group are modified, the fluorescent group and the fluorescence quenching group are close to each other to cause fluorescence quenching, the sheared two half single-chain dumbbell rings can be combined with the hairpin at each top end of the tetrahedron to generate fluorescence, and the Dam methyltransferase catalytic reaction with different concentrations can generate fluorescence signals with different intensities.

Further, the sequence of the symmetrical double-ring dumbbell is shown as SEQ ID NO. 1.

Further, the DNA methyltransferase is Dam methyltransferase.

Further, the cleavage reaction uses a DpnI endonuclease as a cleavage enzyme.

Further, the fluorescent group is FAM and the fluorescence quenching group is BHQ1.

In a second aspect, the present invention provides a method for preparing a fluorescent biosensor for detecting DNA methyltransferase, comprising the steps of:

(1) Symmetrical Double Ring Dumbbells (SDRDs) were prepared: adopting a dumbbell single chain with a sequence shown as SEQ ID NO.1, wherein a phosphate group P is modified at the 5 'end of the dumbbell single chain, an OH is connected to the 3' end of the dumbbell single chain, under the action of T4DNA ligase, the 5 'phosphate group P and the 3' end OH are connected to form a closed loop, a 5'-GATC-3' DNA methylation site is arranged at a connection port, and the site is a recognition site of DNA methylation transferase;

(2) 3D tetrahedral fluorescent scaffold (DTFS): 4 long chains with the sequence shown as SEQ ID NO.2-5 are adopted to synthesize a 3D tetrahedron fluorescent scaffold (DTFS), and fluorescent groups and fluorescence quenching groups are modified at four corners of the tetrahedron.

Further, in the step (1), the DNA methyltransferase is Dam methyltransferase.

Further, in the step (1), the preparation method of the Symmetrical Double Ring Dumbbell (SDRDs) comprises the following steps: dissolving dumbbell single-chain dry powder with TE buffer solution, adding 10×T4DNA ligase reaction buffer solution, reacting, adding T4DNA ligase, and reacting to form dumbbell ring.

Alternatively, in the step (1), after adding 10×T4DNA ligase reaction buffer, heating is performed for 5 minutes at 95℃and then reaction is performed for 30 minutes at 37 ℃.

Alternatively, in the step (1), after adding the T4DNA ligase, the reaction is performed for 1 hour at 16℃and then for 10 minutes at 70℃to form a dumbbell ring.

Optionally, in the step (1), after the dumbbell ring is formed, exonuclease I and exonuclease III are added to react for 1 hour at 37 ℃, and then the exonuclease is completely inactivated after reacting for 30 minutes at 90 ℃, so that only the dumbbell ring exists in the system, and the existence of non-looped single and double chains is avoided.

Further, in the step (2), the synthesis method of the 3D tetrahedral fluorescent scaffold (DTFS) includes the following steps: dissolving 4 long-chain dry powders with TE buffer solution respectively, heating at 95deg.C, and standing at room temperature to form hairpin completely; then, the solution of each long chain is fully and evenly mixed with TM buffer solution, reacted at 95 ℃, then quickly cooled to 4 ℃, incubated, finally taken out and placed on ice for standby.

Optionally, in the step (2), the heating and/or reaction time at 95 ℃ is 5 minutes, and the incubation time is 30 minutes.

Further, in the step (2), a light-shielding operation is required in synthesizing the 3D tetrahedral fluorescent scaffold.

Further, in the step (2), the fluorescent group is FAM and the fluorescence quenching group is BHQ1.

Further, the detection range of the fluorescent biosensor is 0.002U/mL-100U/mL, and the lowest detection limit is 0.00036U/mL.

In a third aspect, the present invention provides a fluorescent biosensor according to the first aspect and/or a fluorescent biosensor prepared by a method according to the second aspect for detecting DNA methyltransferase.

According to a fourth aspect of the present invention, there is provided a method for detecting DNA methyltransferase using the fluorescent biosensor of the first aspect and/or the fluorescent biosensor prepared by the method of the second aspect, the method comprising the steps of: taking symmetrical double-ring dumbbell, adding DNA methylation transferase and S-adenosylmethionine (SAM) to carry out methylation reaction, and then carrying out DNA methylation specific enzyme digestion reaction; then adding a fluorescence detection system prepared from a 3D tetrahedral fluorescent scaffold (DTFS) into a product obtained by the enzyme digestion reaction, reacting in a dark place, and then detecting a fluorescence signal.

Further, the DNA methyltransferase is Dam methyltransferase.

Further, in the fluorescence detection system, the concentration of the 3D tetrahedral fluorescent scaffold (DTFS) is 0.02 to 0.2. Mu.M, preferably 0.06 to 0.2. Mu.M, more preferably 0.1. Mu.M.

Further, the DNA methyltransferase concentration is 0.002 to 100U/mL, preferably 0.008 to 4U/mL, more preferably 0.002 to 0.01U/mL.

Alternatively, when the DNA methyltransferase concentration is in the range of 0.008-4U/mL, the relationship equation of the fluorescence intensity (y) and the DNA methyltransferase concentration (x) is y=33.98x+266.55, and the correlation coefficient is 0.9931; when the DNA methyltransferase concentration was in the range of 0.002-0.01U/mL, the equation of the relationship between the fluorescence intensity (y) and the DNA methyltransferase concentration (x) was y=90.79x+388.26, and the correlation coefficient was 0.9891.

