CN114561442A

CN114561442A - Helicase activity determination method based on double-stranded DNA and application thereof

Info

Publication number: CN114561442A
Application number: CN202210278487.4A
Authority: CN
Inventors: 黄志纾; 王佳恩; 陈硕斌; 谭嘉恒; 吴璧含; 韦力源; 刘培庆
Original assignee: Sun Yat Sen University; Guangzhou Zhongda Nansha Technology Innovation Industrial Park Co Ltd
Current assignee: Sun Yat Sen University; Guangzhou Zhongda Nansha Technology Innovation Industrial Park Co Ltd
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-05-31

Abstract

The invention discloses a helicase activity determination method based on double-stranded DNA and application thereof. The tail end of at least one DNA single strand in the nucleic acid secondary structure is connected with a tail sequence, and the tail ends of any two different DNA single strands in the nucleic acid secondary structure are respectively connected with two non-interfering specifically combined substances or groups. Compared with other helicase detection methods, the detection method disclosed by the invention has a more accurate helicase activity quantification effect, does not need complicated steps such as electrophoresis and membrane rotation, shortens the detection time by 3-4 hours, allows ATP consumption to be detected in the same solution system, and enables the assessment of the helicase activity to be more comprehensive.

Description

Helicase activity determination method based on double-stranded DNA and application thereof

Technical Field

The invention belongs to the field of enzyme activity detection methods, and particularly relates to a helicase activity determination method based on double-stranded DNA and application thereof.

Background

At the molecular level, helicases are motor proteins that move directionally along the nucleic acid phosphodiester backbone and are capable of utilizing energy from Adenosine Triphosphate (ATP) hydrolysis to unfold folded nucleic acid strands. In the related art, helicase activity has been shown to affect most nucleic acid metabolic processes within the cell, such as DNA replication and repair, gene transcription and translation, chromosome segregation and telomere maintenance, and the like. Deletions or mutations in helicases may also contribute to various diseases and cancer susceptibility.

There are a number of important secondary nucleic acid structures and corresponding helicases in organisms and the search for these different helices requires specific research tools. Among the related arts, the most common assay methods for measuring in vitro helicase activity are the gel electrophoresis migration assay (EMSA) method, the fluorescence-based helicase activity assay method, and the single molecule technique. These methods may be advantageous, such as gel electrophoresis, which has the ability to reflect the relative abundance of various DNA substrates during unwinding; fluorescence-based assays enable real-time dynamic detection of unfolding events; single molecule technology (such as magnetic tweezers, optical tweezers, single molecule FRET (smFRET) and the like) can dynamically detect the unwinding process of single nucleic acid molecules. However, these methods also have a number of limitations and drawbacks. EMSA can only achieve semi-quantitative detection, accurate quantification cannot be further realized, and the detection method is relatively complex, low in flux and incapable of realizing rapid detection of large-batch samples. The fluorescence method is limited by the use of fluorescence labels, the fluorophores can only be labeled at specific positions on two DNA chains, the distance between the two fluorophores must be accurately designed, the necessary technical requirements greatly limit the design of a developed DNA substrate model, the universality is poor, and the measurement based on the fluorescence method is easily interfered by proteins in a sample, the proteins can interfere with the fluorophores and even directly cause fluorescence quenching, so that the problem of inaccurate detection exists. However, the unimolecular technology has the disadvantages of high use requirement, high requirement on operators, high technical difficulty, high detection cost and low flux, and is difficult to meet the use requirements of most common laboratories or simple detection, so that the development of a new detection method for conveniently and accurately quantitatively detecting the helicase activity is urgently needed.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a helicase activity determination method based on double-stranded DNA and application thereof. The method can detect the helicase activity and the ATP hydrolytic activity on different DNA substrates, can realize high-sensitivity quantitative analysis, and has the advantages of high detection speed, low cost, simpler operation and high practical value.

In a first aspect of the invention, a detection reagent is provided.

According to the first aspect of the present invention, in some embodiments of the present invention, the detection reagent comprises a nucleic acid secondary structure obtained by hybridization of at least two DNA single strands, wherein the tail sequence is connected to the end of at least one DNA single strand in the nucleic acid secondary structure, and two non-interfering specifically binding substances or groups are respectively connected to the ends of any two different DNA single strands in the nucleic acid secondary structure.

In some embodiments of the invention, the specific binding substance or group comprises at least one of an antibody, an antigen, biotin, avidin, complementary paired bases, a magnetic bead, a transcription activation domain, a DNA binding domain, an amino acid, a tRNA.

In some preferred embodiments of the invention, the specific binding substances or groups are biotin and digoxin.

Biotin is used to immobilize the nucleic acid secondary structure to the avidin coated reaction vessel. In some preferred embodiments of the invention, the avidin is streptavidin.

Digoxin is used as an intermediate linking substance to specifically bind digoxin antibody, thereby linking the separable B chain with a recognition marker for subsequent qualitative or quantitative determination. In some preferred embodiments of the invention, the recognition marker is HRP, which is conjugated to a digoxin antibody, which is linked to the B chain based on the specific binding of the digoxin antibody to digoxin on the B chain. And the secondary structure of nucleic acid which is not helicized can be quantified by using an HRP substrate (such as TMB) for color reaction, so that the helicase hydrolysis amount can be calculated.

Other alternatives for the detection of digoxin and digoxin-HRP conjugated antibodies may also be employed, such as:

(1) replacing digoxin and digoxin antibodies with FITC labels and corresponding FITC antibodies, or ROX labels and corresponding ROX antibodies and the like, and then monitoring the antigen antibodies by incubating HRP secondary antibodies;

(2) other fluorescent labels may also be used instead of digoxigenin. For example, a fluorescent label such as FAM, TAMRA, Cy3 can be used, and the level of unwinding can be detected and quantified by detecting the intensity of fluorescence in the well after the unwinding reaction is completed.

(3) Detection may also be performed using a recognition means such as magnetic bead coupling.

In some preferred embodiments of the invention, the nucleotide sequence of the tail sequence is: 5'-TTTTTTTTTT-3' are provided.

