CN110818757A - Nucleotide analogs and method for screening DNA polymerase - Google Patents
Nucleotide analogs and method for screening DNA polymerase Download PDFInfo
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- CN110818757A CN110818757A CN201810892628.5A CN201810892628A CN110818757A CN 110818757 A CN110818757 A CN 110818757A CN 201810892628 A CN201810892628 A CN 201810892628A CN 110818757 A CN110818757 A CN 110818757A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention provides a nucleotide analogue. The nucleotide analog has the following structure,
Description
Technical Field
The present invention relates to the field of biotechnology, in particular, the present invention relates to nucleotide analogs and methods of screening for DNA polymerases.
Background
With the successful decoding of the human genome, gene sequencing technology is becoming more and more widely used, and sequencing-by-synthesis (SBS) is favored because of its high throughput and low price. SBS sequencing technology utilizes a polymerase to introduce each fluorescently labeled nucleotide into a DNA primer, and then measures the fluorescent signal from the nucleotide to identify the base of the nucleotide and simultaneously analyze its complementary base. However, since nucleoside triphosphates for SBS are essentially double-modified reversible terminators (DRTs) that have reversible blocking groups at the 3' -OH portion of nucleoside triphosphates and fluorophore modifications at the bases, this double modification does not allow the linkage linking the fluorophore to the base to be completely removed after sequencing, thus leaving so-called molecular scars, as sequencing proceeds, newly polymerized strands contain more molecular scars, and eventually accumulation of molecular scars reduces polymerase activity and fidelity, resulting in limited number of bases sequenced.
The single Modified Reversible Terminator (MRT) is on the 3' -OH group, the reversible blocking group has double functions, and can be used as a reporter of a fluorescence signal and a reversible terminator, and the modification mode can successfully avoid molecular scars generated in SBS when DRT modification is adopted, so that the influence on polymerase is reduced, and the reading length of sequencing is improved.
However, conventional DNA polymerases bred under natural conditions cannot utilize MRT-modified nucleotide triphosphates to perform efficient polymerization, and appropriate artificial modification or modification of the polymerase is required to make MRT-modified nucleotide triphosphates widely applicable to SBS sequencing technology. In addition, the size of the modified nucleotide triphosphate can also be reduced to facilitate enzymatic polymerization.
However, the DNA polymerases that can utilize MRT technology are lacking in a direct, rapid and efficient screening system and utilization system.
Disclosure of Invention
The present application aims to solve at least to some extent one of the technical problems of the prior art:
the invention provides a method for in vitro high-throughput screening of DNA polymerase by utilizing Fluorescence Resonance Energy Transfer (FRET) technology, wherein the DNA polymerase can catalyze nucleoside triphosphate which is used as a reversible terminator (MRT) and has a Biotin (Biotin) blocking group at the 3' -OH end to carry out polymerization reaction and can be applied to SBS.
In a first aspect of the invention, a nucleotide analog is provided. According to an embodiment of the present invention, the nucleotide analog has the following structure,
wherein L represents a cleavable linking group; label represents a first detectable Label; p represents a triphosphate group; base represents adenine, guanine, cytosine or thymine. Wherein L and Lable constitute a single-Modified Reversible Terminator (MRT). According to embodiments of the present invention, by detecting a change in the polymerization reaction of the first detectable label in the nucleotide analog, it can be accurately determined whether the polymerase to be screened has polymerization activity. The nucleotide analogs according to the embodiments of the present invention can be effectively used for screening polymerases having activities of catalyzing polymerization of nucleotide triphosphates having a reversible terminator (MRT) at the 3' -OH terminal and can be used for SBS.