Further, the methylation reaction was performed in 10 Xdam methyltransferase buffer.

Further, the methylation reaction time is 30 to 120 minutes, preferably 45 to 90 minutes, more preferably 60 minutes.

Further, the shearing enzyme used in the DNA methylation specific cleavage reaction is DpnI endonuclease; alternatively, the concentration of the DpnI endonuclease is 4-12U, preferably 6-10U, more preferably 8U.

Further, the cleavage reaction time is 30 to 120 minutes, preferably 45 to 90 minutes, more preferably 60 minutes.

Further, the photophobic reaction time (i.e., the time for the dumbbell to bind to the tetrahedra) is 15 to 120 minutes, preferably 30 to 90 minutes, more preferably 60 minutes.

Further, the reaction temperature of the methylation reaction, the cleavage reaction and the light-shielding reaction is 37 ℃.

In a fifth aspect, the present invention provides a fluorescent biosensor according to the first aspect and/or a fluorescent biosensor prepared by a method according to the second aspect, for use in screening an MTase inhibitor.

Further, the MTase inhibitor is 5-fluorouracil.

As described above, the fluorescent biosensor for detecting DNA methyltransferase of the present invention, and its preparation and application, have the following beneficial effects:

the invention designs a fluorescent biosensor based on a symmetrical double-ring dumbbell auxiliary 3D tetrahedron space structure, which is used for carrying out ultrasensitive detection on DNA methyltransferase (Dam methyltransferase), and carrying out experiment to synthesize a symmetrical dumbbell structure containing 5'-GATC-3' DNA methylation sites, wherein the sites are specifically identified by the Dam methyltransferase, a methylated double chain is formed after catalysis, and then Dpn I enzyme digestion reaction is carried out to form two half dumbbell rings with completely consistent structures, a fluorescent carrier 3D tetrahedron fluorescent bracket is quickly synthesized through 4 long chains, each top end of the tetrahedron contains the same hairpin and simultaneously modifies a fluorescent group and a quenching group, fluorescence is quenched, the dumbbell is combined with each top end hairpin of the tetrahedron to generate fluorescence after being sheared, and the two single chains can be combined with 4 top end fluorescent hairpins of the tetrahedron after being sheared, so that the identification efficiency of the 3D tetrahedron fluorescent hairpin is improved, and the scheme realizes the detection range from 0.002U/mL to 100U/mL and the lowest detection concentration reaches 0.00036U/mL. Meanwhile, the specificity experiment of the M.SssI methyltransferase and the Hpa II methylation restriction enzyme, the serum labeling experiment and the 5-fluorouracil inhibition experiment also provide a vital reference value for the early sensitive detection of the Dam methyltransferase and the early diagnosis and treatment of cancers.

Drawings

FIG. 1 shows a schematic diagram of ultrasensitive detection of DNA methyltransferase based on fluorescent amplification effect of a symmetrical double-ring dumbbell-assisted 3D tetrahedral space structure support in the present invention.

FIG. 2 shows a synthetic representation of 3D tetrahedra in the present invention, wherein (A) 8% native PAGE images and (B) 3D tetrahedra of AFM are structured.

FIG. 3 is a graph showing the results of verifying the feasibility of the biosensor construction in the present invention.

FIG. 4 shows a graph of the results of optimization of the important parameters in the experiments of the present invention.

FIG. 5 is a graph showing The results of concentration analysis of The SDRDs-assisted DTFS detection Dam methyltransferase in The present invention.

FIG. 6 is a graph showing the results of the specificity analysis in the present invention.

FIG. 7 is a graph showing the results of the inhibition of 100U/mL Dam MTase activity by the change in the concentration of 5-fluorouracil inhibitor according to the present invention.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Fluorescent biosensor for detecting DNA (deoxyribonucleic acid) methyltransferase and preparation and application of fluorescent biosensor

1.1 reagents

Dam methyltransferase, 10 Xdam methyltransferase buffer, SAM, dpn I methylation restriction enzyme, hpa II methylation restriction enzyme, 10 XCutSmart buffer, T4DNA ligase, 10 XT 4DNA ligase reaction buffer and exonuclease I (E.coli), exonuclease III (E.coli), cpG methyltransferase (M.SssI), 1 XNEBuffer 2 were all purchased from NEB (Beijing) Inc. 5-fluorouracil is purchased from Soy Biolabs. TE buffer, tris (hydroxymethyl) aminomethane (Tris) powder, magnesium chloride hexahydrate (MgCl2.6H2O) reagent, ammonium Persulfate (APS), acrylamide/methylene bisacrylamide 30% solution (29:1), N, N, N ', N' -tetramethyl ethylenediamine (TEMED), 5 XTBE buffer were purchased from Bio-company. 1000DNA Marker,20bp DNA Ladder and 6 Xloading buffers were purchased from Takara Bio Inc. The third generation Gelred nucleic acid dye solution was purchased from beijing polymeric biotechnology limited. The fluorescence modified nucleotide sequence is synthesized in Shenzhen large gene technology Co. The common oligonucleotide sequences were synthesized by Bio Inc. The experimental ultra-pure water (18.2 M.OMEGA.cm) was obtained from the Millipore water purification system (Millipore Co.).