In the present invention, the tail sequence mainly serves to facilitate the recognition and binding of a DNA sample by partial helicases (e.g., BLM, WRN, Pif1, etc.) which initiate a helicase reaction by binding to the tail sequence, and gradually shift forward along the tail sequence to unwind the DNA strand.

In some embodiments of the invention, the DNA single strands are 12-60 bp, and the complementary pairing length between the DNA single strands is greater than or equal to 12 bp.

In the present invention, the shorter the duplex portion obtained by hybridization of a single DNA strand, the faster the unwinding speed of helicase, and therefore, the detection speed can be effectively controlled based on the designed secondary structure of nucleic acid, and the unwinding speed of helicase can be accurately reflected.

In some embodiments of the invention, the nucleic acid secondary structure comprises a complementary DNA duplex, a replication fork structure, a Holliday Junction structure, a babble structure, a G4 structure, a hairpin structure.

In the invention, the inventors verify through experiments that the detection reagent according to the first aspect of the invention can detect various conventional secondary structures of nucleic acids, and has high detection sensitivity.

Of course, one skilled in the art can select other conventional secondary nucleic acid structures that can be helicized by helicases in the art as a reasonable alternative according to practical use requirements.

In some preferred embodiments of the invention, the nucleotide sequence of each single strand of DNA in the secondary structure of the nucleic acid is:

complementary DNA double strand:

Model A：5’-TTTTTTTTTTCGTCGAGCAGAG-3’(SEQ ID NO:4)；

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

replicating the fork structure:

fork1：

fork1 A：5’-TTTTTTTTTTCGTACCCGATGTGTTCGTTC-’3(SEQ ID NO：1)；

fork1 B：5’-GAACGAACACATCGGGTACGTTTTTTTTTT-’3(SEQ ID NO：2)。

fork2：

fork2 A：5’-TTTTTTTTTTCGTACCCGATGTGTTCGTTC-3’(SEQ ID NO:1)；

fork2 B：5’-GAACGAACACATCGGGTACG-3’(SEQ ID NO:3)。

fork3：

Fork3 A：5’-TTTTTTTTTTCGTCGAGCAGAG-3’(SEQ ID NO:4)；

Fork3 B：5’-CTCTGCTCGACGTTTTTTTTTT-3’(SEQ ID NO:5)。

Fork4：

Fork4 A：5’-TTTTTTTTTTCGTACCCGATGTGTTCGTTCAACTTAGC-3’(SEQ ID NO:6)；

Fork4 A：5’-GCTAAGTTGAACGAACACATCGGGTACGTTTTTTTTTT-3’(SEQ ID NO:7)。

in some more preferred embodiments of the present invention, the replication fork structure is fork3, fork1, and fork 4.

Holliday Junction structure:

HJ-A：5’-TTTTTTTTTTCACCCGTTTCTACAGGATCGTTCGGTCTTAAG-3’(SEQ ID NO:10)；

HJ-B：5’-GATCTTGTCGTACAGGATCGTTACATTAGCAG-3’(SEQ ID NO:9)；

HJ-C：5’-CTTAAGACCGAACGATCCTGTACGACAAGATCTTTTTTTTTT-3’(SEQ ID NO:11)；

HJ-D：5’-CTGCTAATGTAACGATCCTGTAGAAACGGGTGTTTTTTTTTT-3’(SEQ ID NO:12)。

a bubble structure:

Bubble A：5’-CTGCTAATGTAATTTTTTTTTTTTTACGACAAGATCTTTTTTTTTT-3’(S EQ ID NO:8)；

Bubble B：5’-GATCTTGTCGTACAGGATCGTTACATTAGCAG-3’(SEQ ID NO:9)。

structure G4:

GQ1(G3T3) unwinding detection model:

GQ1-A：5’-TTTTTTTTTTCGTCGAGCAGAGATGGGTTTGGGTTTGGGTTTGGGTTTTTTTTTT-3’(SEQ ID NO:14)；

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

GQ2(G3T1) unwinding detection model:

GQ2-A：5’-TTTTTTTTTTCGTCGAGCAGAGATTGGGTTTGGGTGGGTTTGGGTTTTTTTTTTT-3’(SEQ ID NO:15)；

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

GQ3(G4T3) unwinding detection model:

GQ3-A：5’-TTTTTTTTTTCGTCGAGCAGAGATGGGGTTTGGGGTTTGGGGTTTGGGGTTTTTTTTTT-3’(SEQ ID NO:16)；

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

as shown in the attached figure 1 of the specification, the detection principle of the detection reagent is as follows:

since complementary paired parts exist between different single strands in constituting the nucleic acid secondary structure, a stable nucleic acid secondary structure can be obtained after annealing treatment. Here, for example, fork1, one strand (fork 1A) constituting the structure is used for fixing the secondary structure of nucleic acid, and the other strand (fork 1B) is used for quantification. When no helicase is present, since the two chains are not separated, the amount of these nucleic acid secondary structures with multiple complex folds can be detected by the chromogenic reaction of HRP with TMB after the conjugate a (digoxin) on the other chain (fork 1B) is bound to a digoxin antibody conjugated with horseradish peroxidase (HRP). When helicase and ATP are added, helicase reaction occurs, and complex nucleic acid substances can be subjected to helicase enzymatic folding, so that a DNA chain (fork 1B) with non-immobilization effect is liberated in a reaction system and is sufficiently removed after washing and other steps, and at the moment, the helicase activity can be determined according to the change of the detected absorbance values before and after helicase reaction (the quantification is carried out according to a standard curve of the absorbance and substrate concentration gradient measured by a standard solution). Meanwhile, since most of the unwinding reaction depends on ATP, the ATP consumption in the unwinding reaction process may be detected by using an ATP detection kit after recovering the reaction solution obtained by the unwinding reaction.

In a second aspect of the invention, a test kit is provided.

According to the second aspect of the present invention, in some embodiments of the present invention, the detection kit comprises the detection reagent according to the first aspect of the present invention, and a substance targeting two specific binding substances or groups on a single DNA strand in the detection reagent according to the first aspect of the present invention, respectively.

