CN111394421A - Kit for detecting activity of Cas3 protein and application thereof - Google Patents
Kit for detecting activity of Cas3 protein and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly discloses a kit for detecting activity of Cas3 protein and application thereof. The kit comprises avidin and double-stranded DNA fixed on a reaction vessel; the double-stranded DNA contains a non-complementary region which can be recognized by Cas 3; the two ends of the duplex DNA are respectively marked by a fluorescent group and biotin; one end of the double-stranded DNA may be immobilized on the reaction vessel by the binding of the biotin to the avidin. The kit and the detection method can quickly, effectively and high-flux detect the activity of the Cas3 protein, and further can be applied to large-scale screening of an inhibitor of Cas3, the Z factor of the screening method can reach 0.58, and the requirement (between 0.5 and 1.0) of a high-flux screening model is met. By using the selected inhibitor, the activity of the Cas3 protein can be controlled, and the influence on the immune system of bacteria can be generated.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a kit for detecting activity of Cas3 protein and application thereof.
Background
The CRISPR system is an adaptive immune system for most bacteria and archaea, designed to combat exogenously invading nucleic acids. CRISPR sites are composed of the repeat (repeat) of the bacterium itself, and a spacer (spacer) for recognition of foreign nucleic acids. The CRISPR immune system performs a function, requiring 3 stages: adaptation, expression and interference. During the adaptation phase, the bacteria integrate nucleic acid sequences derived from the phage or foreign plasmid as spacer sequences between the repeated sequences of the CRISPR site. These spacer sequences perform a sequence memory function to ensure a targeted defense during subsequent invasion of the corresponding phage or plasmid. During the expression phase, all repeats and spacers as a whole are transcribed into crRNA precursors, which are finally processed by the corresponding Cas protein into mature crRNA. During the interference phase, the crRNA serves as a guide, complementarily pairs with the invading nucleic acid, while the corresponding Cas protein recognizes and cleaves the exogenous invading nucleic acid.
CRISPR systems are currently divided into two classes (class 1 and 2) based on Cas genetic composition, sequence similarity between Cas proteins and gene structures, where class 1 includes 3 types (I, III and IV), class 2 also includes 3 types (II, V and VI) and 19 subtypes. Cas3 is involved as an important protein during the interference phase of the application of the CRISPR system of pseudomonas aeruginosa belonging to type I-F. The RNA obtained in the first two stages was used as guide RNA to form a Csy effect complex with 4 Csy proteins. This complex can recognize and unwind the re-invaded homologous nucleic acid and form the R-loop structure of the homologous nucleic acid. The Csy complex recruits Cas3 nuclease protein while recognizing homologous nucleic acids, and Cas3 can recognize an R-loop structure helicized by the Csy effector complex and perform nucleic acid degradation.
It is reported in the literature that Cas3 can recognize a non-complementary region and cleave it by constructing a double-stranded DNA with this region without the help of the recruitment of Csy complex.
The study on the activity of the Cas3 protein is beneficial to the study on the mechanism of a bacterial CRISPR system, and the activity of the Cas3 protein can be controlled to influence the immune system of bacteria. Therefore, there is a need to provide a kit for detecting Cas3 protein and applications thereof.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a simple, sensitive and rapid kit for detecting the activity of Cas3 protein and an application thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a kit for detecting Cas3 protein activity, comprising:
(1) avidin immobilized on the reaction vessel;
(2) double-stranded DNA; the double-stranded DNA contains a non-complementary region which can be recognized by Cas 3; the two ends of the duplex DNA are respectively marked by a fluorescent group and biotin;
one end of the double-stranded DNA may be immobilized on the reaction vessel by the binding of the biotin to the avidin.
In the "avidin immobilized on the reaction vessel" mentioned in the present invention, the immobilization method is generally coating, and in order to immobilize the avidin on the reaction vessel without affecting the binding effect of the avidin and biotin, other methods may be selected by those skilled in the art, and are not further limited herein.
As long as there is a PAM site, non-complementary regions, can be recognized by Cas 3.
Preferably, the length of the non-complementary region in the double-stranded DNA is 9 to 11nt, preferably 10 nt.
The invention designs the double-stranded DNA, and preferably, the nucleotide sequences of two single strands are respectively shown as SEQ ID No.1 and SEQ ID No. 2.