The relevant nucleotide sequences used in this experiment are shown in table 1.

TABLE 1 nucleotide sequence listing

1.2 instruments and apparatus

Fluorescence detection was performed on a Cary Eclipse fluorescence spectrophotometer (Agilent Co.) with excitation wavelength of 492nm, emission wavelength range of 505nm-650nm, excitation bandwidth and emission bandwidth of 5nm. The optical path of the quartz fluorescent cuvette is 1.0cm, and the detection amount is 100 mu L. All temperature control and heating experiments were performed in a T100 thermal cycler (Bio-Rad Co.). DNA tetrahedral hairpins were formed in HH-ZK420 intelligent thermostated water tank (Ware instruments Co.). DYY-6C electrophoresis tanks were purchased from six instrument factories, beijing, and electrophoresis imaging was performed in a ChemDoc XRS gel imaging system (Bio-Rad). The oligonucleotide sequence dry powder was dissolved in a Thermo Heraeus Fresco high-speed centrifuge (Semer Feishmania technology Co.) and centrifuged at a temperature of 4 ℃. Atomic force microscopy imaging was performed in a Dimension Icon instrument (bruk, germany).

1.3 3D tetrahedral (DTS) synthesis

The 3D tetrahedron (DTS) synthesis procedure is as follows: firstly, 4-chain dry powder of DTS is placed on ice after being centrifuged for 10 minutes at 12000g in a high-speed centrifuge with pre-cooled 4 ℃, a certain amount of TE buffer is accurately added for repeated shaking and rotation for 3 times to fully dissolve the DTS to 100 mu M, and after heating in a water bath box with 95 ℃ for 5 minutes, the dry powder is left overnight, the hairpin is completely formed at the room temperature, and then the hairpin is reserved at-20 ℃. Then 0.5. Mu.L and 48. Mu.L of TM buffer (20 mM Tris and 50mM MgCl2.6H2O, pH=8.0) were mixed well for each strand, and the mixture was put into a T100 thermocycler for reaction at 95℃for 5 minutes, then rapidly cooled to 4℃for incubation for 30 minutes, and finally taken out and put on ice for standby. The 3D fluorescent tetrahedron is synthesized while being protected from light.

Successful synthesis of DTS structures was characterized in 8% native PAGE and Atomic Force Microscopy (AFM).

1.4 Electrophoretic characterization of 3D tetrahedral (DTS) synthesis

The single strands of the dissolved dry powder DTS were diluted to 1. Mu.M, and 3 strands were synthesized with TM buffer to synthesize 0.1. Mu.M of DTS123, DTS124, DTS134, DTS234, and 4 strands of DTS1234, respectively, to confirm successful synthesis of DTS. Meanwhile, single-stranded dilution is 0.1 mu M, 10 mu L of each single-stranded dilution of 0.1 mu M is taken, and synthesized 0.1 mu M DTS123, DTS124, DTS134, DTS234 and DTS1234 10 mu L are added with 2 mu L of 6 x loading buffer, mixed evenly, added into an electrophoresis tank, set at voltage of 100V, electrophoresed for 35 minutes, taken out, and fully stained with 2 mu L of Gelred nucleic acid stain in 50mL of 1 XTBE buffer for 50 minutes to form an image.

1.5 fluorescent biosensor construction

And (3) preparing a reaction dumbbell product, wherein a phosphate group P is modified at the 5 'end of the dumbbell single chain, and the dumbbell single chain can be connected with a 3' end OH to form a ring when the structure is formed. Firstly, dissolving dry powder into 100 mu M by using TE buffer solution, preserving at-20 ℃, preparing 40 mu L of reaction solution by using 2 mu L of dumbbell and 4 mu L of 10 xT 4DNA ligase reaction buffer solution during experiments, reacting at 95 ℃ for 5 minutes, then reacting at 37 ℃ for 30 minutes, then adding 100U of T4DNA ligase, reacting at 16 ℃ for 1 hour, reacting at 70 ℃ for 10 minutes to form a dumbbell ring, adding 1 mu L of exonuclease I and 1 mu L of exonuclease III for 1 hour, and reacting at 90 ℃ for 30 minutes to completely inactivate the exonuclease, wherein only dumbbell rings exist in the system.

DNA methylation reaction, preparing 100 mu L of total reaction system by taking 10 mu L of formed dumbbell ring reaction product, wherein the reaction concentration of Dam methyltransferase in the system is 100U/mL, the concentration of SAM is 160 mu M, the reaction is completed in 10 x Dam methyltransferase buffer solution, the reaction is carried out for 1 hour at 37 ℃ after fully mixing, then DNA methylation specific enzyme digestion reaction is carried out, 50 mu L of reaction product is prepared into 100 mu L of reaction system, 10 mu L of 10 x CutSmart buffer solution and 8U of Dpn I endonuclease are added, and the reaction is carried out for 1 hour at 37 ℃ after fully mixing. The preparation of DNA fluorescent tetrahedron should be kept at minus 20 ℃ in dark, 100 mu L of fluorescent detection system is prepared, 80 mu L of the product after Dpn I enzyme digestion is added into 10 mu L of 1 mu M DNA fluorescent tetrahedron, and after being uniformly mixed in dark, the reaction is completed at 37 ℃ for 1 hour, and the detection is carried out as soon as possible.