In some embodiments of the invention, the substance targeting two specific binding substances or groups on a single DNA strand in the detection reagent according to the first aspect of the invention comprises at least one of an antibody, an antigen, biotin, avidin, complementary paired bases, a magnetic bead, a transcription activation domain, a DNA binding domain, an amino acid, a tRNA.

In some embodiments of the invention, the substance targeting two specific binding substances or groups on the single strand of DNA in the detection reagent according to the first aspect of the invention is a digoxin antibody and avidin.

In some preferred embodiments of the invention, the digoxin antibody is conjugated with HRP.

In some preferred embodiments of the invention, the avidin is streptavidin.

In a third aspect of the invention, there is provided a method for qualitatively and/or quantitatively detecting helicase activity, comprising the steps of:

(1) taking the DNA single strand of each nucleic acid secondary structure in the first aspect of the invention, and heating and annealing to obtain the corresponding nucleic acid secondary structure;

(2) fixing the nucleic acid secondary structure on the surface of a container through a specific binding substance or group contained in the nucleic acid secondary structure, adding a sample to be detected to react with ATP for 25-40 min, washing off all solutions, adding a substance which is coupled with a labeling group and targets the nucleic acid secondary structure specific binding substance or group, and qualitatively and/or quantitatively determining helicase activity by comparing parameter change conditions of a blank control.

According to a third aspect of the invention, in some embodiments of the invention, the parameter is an absorbance value.

In some embodiments of the present invention, the variation is calculated based on the following formula:

wherein A (f) is the absorbance value of the secondary structure of the nucleic acid before helicase is added, A (t) is the absorbance value of the secondary structure of the nucleic acid after helicase is added, A (u) is the absorbance value of the mixture of single strands of DNA before the secondary structure of the nucleic acid is not formed, and Q (ds) is the loading amount of the original DNA strand.

According to the third aspect of the present invention, in some embodiments of the present invention, in each of the DNA single strands in step (1), the molar ratio of the separable strand to the strand for immobilization in constructing the secondary structure of the nucleic acid is 1-2: 1.

In the present invention, the amount of the separable strand used is larger than that of the strand used for immobilization, so that the hybridization ratio of the system reaches 100%, to further improve the accuracy of detection.

In some embodiments of the invention, in each of the single DNA strands in step (1), the molar ratio of the separable strand to the strand used for immobilization in constructing the secondary structure of the nucleic acid is 1.2: 1.

In the present invention, the inventors verified through experiments that under the above-described method for measuring helicase activity based on double-stranded DNA, the absorbance gradually increased with the increase in the degree of structural folding, and a good linear correlation (linear correlation coefficient R) was exhibited²0.96), indicating that the absorbance signal is linearly related to the degree of folding of the replication fork. Therefore, it can be shown that the above-mentioned double-stranded DNA-based helicase activity assay method can be used to determine the unfolding process of nucleic acid structures of different degrees and can quantify the structures of different folding degrees.

In some preferred embodiments of the present invention, taking a replication fork model as an example, the detection method specifically includes:

(1) two single-stranded DNAs with biotin and digoxin were expressed as follows: 1.2, placing in a metal bath at 95 ℃ for 10min, heating and then slowly lowering to room temperature (annealing). And (3) diluting the annealed DNA double strands to the final concentration of 2.5-15 nM, adding the DNA double strands to a 96-well plate coated with streptavidin (the addition amount of the DNA double strands is 60 muL/well), and coating in a 37 ℃ thermostat for 30 min.

Wherein, for the annealing in the step (2), different annealing buffers should be selected as solvents of the DNA substrate according to different DNA substrates, and the annealing buffers are specifically: the final concentration was 30mM NaCl and 10mM Tris-HCl, pH 7.4. In the step, the DNA substrate formed after annealing is diluted to specific final concentration (used for coating a 96-well plate, and the final concentration range is 2.5-15 nM) according to different detection standard curves of the DNA substrate.

(2) ELISA buffer (containing final concentrations of 50mM KCl and 50mM KH) was used₂PO₄pH 7.4) washing of the coated 96-well plates(use amount about 200. mu.L/well), three times, each for 5 min.

(3) The well plates were blocked with ELISA buffer containing 3% (v/v) Bovine Serum Albumin (BSA) for 30min, after which the well plates were washed three times for 5min each with ELISA buffer containing 0.05% (v/v) Tween 20.

(4) To a 96-well plate, 60. mu.L/well of helicase buffer (containing 10mM Tris-HCl, 50mM NaCl, 2mM MgCl) was added₂200 μ M ATP, pH 7.4) and reacted at 37 ℃ for 20 min.

(5) 10 μ L of the reaction mixture was aspirated from each well, transferred to a 384-half well plate, and the remainder of the reaction mixture in the original 96-well plate was discarded. Add 10. mu.L luciferase ATP detection reagent to 384 half-well plate, blow and mix well, then use the microplate reader for chemiluminescence detection.

Wherein, after the luminescence detection, the ATP consumption during the unwinding may be calculated according to the following formula:

wherein Q (h) is the amount of ATP consumed by the protein during helication, Q (ds) is the amount of raw ATP, L (ds) is the luminescence value of the reaction mixture without helicase, L (p) is the luminescence value of the reaction mixture with helicase, and L (b) is the luminescence value of the reagent without ATP.

(6) The 96-well plate from which the reaction mixture was discarded was washed three times (using about 200. mu.L/well) with ELISA buffer containing 0.05% (v/v) Tween 20 for 5min each.

(7) HRP-conjugated digoxin antibody was reacted with an ELISA buffer containing 3% (v/v) BSA at a 1: the cells were diluted at a dilution ratio of 1000, and then incubated in washed 96-well plates (40. mu.L/well), and incubated at 37 ℃ for 1 hour in an incubator. The well plates were washed three times (approximately 200. mu.L/well) with ELISA buffer containing 0.05% (v/v) Tween 20 for 5min each time.

(8) 100. mu.L of a Tetramethylbenzidine (TMB) color developing solution as a substrate of HRP was placed in each well of a 96-well plate and placed in a 37 ℃ incubator for 5min for color development (protected from light). After the color development is complete, 5 is added0 μ L/well of 1M H₂SO₄The reaction was terminated. Absorbance measurements were then performed using a microplate reader at a wavelength of 450 nm.