In a second aspect of the present invention, the present invention provides a method of screening for a DNA polymerase. According to an embodiment of the invention, the method comprises: subjecting a reaction mixture comprising a nucleic acid molecule, a nucleotide analog, and a candidate DNA polymerase to first conditions suitable for a DNA polymerization reaction to occur; detecting the first detectable label in the reaction mixture; and determining a target DNA polymerase based on the detection result, wherein the nucleic acid molecule has a double-stranded region and a 5 '-single-stranded region, the 5' -single-stranded region having double-stranded region contiguous nucleotides; the nucleotide analog is a nucleotide analog as described previously, and the base of the nucleotide analog matches the base of the adjacent nucleotide of the double-stranded region. By using the method according to the embodiment of the present invention, using a nucleic acid molecule as a template, a candidate DNA polymerase or a DNA polymerase that can catalyze the nucleotide analog of the present invention to perform a polymerization reaction at the 3 ' end of the nucleic acid molecule, and detecting a first detectable label in a reaction mixture, a DNA polymerase having a function of catalyzing the polymerization reaction of the nucleotide analog of the present invention is determined based on a change of the first detectable label during the reaction or a difference between before and after the reaction, and the 3 ' -OH end of the nucleic acid analog of the present invention is linked with MRT, so that the DNA polymerase having a function of catalyzing the polymerization reaction of the nucleotide triphosphate having a reversible terminator (MRT) at the 3 ' -OH end and being applicable to SBS can be efficiently screened by using the method according to the embodiment of the present invention.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic diagram of the structure of a nucleic acid molecule according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a nucleic acid molecule according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of a nucleic acid molecule according to an embodiment of the present invention;
FIG. 4 is a schematic diagram illustrating the principle of detecting the activity of a screening body using FRET technique according to an embodiment of the present invention; and
FIG. 5 is a graph of enzyme activity detected by a microplate reader according to an embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Interpretation of terms
It should be noted that, unless otherwise specified, the term "conditions suitable for DNA polymerization" as used herein refers to conventional conditions for allowing nucleotide triphosphates to undergo polymerization under catalysis of DNA polymerase, including reaction system conditions, temperature conditions, pH conditions, etc., for example, the reaction buffer in the reaction system includes 20mM Tris-HCl, 10mM (NH)4)2SO4、10mM KCl、30mM MgSO4The pH value of the reaction buffer solution is 8.8, and the reaction temperature is 40-60 ℃.
The term "5 ' -single-stranded region" in a nucleic acid molecule as used herein refers to a single-stranded region located at the 5 ' end of a nucleic acid molecule, such as the structure of a nucleic acid molecule shown in FIG. 1, FIG. 2 or FIG. 3, the 5 ' -single-stranded region of which is shown.
As used herein, unless otherwise specified, the terms "first," "second," "third," and the like are used for descriptive purposes and are not intended to imply or imply any differences in order or importance between the terms and the like, and are not intended to imply that the terms "first," "second," "third," and the like, are limited to only one component.
Nucleotide analogs
In a first aspect of the invention, a nucleotide analog is provided. According to an embodiment of the present invention, the nucleotide analog has the following structure,
wherein L represents a cleavable linking group; label represents a first detectable Label; p represents a triphosphate group; base represents adenine, guanine, cytosine or thymine. Wherein L and Label constitute a single Modified Reversible Terminator (MRT). According to embodiments of the present invention, by detecting a change in the polymerization reaction of the first detectable label in the nucleotide analog, it can be accurately determined whether the polymerase to be screened has polymerization activity. The nucleotide analogs according to the embodiments of the present invention can be effectively used for screening polymerases having activities of catalyzing polymerization of nucleotide triphosphates having a reversible terminator (MRT) at the 3' -OH terminal and can be used for SBS.
According to an embodiment of the invention, the linking group L has the following structure,
after the nucleotide analog according to the embodiment of the present invention completes the base detection, the above-mentioned linking group can be cleaved, for example, by the action of an organic phosphonate, to realize sequencing-by-synthesis (SBS).