2 results and discussion

2.1 construction principle of fluorescent biosensor

FIG. 1 shows a schematic diagram of ultrasensitive detection of DNA methyltransferase based on fluorescent amplification effect of a symmetrical double-ring dumbbell assisted 3D tetrahedral spatial structure support.

As shown in FIG. 1, the fluorescent signal amplification system of the symmetrical double-ring dumbbell (SDRDs) auxiliary 3D tetrahedral fluorescent support (DTFS) structure constructed in the experiment is composed of two parts. Firstly, preparing a symmetrical dumbbell ring, wherein the dumbbell ring structure designed in the experiment is a 40-base oligonucleotide single chain with a 5' -end modified P, the single chain is divided into two parts of 20 bases, after the dumbbell ring structure is formed, the dumbbell ring structure can be symmetrically formed, under the action of T4DNA ligase, the 5' -end P and the 3' -end OH are connected to form a closed ring, and the connection port is 5' -GATC-3', which is a recognition site of Dam methyltransferase, and the subsequent methylation reaction cannot be carried out without catalytic connection of the T4DNA ligase. When dumbbell rings are formed, exonuclease I and exonuclease III are added to eliminate the influence of other interfering chains, and the dumbbell rings are completely inactivated at 90 ℃ without influencing the formation of the rings. DNA methyltransferase is added, under the catalysis of SAM, methyl is combined to double-chain 5'-GATC-3' site, and Dpn I endonuclease is added to recognize and cut the DNA methyltransferase, so that a chain with two identical parts and 20 bases can be formed.

Following the synthesis of DTFS, the tetrahedral scaffold is synthesized from 4 strands, each strand being 91 bases in total and divided into two parts, the tetrahedral part being 55 bases, each 17 bases being capable of base complementary pairing with the other strands to form a tetrahedral structure. The end parts of the tetrahedron are of the same hairpin structure, the four corners of the 3D tetrahedron are modified with FAM fluorescent groups and BHQ1 fluorescent quenching groups, the FAM fluorescent groups and the BHQ1 fluorescent quenching groups are close to each other to generate no fluorescence when the fluorescent tetrahedron is formed, and when a sheared dumbbell single chain appears, the fluorescent signal can be generated by quickly hybridizing with the tetrahedron hairpin to open the hairpin, and the fluorescent signal with different intensities can be generated by the Dam methyltransferase catalytic reaction with different concentrations.

2.2 Synthetic characterization of 3D tetrahedra (DTS)

Characterization of DTS structures in electrophoresis and Atomic Force Microscopy (AFM).

FIG. 2 shows a synthetic characterization of 3D tetrahedra, wherein (A) 8% native PAGE images and (B) 3D tetrahedra of AFM are structured.

As shown in FIG. 2A, the molecular weight difference of different target products in 8% native PAGE electrophoresis fully demonstrates the successful synthesis of DTS, lanes M are 1000bp DNA markers from left to right, lanes 1-4 are DTS 4 single strands DTFS-1, DTFS-2, DTFS-3 and DTFS-4 in sequence, lane 5 is a trihedron successfully formed by the DTFS-123,3 strands, the molecular weight is smaller than that of DTFS-1234 of lane 9, the DTS is fully successfully synthesized, and the single strand and the compound concentration are all 0.1 mu M. The arrow of the ultra-clean structure in the AFM of fig. 2B marks a tetrahedral three-dimensional structure.

2.3 fluorescent biosensor feasibility analysis

DNA methyltransferases were detected based on a fluorescence strategy of symmetric dumbbell (SDRDs) assisted 3D tetrahedral fluorescence scaffolds (DTFS) and verified by different fluorescence spectral changes.

FIG. 3 shows a graph of results for verifying the feasibility of fluorescent biosensor construction. (A) SDRDs-assisted DTFS detects changes in fluorescence intensity of Dam methyltransferase: the fluorescence intensity of the simple synthesis of DTFS (a), the fluorescence intensity of the reaction of SDRDs and DTFS under the catalysis of the Dam methyltransferase and the recognition and cleavage of the Dpn I endonuclease (b), the fluorescence intensity of the reaction of SDRDs and DTFS under the catalysis of the Dam methyltransferase and the cleavage of the Dpn I endonuclease (c), the fluorescence intensity of the reaction of SDRDs and DTFS under the catalysis of the Dam methyltransferase and the cleavage of the Dpn I endonuclease (d), and the fluorescence intensity of the binding of SDRDs and DTFS after the catalysis of the Dam methyltransferase and the cleavage of the Dpn I endonuclease (e). (B) 12% PAGE electrophoresis of SDRDs: lane M,20bp DNA Ladder. Lane 1, SDRDs without exonuclease formed. Lane 2, SDRDs with added exonuclease to eliminate interfering chain effects. Lane 3, SDRDs are completely catalytic sheared after addition of Dam methyltransferase and Dpn I endonuclease. (C) AFM mapping of successful synthesis of dumbbell ring.