After reading the absorbance at a wavelength of 450nm, the amount of derotation can be calculated according to the following formula:

wherein A (f) is the absorbance value of the secondary structure of the nucleic acid before helicase is added, A (t) is the absorbance value of the secondary structure of the nucleic acid after helicase is added, A (u) is the absorbance value of the mixture of DNA single strands before the secondary structure of the nucleic acid is not formed, and Q (ds) is the original DNA strand loading amount (i.e., coating amount).

Taking the replication fork model as an example, the linear detection range of the detection method in the invention is 0.8 to 5nM, and the limit of detection (LOD) can reach 0.17 nM. Compared with the EMSA method, the method has similar LOD (0.16nM) obtained by chemiluminescence immunoassay of membrane transfer signal amplification, but the sensitivity of the helicase activity determination method based on the double-stranded DNA is far better than that of the common EMSA (0.17 mu M) or FRET (2.86nM) detection method in terms of sensitivity.

In a fourth aspect of the present invention, there is provided a method for detecting ATP consumption of helicase, comprising the steps of:

(2) fixing a nucleic acid secondary structure on the surface of a container through a specific binding substance or group contained in the nucleic acid secondary structure, and adding a sample to be detected to react with ATP for 15-30 min to obtain a mixed reaction solution;

(3) the consumption of the helicase ATP is calculated by detecting the residual amount of ATP in the mixed reaction solution.

(2) ELISA buffer (containing final concentrations of 50mM KCl and 50mM KH) was used₂PO₄pH 7.4) the coated 96-well plates (approximately 200 μ L/well) were washed three times for 5min each.

(4) mu.L/well of helicase buffer (containing 10mM Tris-HCl, 50mM NaCl, 2mM MgCl) was added to 96-well plates ₂200 μ M ATP, pH 7.4) and reacted at 37 ℃ for 20 min.

(5) 10 μ L of the reaction mixture was aspirated from each well and transferred to a 384-half well plate. Add 10. mu.L luciferase ATP detection reagent to 384 half-well plate, blow and mix well, then use the microplate reader for chemiluminescence detection.

In a fifth aspect of the present invention, there is provided a use of the detection reagent according to the first aspect of the present invention in any one of the following (1) to (4);

(1) qualitatively or quantitatively detecting helicase activity;

(2) qualitatively or quantitatively detecting ATP consumption of helicase;

(3) screening a substance for regulating and controlling the helicase or the helicase activity;

(4) screening for a substance having helicase activity for helicating a specific nucleic acid secondary structure.

The beneficial effects of the invention are:

(1) compared with other helicase detection methods, the detection method disclosed by the invention has a more accurate helicase activity quantification effect, does not need complicated steps such as electrophoresis and membrane rotation, and shortens the detection time by 3-4 hours; furthermore, compared with the FRET method, biotin and digoxigenin or other nucleic acid tags used in the method can be labeled at any position on a DNA single strand without worrying about mutual interference between the labels, so that the design range of the unwinding substrate is wider and the degree of freedom is higher.

(2) The detection reagent and the detection kit have wide application range, allow ATP consumption to be detected simultaneously in the same solution system, and enable the assessment of helicase activity to be more comprehensive.

(3) The detection method of the invention can be used for unwinding tests based on various DNA substrate models, such as secondary structure intermediates or barriers frequently occurring in the processes of cell growth and metabolism of replication forks, HJ, G4 and the like, so that the detection method can be further used for researching and comparing the selectivity of helicases on various secondary structures.

(4) The assay of the invention can also be used to screen helicase inhibitors and the effect of the inhibitors varies significantly when different DNA substrates are used. The method is helpful for developing small molecule inhibitors with stronger selectivity according to different research purposes, and provides a new tool for the research of diseases taking helicase as a target.

Drawings

FIG. 1 is a schematic diagram showing the measurement method of helicase activity in the example of the present invention.

FIG. 2 is a schematic diagram of the secondary structure of a replication fork (fork1) according to an embodiment of the present invention.

FIG. 3 shows the result of the measurement of fork1 by the helicase activity measurement method in the example of the present invention.

FIG. 4 is a standard graph showing the method for measuring helicase activity in the example of the present invention.

FIG. 5 shows the result of detection of double strand by FRET method.

FIG. 6 shows the detection results of the EMSA method on double strands, wherein A and B are the detection results without FAM fluorophore added, and C and D are the detection results after FAM fluorophore was added.

FIG. 7 shows the results of the helicase activity assay based on the presence of replication forks at different hybridization levels in the examples of the invention.

FIG. 8 shows the results of detection of the presence or absence of replication forks for the 3' tail sequence by the helicase activity assay method in the examples of the present invention.

FIG. 9 shows the results of the helicase activity measurement method based on the presence of replication forks of different lengths in the examples of the present invention.

Fig. 10 is a schematic design diagram of a bubble and horizon Junction unwinding detection model in the embodiment of the present invention.

FIG. 11 is a comparative detection result of the helicase activity determination method in the embodiment of the present invention based on the forking 1, bubble and family Junction helicase detection models.

FIG. 12 is a schematic design diagram of a G-quadruplex de-rotation detection model in an embodiment of the invention.

FIG. 13 shows the results of the helicase activity assay based on the different G-quadruplexes in the examples of the present invention.

FIG. 14 shows the results of measurement of ATP consumption in the process of unwinding by the helicase activity measuring method according to the example of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Helicase activity determination method based on double-stranded DNA

The method for measuring helicase activity based on double-stranded DNA in this example comprises the steps of:

(1) design of double-stranded DNA:

in this example, the double-stranded DNA used was a replication fork (replication fork) double-stranded DNA.

The replication fork (fork1) is selected from the group consisting of forked double-stranded DNA (fork1 a and fork 1B) of 30 base pairs in length (including a tail sequence of 10 base pairs), the nucleotide sequences of which are shown below.

fork1 A：5’-TTTTTTTTTTCGTACCCGATGTGTTCGTTC-’3(SEQ ID NO：1)；

fork1 B：5’-GAACGAACACATCGGGTACGTTTTTTTTTT-’3(SEQ ID NO：2)。

Wherein the underlined part is the double-stranded binding part of the replication fork. At the same end (5 ' end or 3' end, in this example, 5' end) of fork1 a and fork 1B, conjugate a and conjugate B are attached, respectively (in this example, conjugate a and conjugate B are digoxin and Biotin (Biotin), respectively).