Preferably, the 3' ends of the two single strands of the double-stranded DNA are labeled with a fluorophore and biotin, respectively.
Preferably, the fluorophore is FAM.
Preferably, the avidin is streptavidin.
Preferably, the 20 μ L reaction system of the kit comprises:
cas3 protein liquid with the concentration of 0.8-1.2 mg/mu L is 2 mu L, and more preferably 1 mg/mu L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
the balance being water.
In some embodiments, the Cas3 protein solution further contains 20mM Tris-HCl, 500mM NaCl, and glycerol at a concentration of 40% by volume.
When the kit is used for detecting and screening the inhibitor of the Cas3 protein, the 20 mu L reaction system of the kit also contains the inhibitor 2 mu L, preferably, the concentration of the inhibitor is not higher than 2mM, and the inhibitor higher than the concentration can cause the stripping of streptavidin in a coated plate, thereby causing false negative of experimental results.
The invention further provides application of the kit in detecting the activity of the Cas3 protein.
The invention further provides a method for detecting the activity of the Cas3 protein, and the kit is used, and the specific steps comprise:
(1) adding double-stranded DNA, Cas3 protein liquid and reaction liquid into the reaction vessel, and incubating at 36-38 ℃;
preferably, the 20. mu. L reaction system contains:
cas3 protein liquid with the concentration of 0.8-1.2 mg/mu L being 2 mu L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
the balance of water;
(2) and after the incubation is finished, cleaning the reaction vessel, and then detecting a fluorescent signal.
When the Cas3 is functional, the end with the fluorescent label is cut off, and then the reaction vessel is cleaned, the fluorescent signal is washed away, on the contrary, when the Cas3 is not functional or the function is inhibited, the end with the biotin is combined with the avidin fixed on the reaction vessel, and the fluorescent signal at the other end is not washed away.
The invention further provides application of the kit in screening inhibitors of Cas3 protein.
The invention further provides a method for screening an inhibitor of Cas3 protein, and the kit is used for the method, and comprises the following specific steps:
(1) adding double-stranded DNA, Cas3 protein liquid, an inhibitor and reaction liquid into the reaction vessel, and incubating at 36-38 ℃;
preferably, the 20. mu. L reaction system contains:
cas3 protein liquid with the concentration of 0.8-1.2 mg/mu L being 2 mu L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
inhibitor 2 mu L, preferably the concentration of the inhibitor is not higher than 2 mM;
the balance of water;
(2) and after the incubation is finished, cleaning the reaction vessel, and then detecting a fluorescent signal.
In screening for inhibitors, a negative control group for detecting the activity of Cas3 protein can be set, and the effect of the inhibitor can be known by comparing the results of the inhibitor-added group and the inhibitor-not-added group.
In screening for inhibitors, a reaction system containing no Cas3 protein solution can be set as a positive control group.
When the inhibitor is screened and the stability of the screening system is verified, the negative and positive controls are opposite, when the inhibitor is screened, the reaction system with the Cas3 protein liquid is a negative control group, and the reaction system without the Cas3 protein liquid is a positive control group; when the stability of the system is verified, the reaction system with the Cas3 protein liquid is a positive control group, and the reaction system without the Cas3 protein liquid is a negative control group.
When screening the inhibitor, the variance of the deviation from the main body can be calculated according to the fluorescence signal value of the group to be detected, and the compound with the deviation larger than the main body is selected, namely the effective inhibitor.
In the "incubation" mentioned in the method of the present invention, the incubation time can be adjusted according to the choice of the reagent and the actual situation, and in the system of the present invention, the incubation time is preferably 1.8-2.5 h, and more preferably 2 h.
In the "washing the reaction vessel" mentioned in the method of the present invention, the washing solution is typically PBS, and the main purpose of this step is to wash away DNA fragments cut off in the function of Cas 3.
The invention has the following beneficial effects:
the kit and the detection method can quickly, effectively and high-flux detect the activity of the Cas3 protein, and further can be applied to large-scale screening of an inhibitor of Cas3, the Z factor of the screening method can reach 0.58, and the requirement (between 0.5 and 1.0) of a high-flux screening model is met. By using the selected inhibitor, the activity of the Cas3 protein can be controlled, and the influence on the immune system of bacteria can be generated.