As shown in FIG. 3A, curve a shows that the fluorescent hairpin is not opened in the presence of DTFS only, no obvious fluorescence is generated, curve b shows that after the dumbbell ring is formed, no catalysis of Dam methyltransferase and recognition shearing of Dpn I endonuclease are directly combined with DTFS, no strong fluorescence signal is generated at the moment, curve c shows that the dumbbell ring is sheared under the condition that no catalysis of Dam methyltransferase is performed, but Dpn I enzyme is added for shearing, then the dumbbell ring is directly reacted with synthesized DTFS, the fluorescent hairpin cannot be opened by the complete dumbbell ring, and a trace of single-stranded dumbbell does not form a ring to react with the hairpin, so that weak fluorescence signal is generated but high signal to noise ratio F/F0 is not influenced, and the experiment has better feasibility and stability due to higher signal to noise ratio. And after the formation of the curve D dumbbell ring, single chains reacting with the 3D tetrahedral fluorescent support cannot be formed through the catalysis of the Dam methyltransferase without adding the Dpn I endonuclease reaction, so that no fluorescent signal is generated, and finally, as in the curve e, when the Dam methyltransferase catalysis and the Dpn I endonuclease are reacted, the sheared dumbbell can be combined with the 3D tetrahedral fluorescent support, so that extremely high fluorescent signals are generated, and the good feasibility of the symmetrical double-ring dumbbell auxiliary 3D fluorescent tetrahedral system is verified.

The characterization of FIGS. 3B and 3C is a 12% native PAGE and AFM image of the dumbbell ring, respectively, showing successful synthesis of symmetrical dumbbell rings under enzyme catalysis. FIG. 3B shows that lane M is a 20bp DNA Ladder, lane 1 is a dumbbell ring with a single strand of dumbbell formed by the action of T4DNA ligase, lane 2 is a dumbbell ring after the addition of exonuclease to eliminate the effect of a trace of non-specific strand, while demonstrating that no band will be generated by the addition of exonuclease when the dumbbell ring is not formed successfully, and finally lane 3 is a dumbbell ring that is completely recognized and sheared after the addition of Dam methyltransferase and Dpn I endonuclease. FIG. 3C is an AFM imaging of dumbbell, with the chainless fine ring structure demonstrating successful dumbbell synthesis.

2.4 optimization of experimental conditions

The important conditions in the experiment are optimized. Fig. 4 shows the results of optimization of important parameters in the experiment. (A) concentration optimization of 3D tetrahedral fluorescent scaffold. (B) And the reaction time of the 3D tetrahedral fluorescent scaffold and the enzyme digestion dumbbell ring is optimized. (C) Dpn I endonuclease concentration optimization. (D) Dpn I endonuclease cleavage time optimization. (E) optimization of Dam methyltransferase catalytic reaction time.

The stable spatial structure of the 3D tetrahedral fluorescence scaffold (DTFS) plays an important role in capturing the sheared dumbbell single strand, and ensures that the detection efficiency is guaranteed to ensure that the nonspecific fluorescence signal is the lowest, and the optimal signal to noise ratio F/F0 is achieved, as shown in fig. 4A, the tetrahedral concentration is optimized first, the fluorescence signal is gradually enhanced with increasing concentration, the fluorescence intensity is the highest when the concentration reaches 0.1 μm, and the fluorescence signal is consistent with the result that the double concentration dumbbell single strand fully binds with 0.1 μm tetrahedron after shearing the 0.2 μm dumbbell ring, so that when the concentration of the fluorescence tetrahedron scaffold gradually increases, the detection F/F0 decreases, the higher the fluorescence tetrahedron concentration is, the higher the background interference signal is, the 0.1 μm DTFS is selected as the optimal experimental detection concentration, and F0 is the blank fluorescence signal without Dam methyltransferase.

The hybridization time of the single-stranded dumbbell and the fluorescent tetrahedron after the Dpn I endonuclease is sheared is also important to perform high-efficiency detection under the condition of minimum background interference, as shown in FIG. 4B, when the single-stranded dumbbell and the tetrahedron are combined in a homogeneous solution, a fluorescent signal is generated by rapid reaction, the reaction is performed within 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes and 120 minutes after the time is optimally set, the reaction can occur within 15 minutes, and when the reaction time reaches 60 minutes, the strongest fluorescence is generated, namely the highest value of F/F0, and the fluorescent signal has no obvious change after the reaction time is continuously increased, so that the reaction is completed. Thus 60 minutes was chosen as the optimal time for dumbbell to tetrahedron bonding.

Dpn I endonuclease plays an important role in the shearing efficiency of symmetrical dumbbell, the ultrasensitive detection of Dam methyltransferase is affected by the full shearing of dumbbell ring, and the shearing efficiency of strand is also affected by the excessive concentration of enzyme, as shown in FIG. 4C, the concentration of Dpn I endonuclease is continuously increased to make the fluorescence intensity higher and higher, when the strand concentration reaches 8U, the fluorescence signal is stabilized to the highest value, and the concentration fluorescence is increased to be unchanged or reduced, so that 8U is the optimal shearing concentration of Dpn I endonuclease for experiments.

The Dpn I cleavage time was also optimized when the enzyme concentration was fixed, as shown in FIG. 4D, as the cleavage time increased, the binding rate of the dumbbell cleavage single strand to the fluorescent tetrahedron increased, generating a progressively higher fluorescent signal, and when the Dpn I cleavage time reached only 60 minutes, fmax, continued to increase the cleavage time, with no significant change in fluorescent intensity, thus selecting 60 minutes as the optimal reaction time for Dam methyltransferase detection.