(2) Two single-stranded DNAs with biotin (fork1 a) and digoxin (fork 1B) were expressed as follows: 1.2 ratio (molar ratio) mixing (digoxigenin labeled fork 1B should be in excess relative to fork 1A to ensure as complete pairing of the two strands as possible, resulting in about 40. mu.L of a DNA mixture at a concentration of about 10. mu.M), heating in a 95 ℃ metal bath for 10min, and then slowly lowering to room temperature (annealing). And (3) diluting the annealed DNA double strands to the final concentration of 2.5-15 nM, adding the DNA double strands to a 96-well plate coated with streptavidin (the addition amount of the DNA double strands is 60 muL/well), and coating in a 37 ℃ thermostat for 30 min.

Wherein, for the annealing in the step (2), different annealing buffers should be selected as the solvents of the DNA substrates according to different DNA substrates; when the annealed DNA substrate is in a double helix structure, the annealing buffer can be selected as: final concentrations were 30mM NaCl and 10mM Tris-HCl, pH 7.4; when the annealed DNA substrate is a G4 structure, the annealing buffer can be selected as: 100mM KCl and 10mM Tris-HCl, pH 7.4. In the step, the DNA substrate formed after annealing is diluted to specific final concentration (used for coating a 96-well plate, and the final concentration range is 2.5-15 nM) according to different detection standard curves of the DNA substrate.

(3) ELISAbuffer (containing final concentrations of 50mM KCl and 50mM KH) was used₂PO₄pH 7.4) the coated 96-well plates (used in about 200 μ L/well) were washed three times for 5min each.

(4) The well plates were blocked with ELISA buffer containing 3% (v/v) Bovine Serum Albumin (BSA) for 30min, after which the well plates were washed three times for 5min each with ELISA buffer containing 0.05% (v/v) Tween 20.

(5) mu.L/well helicase buffer (suitable for BLM or WRN helicase containing 10mM Tris-HCl, 50mM NaCl, 2mM MgCl final concentration) was added to 96-well plates ₂200 μ M ATP, pH 7.4) and reacted at 37 ℃ for 20 min.

(6) 10 μ L of the reaction mixture was aspirated from each well, transferred to a 384-half well plate, and the remainder of the reaction mixture in the original 96-well plate was discarded. Add 10. mu.L luciferase ATP detection reagent to 384 half-well plate, blow and mix well, then use the microplate reader for chemiluminescence detection.

(7) The 96-well plate from which the reaction mixture was discarded was washed three times (using about 200. mu.L/well) with ELISA buffer containing 0.05% (v/v) Tween 20 for 5min each.

(8) HRP-conjugated digoxin antibody was reacted with an ELISA buffer containing 3% (v/v) BSA at a 1: the cells were diluted at a dilution ratio of 1000, and then incubated in washed 96-well plates (40. mu.L/well), and incubated at 37 ℃ for 1 hour in an incubator. The well plates were washed three times (approximately 200. mu.L/well) with ELISA buffer containing 0.05% (v/v) Tween 20 for 5min each time.

(9) 100. mu.L of a Tetramethylbenzidine (TMB) color developing solution as a substrate of HRP was placed in each well of a 96-well plate and placed in a 37 ℃ incubator for 5min for color development (protected from light). After the color development was completed, 50. mu.L/well of 1M H was added₂SO₄The reaction was terminated. Absorbance measurements were then performed using a microplate reader at a wavelength of 450 nm.

wherein A (f) is the absorbance value of the secondary structure of the nucleic acid before helicase is added, A (t) is the absorbance value of the secondary structure of the nucleic acid after helicase is added, A (u) is the absorbance value of the mixture of single strands of DNA before the secondary structure of the nucleic acid is not formed, and Q (ds) is the loading amount (i.e., coating amount) of the original DNA strand.

The detection principle of the helicase activity determination method based on the double-stranded DNA is as follows:

as shown in FIG. 1, the helicase activity measurement method described above is actually based on the formation of multiple paired single strands (double strands in this example), and a stable nucleic acid secondary structure can be obtained after annealing treatment by the presence of complementary paired parts between different single strands. Wherein one strand (fork 1A) constituting the structure is used for fixing the secondary structure of the nucleic acid, and the other strand (fork 1B) is used for quantification. When no helicase is present, since the two chains are not separated, the conjugate a (digoxin) on the other chain (fork 1B) can be bound to a digoxin antibody conjugated with horseradish peroxidase (HRP), and then the amount of these nucleic acid secondary structures with multiple complex folds can be detected by the color reaction of HRP with TMB. When helicase and ATP are added, a helicase reaction occurs, and complex nucleic acid substances can be subjected to helicase folding, so that a DNA chain (fork 1B) with non-immobilization effect is liberated in a reaction system and is sufficiently removed after washing and other steps, and at the moment, the helicase activity can be determined according to the change of the detected absorbance values before and after helicase (the quantification is carried out according to a standard curve of the absorbance measured by a standard solution and the substrate concentration gradient). Meanwhile, since most of the unwinding reaction depends on ATP, the ATP consumption in the unwinding reaction process may be detected by using an ATP detection kit after recovering the reaction solution obtained by the unwinding reaction.

The schematic diagram of the secondary structure of the nucleic acid of the replication fork (fork1) in this example is shown in FIG. 2, and the detection results are shown in FIGS. 3 and 4.

It was found that by controlling the final concentration of the DNA substrate used for coating, the absorbance of TMB (HRP substrate) increased with the increase of the DNA substrate in the system. FIG. 4 is a standard graph of the above detection method, and it can be found that the linear detection range of the detection method is 0.8 to 5nM, and the limit of detection (LOD) can reach 0.17 nM. Compared with the EMSA method, the LOD (0.16nM) obtained by the chemiluminescence immunoassay with the membrane transfer signal amplification is similar, but the sensitivity of the helicase activity measuring method based on the double-stranded DNA is far better than that of the common EMSA (0.17 mu M) or FRET (2.86nM) detecting method from the aspect of sensitivity (the detection results of the EMSA method and the FRET method are respectively shown in FIG. 5 and FIG. 6, wherein, the EMSA method and the FRET method are both carried out by adopting the routine operation in the field).