According to an embodiment of the invention, the first detectable label is a biotin label or a fluorescent label. Labeling nucleotide triphosphate with biotin, subsequently capturing the biotin-labeled nucleotide triphosphate with avidin, selecting the fluorescein-labeled avidin, and further determining the base by using a fluorescence reaction; the first detectable label is a fluorescent label, and the base can be determined directly by fluorescence reaction of the fluorescent label.
According to an embodiment of the invention, the nucleotide analogue has the following structure:
the nucleotide analogs according to the embodiments of the present invention utilize biotin to label nucleotide triphosphates, which are subsequently captured with avidin. The detection of the trapped or enriched biotin-labeled nucleotide triphosphate lays a foundation for subsequent further detection and determination. For example, according to an embodiment of the present invention, a polymerase having an activity of catalyzing polymerization of a nucleotide triphosphate having a reversible terminator (MRT) at the 3' -OH terminal and being applicable to SBS can be screened by detecting resonance energy coupling transfer between fluorescent labels using the above-described nucleotide analogs according to an embodiment of the present invention.
Method for screening DNA polymerase
In a second aspect of the present invention, the present invention provides a method of screening for a DNA polymerase. According to an embodiment of the invention, the method comprises: subjecting a reaction mixture comprising a nucleic acid molecule, a nucleotide analog, and a candidate DNA polymerase to first conditions suitable for a DNA polymerization reaction to occur; detecting the first detectable label in the reaction mixture; and determining a target DNA polymerase based on the detection result, wherein the nucleic acid molecule has a double-stranded region and a 5 '-single-stranded region, the 5' -single-stranded region having double-stranded region contiguous nucleotides; the nucleotide analog is a nucleotide analog as described previously, and the base of the nucleotide analog matches the base of the adjacent nucleotide of the double-stranded region. By using the method according to the embodiment of the present invention, using a nucleic acid molecule as a template, a candidate DNA polymerase or a DNA polymerase that can catalyze the nucleotide analog of the present invention to perform a polymerization reaction at the 3 ' end of the nucleic acid molecule, and detecting a first detectable label in a reaction mixture, a DNA polymerase having a function of catalyzing the polymerization reaction of the nucleotide analog of the present invention is determined based on a change of the first detectable label during the reaction or a difference between before and after the reaction, and the 3 ' -OH end of the nucleic acid analog of the present invention is linked with MRT, so that the DNA polymerase having a function of catalyzing the polymerization reaction of the nucleotide triphosphate having a reversible terminator (MRT) at the 3 ' -OH end and being applicable to SBS can be efficiently screened by using the method according to the embodiment of the present invention.
According to the embodiment of the present invention, the nucleic acid molecule is linear, and the two ends of the nucleic acid molecule have a 5' -single-stranded region, respectively, and the structure thereof can be seen in FIG. 1.
According to an embodiment of the invention, both 5' -single stranded regions have the same contiguous nucleotides of the double stranded region.
According to an embodiment of the invention, when the nucleic acid molecule has the structure shown in FIG. 1, the 5' -single stranded region carries a second detectable label.
According to an embodiment of the invention, the nucleic acid molecule is linear and has a 5' -single stranded region at one and only one end, the structure of which can be seen in FIG. 2.
According to an embodiment of the present invention, when the nucleic acid molecule has the structure shown in FIG. 2, the 5 ' -single stranded region and/or the 5 ' end of the nucleic acid strand where the non-5 ' -single stranded region is located carries a second detectable label.
According to an embodiment of the present invention, the nucleic acid molecule is in a loop form, and the nucleic acid molecule has a 5' -single-stranded region, the structure of which can be seen in FIG. 3. According to a specific embodiment of the present invention, the nucleic acid molecule of such a structure may be formed by complementary pairing of a single-stranded circular DNA molecule and a primer.
According to a specific embodiment of the present invention, when the nucleic acid molecule has the structure shown in FIG. 3, the non-5 '-single stranded region carries a second detectable label at the 5' end of the nucleic acid strand.