Finally, the full reaction of the Dam methyltransferase plays a vital role in concentration detection, as shown in fig. 4E, when the reaction time reaches 60 minutes, the fluorescence intensity reaches the highest value, the time is continuously increased, the fluorescence intensity has no obvious change, and 60 minutes is the optimal time for the Dam methyltransferase to catalyze the reaction, and long-time reaction is not needed to be performed to avoid high background signal interference.

The above experimental parameters are important conditions in the experiment when enzyme target detection is performed. Under the selected optimized condition, the target detection takes only 3 hours, so that the advantages of high-efficiency and rapid detection of the scheme are fully reflected.

2.5 sensitivity analysis of fluorescent biosensors

After optimization of relevant important conditions, linear analysis of Dam methyltransferase concentration was performed. FIG. 5 shows The results of concentration analysis of The SDRDs-assisted DTFS for The detection of Dam methyltransferase. (A) fluorescence spectra measured at different concentrations: 0. 0.002, 0.004, 0.006, 0.008, 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 25, 100U/mL. (B) Fluorescence scatter plots for different concentrations of Dam methyltransferase. (C) results of enzyme detection linear analysis: 0.008, 0.01, 0.05, 0.1, 0.5, 1, 2, 4U/mL. (D) results of enzyme detection linear analysis: 0.002, 0.004, 0.006, 0.008, 0.01U/mL. Error bars are standard deviations of three parallel experiments.

As shown in FIG. 5A, each fluorescence spectrum corresponding to the different concentrations shows that the Dam methyltransferase was diluted, and when the concentration was 0U/mL,0.002U/mL,0.004U/mL,0.006U/mL,0.008U/mL,0.01U/mL,0.05U/mL,0.1U/mL,0.5U/mL,1U/mL,2U/mL,4U/mL,6U/mL,8U/mL,10U/mL,25U/mL,100U/mL, respectively, the detection was performed, and the result showed that the fluorescence intensity was gradually increased as the enzyme concentration was increased.

FIG. 5B is a graph of fluorescence intensity value F versus target concentration, wherein the fluorescence intensity exhibits a forward trend change at a concentration of 0.002-100U/mL, wherein the correlation coefficient 0.9931 and the linear function equation y=33.98x+266.55 are achieved over the range of 0.008-4U/mL, while following the 3 sigma rule: the standard deviation of the 3 times blank value plus the blank signal value gives a minimum detection limit of 0.00036U/mL, while the correlation coefficient 0.9891 and the linear function equation y=90.79x+388.26 are realized in the range of 0.002-0.01U/mL.

The experimental scheme is established to realize high-sensitivity detection as a primary target, in the scheme, a dumbbell ring is formed by extremely simple and stable DNA oligonucleotides, the dumbbell ring is not easy to degrade in detection, the dumbbell ring is used as a catalytic template of Dam methyltransferase, methylation sites can be sensitively identified, single chains can be formed to be stably combined with tetrahedrons of a 3D structure in detection liquid after shearing, the tetrahedron support has a good space stable structure, so that background interference signals are lower, a certain distance is reserved between the terminals, 4 identical terminal hairpins exist stably, hybridization reaction can only occur when the dumbbell ring is linked to open the hairpins, and the high-signal-to-noise detection in the scheme has high efficiency on enzyme detection, and the high-sensitivity detection can reach the linear detection of a correlation coefficient 0.9931 and the detection limit of 0.00036.

Meanwhile, the detection sensitivity of the scheme is compared with that of the existing research-related methods (papers [1] - [7 ]), and the results are shown in the table 2, so that the advantages established by the method are proved again.

Table 2 comparison of high sensitivity fluorescence detection protocol methodology for target Dam methyltransferase

Note that:

[1]Chen S,Ma H,Li W,et al.An entropy-driven signal amplifying strategy for real-time monitoring of DNA methylation process and high-throughput screening of methyltransferase inhibitors.[J].Analytica Chimica Acta,2017,970:57-63.

[2]Du Y C,Wang S Y,Li X Y,et al.Terminal deoxynucleotidyl transferase-activated nicking enzyme amplification reaction for specific and sensitive detection of DNA methyltransferase and polynucleotide kinase[J].Biosensors&Bioelectronics,2019,145:111700.

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2.6 specificity analysis

To evaluate the specificity of the assay, M.SssI methyltransferase was added as a control assay, the methyltransferase recognition site in this assay was 5'-GATC-'3, and the M.SssI enzyme recognition site was 5'-CG-3', and subsequent reactions were performed similarly with Dpn I endonuclease after catalytic reaction with methyltransferase addition, and FIG. 6 shows the results of the specificity assay: sssI MTase and Dam MTase specific methylation enzyme catalysis conditions compared, hpa II and Dpn I endonuclease recognition of Dam endonuclease enzymatic methylation site cleavage conditions.

As shown in FIG. 6, when 100U/mL of M.SssI methyltransferase was added and compared with 100U/mL of Mldam methyltransferase, fluorescence intensity F after cleavage of the recognition of Dpn I endonuclease was compared, and only Dam methyltransferase was present, the reaction was smooth.