In addition, in the unwinding process of helicase, the folded structure in the secondary structure of nucleic acid will be unfolded to various degrees, the inventors obtained the secondary structure of nucleic acid with different folding degrees by adjusting the molar ratio of two strands constituting the secondary structure of nucleic acid, and further evaluated the effectiveness of the above method for measuring helicase activity based on double-stranded DNA by comparing the results of EMSA method and the above method for measuring helicase activity based on double-stranded DNA for the secondary structure of nucleic acid with different folding degrees.

The detection method is referred to the above embodiment, except that: annealing was performed by adjusting the molar ratio of the two single-stranded DNAs (fork 1A and fork 1B) so that the hybridization ratios were 0%, 10%, 25%, 41%, 66%, 85%, and 100%, respectively. The EMSA was used to detect the change in the amount of folding, and then the above-described double-stranded DNA-based helicase activity assay method was used to quantify the structure of different degrees of folding.

The results are shown in FIG. 7.

It was found that, in the above-mentioned method for measuring helicase activity based on double-stranded DNA, the absorbance gradually increased with the increase in the degree of structural folding, and a good linear correlation (linear correlation coefficient R) was exhibited²0.960), indicating that the absorbance signal is linearly related to the degree of folding of the replication fork. Therefore, it can be demonstrated that the above-described double-stranded DNA-based helicase activity measurement method can be used for determining the development process of nucleic acid structures to various degrees.

Actual detection effect of the above method for measuring helicase activity based on double-stranded DNA

(1) The determination method is based on the detection of the activity of the double-stranded replication fork on the unwinding enzymolysis and the spinning:

in this example, the inventors selected BLM helicase as the helicase species tested. BLM helicase is an ATP-dependent 3 'to 5' directed helicase with a broad spectrum of helicase substrates. This protein may be one of the most desirable choices in the art for validating and optimizing helicase activity assays. BLM helicases are able to unwind DNA duplexes that depend on 3 'tail overhang, whereas DNA duplexes that do not have a 3' tail overhang are difficult to unwind. Thus, replication fork sequences with and without the 10 nucleotide 3' -tail sequence were selected for alignment, respectively.

Wherein, the replication fork sequence with 10 nucleotide 3' -tail sequence is fork 1A and fork 1B. While the replication fork sequence without the 10 nucleotide 3' -tail sequence is shown for fork2, the effect of having only one single stranded DNA in fork2 without the 10 nucleotide 3' -tail sequence is the same for any one of fork2 lacking the 10 nucleotide 3' -tail sequence (5'-TTTTTTTTTT-3' (SEQ ID NO:18)), in this example, the B strand is selected as the deleted strand.

Wherein the nucleotide sequence of fork2 is:

fork2 A：5’-TTTTTTTTTTCGTACCCGATGTGTTCGTTC-3’(SEQ ID NO:1)；

fork2 B：5’-GAACGAACACATCGGGTACG-3’(SEQ ID NO:3)。

the detection method is the same as the above embodiment.

The results are shown in FIG. 8.

By using two different bifurcating duplexes (replication forks) to verify, it was found that fork1 with the complete 3 '-tail folded less after BLM helicase was added, while fork2, which lacks the 3' -tail, folded slightly more in one strand.

To further verify the reliability of the above assay for helicase assays, the inventors again extended the DNA substrate structure types for validation and in this example, the inventors selected bifurcating duplexes (fork3, fork1, and fork4) of 12, 20, and 28bp in length (length here refers to the length after removal of the 3' -tail sequence), respectively.

Wherein the nucleotide sequence of fork3 is:

Fork3 A：5’-TTTTTTTTTTCGTCGAGCAGAG-3’(SEQ ID NO:4)；

Fork3 B：5’-CTCTGCTCGACGTTTTTTTTTT-3’(SEQ ID NO:5)。

the nucleotide sequence of Fork4 is:

Fork4 A：5’-TTTTTTTTTTCGTACCCGATGTGTTCGTTCAACTTAGC-3’(SEQ ID NO:6)；

Fork4 A：5’-GCTAAGTTGAACGAACACATCGGGTACGTTTTTTTTTT-3’(SEQ ID NO:7)。

the results are shown in FIG. 9.

It was found that duplexes with different complementary binding region lengths showed significantly different detection results in the above assay method, where the shorter the complementary binding region length, the faster the unwinding speed of BLM helicase unwinding, and the shorter the reaction time, consistent with the "stepwise" unwinding mechanism of BLM helicase on duplexes. The results in the above examples show that the above measurement method can accurately reflect the unwinding rate of the helicase.

(2) The determination method is based on Holliday Junction and bble nucleic acid secondary structure models to detect the helicase activity:

holliday Junction (HJ), D-loops and corresponding bubble structures are essential secondary structure intermediates in gene recombination and repair processes. BLM helicases can unravel these structures and protect cells from DNA damage and neoplastic transformation. Given the importance of these structures, the suitability of the bubble and HJ models in the above assay methods was designed and validated accordingly in the helicase assay.

In this example, the design concept of the Holliday Junction and bubble nucleic acid secondary structure model used in the above assay method is shown in FIG. 10.

In the bubble model, biotin and digoxin are still respectively marked at the same end of the two single strands, and partial mismatch sequences are introduced into the original complementary regions, so that the two single strands form a bubble structure after combination. To facilitate evaluation of its feasibility by BLM helicase, the inventors ligated the 3' -tail sequence to the end of the non-immobilized B chain distal to digoxin.