According to an embodiment of the invention, the detection system of the first detectable label and the second detectable label are arranged for a resonance energy transfer. Further, when the DNA polymerase is capable of catalyzing the ligation of the nucleotide analog of the present invention to the 3' end of the nucleic acid molecule, the detection system of the first detectable label and the second detectable label are in infinite proximity suitable for resonance energy transfer to occur, thereby enabling detection via FRET technology. In other words, if resonance energy transfer can be detected by FRET technology, which proves that the detection system of the first detectable label and the second detectable label are in infinite proximity, it is further proved that the nucleotide analog of the present invention carrying the first detectable label undergoes a polymerization reaction under the action of the candidate DNA polymerase, which is the target DNA polymerase.
According to an embodiment of the invention, the separation of the detection system of the second detectable label and the first detectable label is not more than 30 nt. In other words, when the nucleic acid molecule has the structure of FIG. 1, the second detectable label on each strand is separated from the detection system for the first detectable label by no more than 30nt or by no more than 30nt in length of the 5' -single-stranded region; when the nucleic acid molecule has the structure of FIG. 2, the 5 '-single stranded region has a length of not more than 30nt, or the nucleic acid strand in which the non-5' -single stranded region is located has a detection system in which the second detectable label is spaced from the first detectable label by not more than 30 nt; when the nucleic acid molecule has the structure shown in FIG. 3, the second detectable label on the nucleic acid strand having the non-5' -single-stranded region is spaced from the detection system for the first detectable label by not more than 30nt, and when the nucleic acid molecule is formed by complementary pairing of a single-stranded circular DNA molecule and a primer, the length of the primer should be not more than 30 nt. The inventors have found that, when the distance between the detection systems of the second detectable label and the first detectable label is not more than 30nt, the detection systems of the second detectable label and the first detectable label are located at positions where resonance energy cotransfer can occur and a resonance energy cotransfer signal can be effectively detected, and whether or not the DNA polymerase to be detected has an activity of catalyzing polymerization of a nucleotide triphosphate having a reversible terminator (MRT) at the 3 '-OH end can be determined by detecting the resonance energy cotransfer signal, and if the resonance energy cotransfer signal can be detected, the DNA polymerase to be detected has an activity of catalyzing polymerization of a nucleotide triphosphate having a reversible terminator (MRT) at the 3' -OH end.
According to an embodiment of the invention, the distance between the detection systems of the second detectable label and the first detectable label is 15 to 30 nt. The inventor finds that the interval between the detection systems of the second detectable label and the first detectable label is 15-30 nt, when the DNA polymerase to be detected has the activity of catalyzing the polymerization reaction of the triphosphate nucleotide of which the 3' -OH end is provided with the reversible terminator (MRT), the signal interference between the two detectable labels can be effectively avoided, and the detected resonance energy continuous transfer signal is more real and reliable.
According to an embodiment of the present invention, the first detectable label is biotin, the detection system of the first detectable label comprises streptavidin carrying a first fluorescent label, and the second detectable label is a second fluorescent label, and the first fluorescent label and the second fluorescent label are suitable for resonance energy coupling transfer.
According to an embodiment of the present invention, the first fluorescent marker is one of cy5 and cy3, and the second fluorescent marker is the other of cy5 and cy 3.
According to a specific embodiment of the present invention, the first fluorescent label is cy3 and the second fluorescent label is cy 5.
According to an embodiment of the invention, the nucleic acid molecule is obtained by: the single-stranded nucleic acid carrying the cy5 at the 5' end was subjected to an annealing reaction at 85 ℃ for 10 min. Further, complementary pairing between single-stranded nucleic acids carrying cy5 at the 5' -end resulted in a nucleic acid molecule having a double-stranded region.
According to a specific embodiment of the present invention, the single-stranded nucleic acid has a nucleotide sequence represented by SEQ ID NO. 1-2.