Meanwhile, the specific digestion reaction of the Dpn I endonuclease is verified, the digestion reaction can be successfully completed only after Dam methyltransferase acts on the corresponding site, 20U Hpa II methylation restriction endonuclease is added for reaction, hpa II recognition site is 5'-CCGG-3' unmethylated site, fluorescence cannot be generated after the reaction with a fluorescent tetrahedron bracket, and finally the experiment is performed in 8UDpn I endonuclease, and the effect generates larger fluorescence intensity, which again indicates that the scheme is established to have good selection specificity.

2.7 serological experiments

To verify the performance of this protocol in human healthy serum for the detection of target enzymes, we diluted the enzymes to 0.05U/mL,0.5U/mL,1U/mL respectively for 3 sets of parallel assays, the results are shown in Table 3, and calculated recovery rates for enzyme assays of 105.9%, 94.6%, 105.26%, respectively, while the relative standard deviations RSD were stable at 7.24%, 0.7%, 1.57%. The results show that the serum detection results have good reproducibility and stability and have great clinical reference value.

TABLE 3 detection effect of Dam methyltransferase in serum

2.8 5-fluorouracil (5-fluorouracil) inhibition assay

Since the mechanism of action of Dam methyltransferase causes various diseases, cancer tumors and the like in human body, effective control of Dam methyltransferase can inhibit abnormal occurrence of DNA methylation, and 5-fluorouracil as an anticancer drug plays an important role in the study of antibiotics, which can play a role in inhibiting Dam methyltransferase, inhibition of Dam methyltransferase is observed by controlling 5-fluorouracil at different concentrations in this experiment, and FIG. 7 shows inhibition of 100U/mL of Dam MTase activity by 5-fluorouracil inhibitor concentration variation.

As shown in fig. 7, when 5-fluorouracil exists, the enzyme inhibitor can play a certain role in inhibiting Dam methyltransferase, and the enzyme inhibitor meeting the corresponding dosage concentration and the relative activity RA (%) show a certain proportion change, and the calculation formula of RA is as follows: RA= (Fi-F0)/(Ft-F0), wherein Fi, ft and F0 respectively represent fluorescence intensities in the presence of 100U/mL of Dam MTase and 5-fluorouracil inhibitors in different concentrations, ft is fluorescence intensity in the presence of 100U/mL of Dam MTase but no 5-fluorouracil, F0 is concentration in the absence of Dam MTase and no 5-fluorouracil is added, and Ft and a reaction blank value F0 are averaged in parallel for 3 groups of experiments to perform subsequent calculation. When the concentration gradually increases to 6. Mu.M, the fluorescence signal does not change any more near blank, so the concentration is the optimal concentration for enzyme inhibition.

Meanwhile, the result shows that the IC50 value of the inhibitor on enzyme (50% of Dam MTase activity inhibition caused by 5-fluorouracil concentration) is 0.847 mu M, and the proposed scheme can be used for screening the inhibition capacity of the MTase inhibitor, and the important clinical reference significance of the scheme on important clinical treatment diagnosis and the like is proved again.

Conclusion 3

According to the research scheme, the Dam methyltransferase is detected by using the symmetrical double-ring dumbbell-assisted 3D tetrahedron fluorescent support, enough concentration dumbbell rings are prepared in experiments, the smooth progress of methylation reaction is ensured, the structure is stable, two symmetrical single chains after enzyme digestion reaction double-amplify tetrahedron fluorescent signals, fluorescent detection background signals are low, interference factors are few, and the tail ends of stable tetrahedron structures 4 are combined with target chains, so that the recognition efficiency of the target chains is greatly improved; the scheme has the advantages of simple experimental operation, short detection time and specific detection, extremely high sensitivity, reaches the detection limit of 0.00036U/mL, simultaneously detects the methylation condition of the corresponding site by the combined specific experiment, utilizes the inhibition effect of 5-fluorouracil on Dam methyltransferase, has important reference significance for developing anti-bacterial drugs and anticancer drugs, and reflects the advantages of the detection system in multiple aspects such as good reproducibility in serum specimen detection, and is beneficial to the early detection diagnosis of clinical cancers, the development of clinical application such as drug research and the like.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

SEQUENCE LISTING

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<120> a fluorescent biosensor for detecting DNA methyltransferase, preparation and application thereof

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Claims (11)

1. A fluorescent biosensor for detecting DNA methylation transferase is characterized by comprising a symmetrical double-ring dumbbell, a 3D tetrahedral fluorescent bracket and Dpn I endonuclease, wherein the symmetrical double-ring dumbbell has a symmetrical dumbbell structure containing 5'-GATC-3' DNA methylation sites, the sequence of the symmetrical double-ring dumbbell is shown as SEQ ID NO.1, the sites can be specifically identified by the DNA methylation transferase, the symmetrical double-ring dumbbell forms a methylation double chain under the catalysis of the DNA methylation transferase, and then a single-chain dumbbell ring with completely consistent two half structures is formed through enzyme digestion reaction; the 3D tetrahedron fluorescent scaffold is synthesized by 4 long chains, the sequences of the 4 long chains are shown as SEQ ID NO.2-5, each top end of the tetrahedron contains the same hairpin structure, meanwhile, a fluorescent group and a fluorescence quenching group are modified, the fluorescent group and the fluorescence quenching group are close to each other to cause fluorescence quenching, the sheared two half single-chain dumbbell rings can be combined with the hairpin at each top end of the tetrahedron to generate fluorescence, and DNA methyltransferase catalytic reactions with different concentrations can generate fluorescent signals with different intensities.