In this example, the nucleotide sequence of the bubble model used is:

Bubble A：5’-CTGCTAATGTAATTTTTTTTTTTTTACGACAAGATCTTTTTTTTTT-3’(SEQ ID NO:8)；

Bubble B：5’-GATCTTGTCGTACAGGATCGTTACATTAGCAG-3’(SEQ ID NO:9)；

among them, only the underlined part is the double-stranded binding part of the bubble model. Only the bold part is the mismatch part of the bubble model to form a bubble structure, and only the italic part is the 3' -tail sequence. At the same end (5 ' end or 3' end, in this example 5' end) of Bubble a and Bubble B, respectively, conjugate a and conjugate B (in this example, conjugate a and conjugate B are digoxin and Biotin (Biotin), respectively) were attached.

The detection method is described in the above embodiments, and is different from the following steps: the two chains of Bubble A and Bubble B of the Bubble model were mixed and heated at 95 ℃ for 10min before being reduced to room temperature (annealing), and the subsequent steps were identical.

In the Holliday Junction model, additional auxiliary chains (HJ-C chain and HJ-D chain) are introduced to form a four-way Junction with the HJ-A and HJ-B chains, resulting in a Holliday Junction structure. To ensure that the HJ-B strand can be separated after the Holliday Junction model is fully developed, a 3' -tail sequence is ligated to the additionally introduced HJ-C and HJ-D strands of the helper strands, so that it has the effect that the B strand can be separated only and only after complete unwinding of the 4-strand hybridization by BLM.

In this example, the nucleotide sequence of the Holliday Junction model used was:

HJ-A：5’-TTTTTTTTTTCACCCGTTTCTACAGGATCGTTCGGTCTTAAG-3’(SEQ ID NO:10)；

HJ-B：5’-GATCTTGTCGTACAGGATCGTTACATTAGCAG-3’(SEQ ID NO:9)；

HJ-C：5’-CTTAAGACCGAACGATCCTGTACGACAAGATCTTTTTTTTTT-3’(SEQ ID NO:11)；

HJ-D：5’-CTGCTAATGTAACGATCCTGTAGAAACGGGTGTTTTTTTTTT-3’(SEQ ID NO:12)。

wherein, only the underlined part is the binding part between different chains in the Holliday Junction model, specifically, HJ-A is complementary paired with partial sequences in HJ-C and HJ-D respectively, and HJ-B is complementary paired with the rest sequences in HJ-C and HJ-D respectively, thereby forming the Holliday Junction structure. Only the italic part is the 3' -tail sequence. To the same ends (5 ' end or 3' end, in this example, 5' end) of HJ-a and HJ-B, respectively, are attached a binder a and a binder B (in this example, a binder a and a binder B are digoxin and Biotin (Biotin), respectively).

The detection method is described in the above embodiments, and is different in that: four chains of HJ-A, HJ-B, HJ-C and HJ-D chains of the Holliday Junction model were mixed, heated at 95 ℃ for 10min and then lowered to room temperature (annealed), and the subsequent steps were identical.

Differences were detected by comparing methods based on the bubble model and the Holliday Junction model, using fork 1A and fork 1B as controls.

The results are shown in FIG. 11. The reaction speed of the bubble model is basically similar to that of fork replication of fork1, while the relative reaction speed of the HJ model is higher, and the result is consistent with the method mechanism of "the shorter the length of the complementary binding region is, the higher the unwinding speed of the BLM helicase and the shorter the reaction time is", and the "step-by-step" unwinding mechanism of the BLM helicase. The results in the above examples show that the above measurement method can accurately reflect the helicase rate based on different nucleic acid secondary structure models.

(3) The determination method is based on the detection of the helicase activity of a G-quadruplex structure model:

since the above-mentioned assay method is compatible with the direct detection of the helicase assay and the process of unwinding the secondary structure of the intermolecular DNA, many of which have important research values in themselves, the assay method can also be used for the study of these intramolecular structures.

In this example, the inventors selected G-quadruplexes, which are biologically important secondary structures, as models and measured them in combination with the above-described measurement method.

The specific design concept is shown in fig. 12.

In this example, the inventors first prepared a reference model with a double-stranded stem in which biotin was labeled at the 5 '-end of the A chain and digoxin was labeled at the 3' -end of the B chain. A 3' -tail sequence was added to the a chain to facilitate recognition by BLM helicase.

Wherein, the specific nucleotide sequence of the double-stranded stem is as follows:

Model A：5’-TTTTTTTTTTCGTCGAGCAGAG-3’(SEQ ID NO:4)；

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

for the G4 melting model, the inventors inserted their G4 forming sequence between the duplex stem and 3' -tail. Specifically, based on the principle, the inventor co-constructs three representative G-quadruplex forming sequences, and respectively names three G4 unwinding detection models as GQ1(G3T3), GQ2(G3T1) and GQ3(G4T3), wherein Model B is used for all B chains of the three G4 unwinding detection models.

Wherein, the concrete nucleotide sequences of the three G4 unwinding detection models are respectively:

GQ1(G3T3) unwinding detection model:

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

GQ2(G3T1) unwinding detection model:

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

GQ3(G4T3) unwinding detection model:

Model B：5’-CTCTGCTCGACG-3’(SEQ ID NO:13)。

before the detection method is used for detection, gel electrophoresis and circular dichroism are used for verifying whether the three G4 unwinding detection models form G-quadruplexes respectively, and the detection is continued after the situation that no errors exist is confirmed.

The results are shown in FIG. 13.

It can be found that, in the case of using BLM helicase, the BLM helicase can better unwind the G4 helicase detection model containing G-quadruplexes than the duplex model used for reference, and at the same time, it can also be found that there is a difference between the unwinding rates of BLM when unwinding different G4 sequences, and the difference is in line with the research conclusion of BLM helicase unwinding mechanism in the prior art. In summary, the detection method shows good sensitivity for the detection of the different models.

Application of detection method in detecting ATP consumption in unwinding process

Most helicases are driven by ATP to carry out the helicase reaction, but most of the existing helicase detection methods can only measure the helicase quantity, and the detection methods can simultaneously detect the ATP consumption in the helicase reaction process by means of recovered reaction liquid, so that the helicase quantity and the ATP consumption in the helicase unwinding process with different structures can be simultaneously obtained by using only one sample, the helicase characteristics and the ATP hydrolysis characteristics are evaluated, and the activity detection of the helicase is more comprehensive.