Compared with the traditional E L ISA, the invention has the advantages that:
(1) compared with direct E L ISA, the invention marks signal on nucleic acid, not protein, and the nucleic acid is easier to operate and lower in cost than protein.
(2) Compared with the indelect E L ISA, the invention does not need to combine a secondary antibody any more, thereby avoiding the occurrence of interaction reaction.
(3) The present invention does not require more than two binding sites for the antigen, as compared to the sandwich E L ISA.
(4) Compared to the competitive E L ISA, the present invention does not require two antigen competitions.
(5) Compared with the cell-based E L ISA, the invention does not need to be based on cells and can directly carry out detection at a cell-free level.
Drawings
FIG. 1 is a schematic diagram of a reagent cartridge in example 1;
FIG. 2 is a graph showing the results of the Z factor experiment in the screening method of example 2;
FIG. 3 is a graph showing the results of the inhibition ratio of the inhibitor to be screened in example 3 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
This example first provides a kit for detecting Cas3 protein activity, comprising:
(1) streptavidin immobilized on 384-well plates, a commercially available microplate purchased from invitrogen corporation was used in this example, and this microplate was a 384-well plate coated with streptavidin and was used as it was;
(2) double-stranded DNA; the double-stranded DNA contains a non-complementary region which can be recognized by Cas3, and the nucleotide sequences of the two single strands are shown as follows:
SEQ ID No.1：
5’-GCTGTACGTCACTATCGAAGCAATACAGGTAGACGCGGACATCAAGCCCGCCGTGAAGGTGCAGCTTCTCTACAGAGTGC-3’；
SEQ ID No.2：
5’-GCACTCTGTAGAGAAGCTGCACCAAGTGCCGCCGCTTGATGTCCGCGTCTACCTGTATTGCTTCGATAGTGACGTACAGC-3’；
the 3' ends of the two single chains are respectively marked by FAM fluorescent group and Biotin (Biotin);
one end of the double-stranded DNA can be immobilized on the 384-well plate by the binding of the biotin to the streptavidin.
The principle of the kit is shown in FIG. 1, which shows a labeled substrate with 10bp non-complementary region, the total length is 80bp, when Cas3 functions, one end with FAM group will be cut, and fluorescence will be washed away when PBS is used to wash the pore plate, thus reducing the fluorescence signal.
The present embodiment further provides a method for screening an inhibitor of Cas3 protein, and the method comprises the following steps:
(1) adding 20 mu L reaction system into a 384-well plate, and incubating for 2h at 37 ℃;
1) the 20 mu L reaction system of the test group contains:
cas3 protein solution at a concentration of 1mg/μ L, 2 μ L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
2 mu L of inhibitor, wherein the concentration of the inhibitor is 2 mM;
the balance being water.
2) A negative control group is arranged for detecting the activity of the Cas3 protein, namely the inhibition rate of the activity of the Cas3 is 0, and a 20 mu L reaction system comprises:
cas3 protein solution at a concentration of 1mg/μ L, 2 μ L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
the balance being water.
3) Setting a positive control group, wherein the reaction system of the positive control group is different from that of the group to be detected in that: the protein solution does not contain Cas3 protein, and the activity inhibition rate of Cas3 is 100 percent.
The Cas3 protein solution used in this example also contained 20mM Tris-HCl, 500mM NaCl, and 40% glycerol by volume.
(2) After the incubation was completed, the 384-well plate was washed with PBS, and then the detection of the fluorescence signal was performed.
Example 2
In this example, the stability of the screening method provided in example 1 was verified (Z factor was calculated), and the specific method was as follows:
48 positive control groups and 48 negative control groups are respectively arranged, and according to the above, when the inhibitor is screened and the stability of the screening system is verified, the negative and positive control groups are opposite, that is, at the moment, the reaction system with the Cas3 protein liquid is the positive control group, and the reaction system without the Cas3 protein liquid is the negative control group.
The fluorescence signal was detected as in example 1, and the Z factor was then calculated according to the following formula:
wherein SD represents the standard deviation of fluorescence values, and X represents the mean of fluorescence values.