2. The fluorescent biosensor of claim 1, wherein:

the DNA methyltransferase is Dam methyltransferase;

and/or the fluorescent group is FAM, and the fluorescence quenching group is BHQ1.

3. A method of preparing a fluorescent biosensor for detecting DNA methyltransferase, the fluorescent biosensor comprising symmetrical double-loop dumbbell and 3D tetrahedral fluorescent scaffold and dpni endonuclease, the method comprising the steps of:

(1) Preparation of symmetrical double ring dumbbell: adopting a dumbbell single chain with a sequence shown as SEQ ID NO.1, wherein a phosphate group P is modified at the 5 'end of the dumbbell single chain, an OH is connected to the 3' end of the dumbbell single chain, under the action of T4DNA ligase, the 5 'phosphate group P and the 3' end OH are connected to form a closed loop, a 5'-GATC-3' DNA methylation site is arranged at a connection port, and the site is a recognition site of DNA methyltransferase;

(2) 3D tetrahedral fluorescent scaffold: 4 long chains with the sequence shown as SEQ ID NO.2-5 are adopted to synthesize the 3D tetrahedron fluorescent scaffold, and fluorescent groups and fluorescence quenching groups are modified at the four corners of the tetrahedron.

4. A method of preparation according to claim 3, characterized in that: in the step (1), the DNA methyltransferase is Dam methyltransferase; in the step (1), the preparation method of the symmetrical double-ring dumbbell comprises the following steps:

dissolving dumbbell single-chain dry powder with TE buffer solution, adding 10×T4DNA ligase reaction buffer solution, reacting, adding T4DNA ligase, and reacting to form dumbbell ring.

5. A method of preparation according to claim 3, characterized in that: in the step (2), the synthesis method of the 3D tetrahedral fluorescent scaffold comprises the following steps: dissolving 4 long chain dry powders with TE buffer solution respectively, heating at 95deg.C, and standing at room temperature to form hairpin completely; then fully and uniformly mixing the solution of each long chain with TM buffer solution, reacting at 95 ℃, then rapidly cooling to 4 ℃, and incubating;

and/or, in the step (2), light-shielding operation is needed when the 3D tetrahedral fluorescent scaffold is synthesized;

and/or, in the step (2), the fluorescent group is FAM, and the fluorescence quenching group is BHQ1.

6. The method of manufacturing according to claim 4, wherein: in the step (1), after adding 10×T4DNA ligase reaction buffer, heating for 5 minutes at 95 ℃ and then reacting for 30 minutes at 37 ℃; and/or, in the step (1), after adding the T4DNA ligase, reacting for 1 hour at 16 ℃ and then reacting for 10 minutes at 70 ℃ to form a dumbbell ring; and/or, in the step (1), after the dumbbell ring is formed, adding the exonuclease I and the exonuclease III to react for 1 hour at 37 ℃, and then, reacting for 30 minutes at 90 ℃ to completely inactivate the exonuclease.

7. The method of manufacturing according to claim 5, wherein: in the step (2), the heating and/or reaction time at 95 ℃ is 5 minutes, and the incubation time is 30 minutes.

8. Use of a fluorescent biosensor according to any one of claims 1-2 and/or a fluorescent biosensor prepared according to the method of any one of claims 3-7 for detecting DNA methyltransferases, said DNA methyltransferases being Dam methyltransferases, said use being for non-disease diagnosis or treatment purposes.

9. A method for detecting DNA methyltransferase for non-disease diagnosis or treatment purposes, characterized by: a fluorescent biosensor prepared by the method of any one of claims 1-2 and/or 3-7, the detection method comprising the steps of: taking symmetrical double-ring dumbbell, adding DNA methylation transferase and S-adenosylmethionine to carry out methylation reaction, and then carrying out DNA methylation specific enzyme digestion reaction by using DpnI endonuclease; then adding a fluorescence detection system prepared from a 3D tetrahedral fluorescent bracket into a product obtained by the enzyme digestion reaction, carrying out light-shielding reaction, and then detecting a fluorescence signal; the DNA methyltransferase is Dam methyltransferase.

10. The method of claim 9, wherein: in the fluorescence detection system, the concentration of the 3D tetrahedral fluorescent scaffold is 0.02-0.2 mu M;

and/or, the concentration of the DNA methylation transferase is 0.002-100U/mL;

and/or, the methylation reaction is performed in 10 x Dam methyltransferase buffer;

and/or the enzyme digestion reaction time is 30-120 minutes;

and/or, the photophobic reaction time is 15-120 minutes;

and/or the reaction temperature of the methylation reaction, the enzyme digestion reaction and the light-shielding reaction is 37 ℃.

11. Use of a fluorescent biosensor according to any one of claims 1-2 and/or a fluorescent biosensor prepared according to the method of any one of claims 3-7 for screening for inhibitors of MTase, which is Dam methyltransferase, for non-disease diagnosis or treatment purposes.