The detection method is the same as the step (6) in the above embodiment. The specific process is as follows: and transferring 10 mu L of reaction mixture to a 384-half-well plate, adding 10 mu L of ATP detection reagent, blowing, uniformly mixing, performing chemiluminescence detection by using a microplate reader, and calculating the ATP consumption of the helicase in different structures by reading chemiluminescence values.

In this example, the enzyme used in the test was BLM, and the enzymatic hydrolysis time was 30 min.

The calculation formula is the same as in step (6) in the above embodiment.

The results are shown in FIG. 14.

It can be found that the detection method is based on the detection of four nucleic acid secondary structure models of Bubble, HJ, GQ1(G3T3) and Fork1, the results of the four models after 30min of unwinding reaction are different, and the results accord with the research conclusion of BLM unwinding and unwinding mechanism in the prior art, so that the detection method can effectively detect unwinding amount and ATP hydrolysis amount of different unwinding proteins for different substrates.

Application of detection method in screening and evaluating helicase inhibitor

Helicases generally play important biological roles in disease. Therefore, the development of helicase inhibitors as candidate therapeutic agents is also an attractive area. And there are few related applications in this respect in the prior art. For example, BLM inhibitors have the potential to treat cancer, however, there is no assay available in the art for evaluating helicase inhibitors, and thus the therapeutic effect thereof cannot be known in a true sense. However, the inventors found that the new helicase assay concept that the detection method can remove small molecules during detection may have good inhibitor evaluation performance, and for this reason, the inventors used BLM inhibitor ML216 known in the art as a detection sample to verify the applicability of the detection method in this respect.

The detection method is different from the above example in that while adding the BLM helicase, ML216 is added at the same time, and the inhibition effect of ML216 on the dissolution of the BLM helicase from various DNA substrates is evaluated by controlling the helicase activity of the control group to which ML216 is not added.

The nucleic acid secondary structure models based on the detection method are set as Fork1, Bubble and GQ1(G3T3), so that the influence of different nucleic acid secondary structure models on the detection result is further analyzed.

The results are shown in Table 1.

TABLE 1 results of enzyme inhibition effect test on ML216 based on different nucleic acid secondary structure models

DNA structural model	IC50(μ M) for ML216 vs BLM
		Fork
1	0.43
		Bubble	0.57
GQ1	7.9

It was found that the IC50 results were different when different substrates were used, indicating that the inhibitory effect of enzyme inhibitors on the inhibition of enzymatic lysis of different DNA substrates may be different. This indicates that in evaluating and screening small molecule inhibitors, the test substrate for helicase can be rationally selected for different research purposes.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Zhongshan university

GUANGZHOU ZHONGDA NANSHA TECHNOLOGY INNOVATION INDUSTRIAL PARK Co.,Ltd.

<120> helicase activity determination method based on double-stranded DNA and application thereof

<130>

<160> 17

<170> PatentIn version 3.5

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Claims

1. A detection reagent is characterized in that the detection reagent contains a nucleic acid secondary structure obtained by hybridization of at least two DNA single strands, the tail end of at least one DNA single strand in the nucleic acid secondary structure is connected with a tail sequence, and the tail ends of any two different DNA single strands in the nucleic acid secondary structure are respectively connected with two non-interfering specifically-combined substances or groups;

the specific binding substance or group comprises at least one of an antibody, an antigen, biotin, avidin, complementary paired bases, magnetic beads, a transcription activation domain, a DNA binding domain, and amino acids.

2. The detection reagent of claim 1, wherein the nucleotide sequence of the tail sequence is: 5'-TTTTTTTTTT-3' are provided.

3. The detection reagent according to claim 1, wherein the DNA single strands are 12-60 bp, and the complementary pairing length between the DNA single strands is 12bp or more.

4. The detection reagent according to claim 1, wherein the nucleic acid secondary structure comprises a complementary DNA double strand, a replication fork structure, a Holliday Junction structure, a bubble structure, a G4 structure, and a hairpin structure.

5. The detection reagent according to any one of claim 4, wherein the nucleotide sequence of each DNA single strand in the secondary structure of the nucleic acid is:

complementary DNA double strand:

SEQ ID NO: 4 and 13;

replicating the fork structure:

SEQ ID NO: 1-2 of the sequence; or

The amino acid sequence of SEQ ID NO:1 and SEQ ID NO: 3, the sequence; or

SEQ ID NO: 4-5 of the sequence; or

The amino acid sequence of SEQ ID NO: 6-7 of the sequence;

holliday Junction structure:

SEQ ID NO: 9-12 of the sequence;

a bubble structure:

SEQ ID NO: 8-9 of the sequence;

structure G4:

SEQ ID NO: 13-16.

6. A detection kit comprising the detection reagent according to any one of claims 1 to 5 and a substance targeting two specific binding substances or groups on a single DNA strand in the detection reagent according to any one of claims 1 to 5.

7. A method for qualitatively and/or quantitatively detecting helicase activity, comprising the steps of:

(1) taking the DNA single strand of each nucleic acid secondary structure in claim 5, and heating and annealing to obtain the corresponding nucleic acid secondary structure;

(2) fixing the nucleic acid secondary structure on the surface of a container through a specific binding substance or group contained in the nucleic acid secondary structure, adding a sample to be detected to react with ATP for 15-30 min, washing off all solutions, adding a substance which is coupled with a labeling group and targets the nucleic acid secondary structure specific binding substance or group, and comparing the parameter change condition of a blank control to determine the activity of the helicase qualitatively and/or quantitatively.

8. The method of claim 7, wherein the parameter is an absorbance value, and the variation is calculated based on the following formula:

9. A method for detecting helicase ATP consumption, comprising the steps of:

(2) fixing a nucleic acid secondary structure on the surface of a container through a specific binding substance or group contained in the nucleic acid secondary structure, and adding a sample to be detected to react with ATP for 25-40 min to obtain a mixed reaction solution;

10. Use of the detection reagent according to any one of claims 1 to 5 in any one of the following (1) to (4);

(1) qualitatively or quantitatively detecting helicase activity;

(2) qualitatively or quantitatively detecting ATP consumption of helicase;