The result is shown in figure 2, the Z factor is an important index for judging whether a high-throughput screening mode has stability, the screening method is calculated, the Z factor is 0.58 and is between 0.5 and 1.0, and the stability requirement of a high-throughput screening model is met within the confidence interval of the Z factor.
Example 3
This example applies the method provided in example 1 to screen for Cas3 protein inhibitors.
A natural product library containing 2000 or more compounds, including a natural product library (1440 compounds) purchased from ceramic Biotechnology Ltd and a natural product library (616 compounds) purchased from Selleck, was screened, the excitation wavelength and the emission wavelength were set to 492nM and 518nM, respectively, the fluorescence signal values were detected, and the average value and the standard deviation were calculated. By calculating the deviation from the subject variance (Z value).
Wherein the formula is: z ═ X (Xi-X)Average) and/SD, wherein Xi is the measured fluorescence signal value of each hole of the group to be detected, X is the average value of the fluorescence signals of all samples of the group to be detected, and SD is the standard deviation of the fluorescence signals of all samples of the group to be detected.
Selecting 13 compounds with a deviation greater than that of the subject (see figure 3 for test results) eases the subsequent screening work for inhibition of the anti-CRISPR system-associated protein Cas 3.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (10)
1. A kit for detecting Cas3 protein activity, comprising:
(1) avidin immobilized on the reaction vessel;
(2) double-stranded DNA; the double-stranded DNA contains a non-complementary region which can be recognized by Cas 3; the two ends of the duplex DNA are respectively marked by a fluorescent group and biotin;
one end of the double-stranded DNA may be immobilized on the reaction vessel by the binding of the biotin to the avidin.
2. The kit according to claim 1, wherein the length of the non-complementary region in the double-stranded DNA is 9 to 11nt, preferably 10 nt.
3. The kit according to claim 2, wherein in the double-stranded DNA, the nucleotide sequences of the two single strands are shown as SEQ ID No.1 and SEQ ID No.2, respectively.
4. The kit according to any one of claims 1 to 3, wherein the fluorophore is FAM;
and/or the avidin is streptavidin.
5. The kit according to any one of claims 1 to 4, wherein the 20 μ L reaction system of the kit comprises:
cas3 protein liquid with the concentration of 0.8-1.2 mg/mu L being 2 mu L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
the balance being water.
6. The kit according to claim 5, wherein the 20. mu. L reaction system of the kit further comprises an inhibitor 2. mu. L, preferably the concentration of the inhibitor is not higher than 2 mM.
7. Use of the kit of any one of claims 1-5 for detecting activity of a Cas3 protein.
8. A method for detecting activity of Cas3 protein, which is characterized in that the kit of any one of claims 1-4 is used, and the specific steps comprise:
(1) adding double-stranded DNA, Cas3 protein liquid and reaction liquid into the reaction vessel, and incubating at 36-38 ℃;
preferably, the 20. mu. L reaction system contains:
cas3 protein liquid with the concentration of 0.8-1.2 mg/mu L being 2 mu L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
the balance of water;
(2) and after the incubation is finished, cleaning the reaction vessel, and then detecting a fluorescent signal.
9. Use of the kit of any one of claims 1-6 for screening for inhibitors of Cas3 protein.
10. A method for screening an inhibitor of Cas3 protein, which is characterized in that the kit of any one of claims 1-4 is used, and the specific steps comprise:
(1) adding double-stranded DNA, Cas3 protein liquid, an inhibitor and reaction liquid into the reaction vessel, and incubating at 36-38 ℃;
preferably, the 20. mu. L reaction system contains:
cas3 protein liquid with the concentration of 0.8-1.2 mg/mu L being 2 mu L;
2 μ L of a 1 μ M double-stranded DNA solution;
reaction solution 2 mu L, which contains 200mM Tris, 3M NaCl and 50mM MgCl 2;
inhibitor 2 mu L, preferably the concentration of the inhibitor is not higher than 2 mM;
the balance of water;
(2) and after the incubation is finished, cleaning the reaction vessel, and then detecting a fluorescent signal.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021149829A1 (en) * | 2020-01-24 | 2021-07-29 | Ｃ４Ｕ株式会社 | Method for detecting specific dna in sample |
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| JP6940086B1 (en) * | 2020-01-24 | 2021-09-22 | Ｃ４Ｕ株式会社 | How to detect specific DNA in a sample |
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