CGTGTATGCGTAATAGGATCCCGACTCACTATGGACG(SEQ ID NO:1)。
CGTGTATCGTCCATAGTGAGTCGGGATCCTATTACGC(SEQ ID NO:2)。
Further, the nucleotide sequence of one strand of the double-stranded region of the nucleic acid molecule formed after annealing of the single-stranded nucleic acid molecule having the above nucleotide sequence was GCGTAATAGGATCCCGACTCACTATGGACG, and the nucleotide sequence of the 5' -single-stranded region of the formed nucleic acid molecule was CGTGTAT.
According to an embodiment of the present invention, the molar ratio of the single-stranded nucleic acids having the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 is 1: 1. Further improves the annealing efficiency and the matching success rate of single-stranded nucleic acid, and obviously improves the proportion of nucleic acid molecules with double-stranded regions and 5' -single-stranded regions in the annealing product.
According to an embodiment of the present invention, the DNA polymerization reaction is performed by: 1) subjecting a reaction mixture comprising a first reaction system to a reaction at 65 ℃ for 10min, 2) contacting the reaction mixture obtained in step 1) with cy3 fluorescently labeled streptavidin to obtain a second reaction system, and 3) reacting the reaction mixture comprising the second reaction system at 40 ℃ for 3 hours; wherein, based on 50 μ L of the first reaction system, the first reaction system comprises the following components: waiting for 20 μ LSelecting DNA polymerase solution, 0.25 μ M nucleotide analogue shown in formula (I), 2.5 μ M dTTP, 2.5 μ M dCTP, 2.5 μ M dGTP, 0.1 μ M nucleic acid molecule and enzyme reaction buffer solution, wherein the enzyme reaction buffer solution comprises 20mM Tris-HCl, 10mM (NH-HCl)4)2SO4、10mM KCl、30mM MgSO4The pH of the enzyme reaction buffer was 8.8. According to the DNA polymerization reaction of the embodiment of the invention, under the reaction conditions and the reaction mode, incubation is carried out at 65 ℃ for 10min, the activity of the DNA polymerase can be effectively excited, the rate of enzyme polymerization MRT modification of dNTP is accelerated, and then the reaction temperature is reduced to 40 ℃ for reaction for 3 hours, so that the reaction rate of the enzyme can be properly reduced, and the signal detection in the reaction process is enhanced.
According to an embodiment of the invention, the detection is performed by: detecting once every 5min in the process of the step 3) of the DNA polymerization reaction under the detection conditions that the excitation wavelength is 530nm and the emission wavelength is 676nm by using a BioTeK microplate reader to obtain a curve of the change of the fluorescence value of the cy5 along with time.
According to an embodiment of the invention, the method further comprises: placing a reaction mixture containing the nucleic acid molecule, dTTP, dCTP, dGTP, dATP with biotin-linked bases, and Taq DNA polymerase under second conditions suitable for DNA polymerization to occur to obtain a positive control reaction system; placing a mixture containing the nucleic acid molecule, dTTP, dCTP, dGTP, the nucleotide analogue represented by the formula (I) and Taq DNA polymerase under a third condition suitable for DNA polymerization to obtain a negative control reaction system; the first, second, and third conditions are the same. Taq DNA polymerase is known to have the activity of catalyzing the polymerization reaction of dNTP with a base connected with biotin and a 3' -OH unconnected group, so that the DNA polymerization reaction is determined to occur in a positive control system; taq DNA polymerase is known to have no activity to catalyze the polymerization of the nucleotide analogs described herein, and therefore, the negative control system was determined not to have a DNA polymerization reaction. In the process of the method for screening the DNA polymerase, a positive control reaction system and a negative control reaction system are simultaneously arranged, so that the screening result is more real and credible.
According to an embodiment of the present invention, a rate of change of the fluorescence value of cy5 that is greater than 0 is indicative that the candidate DNA polymerase is a target DNA polymerase. As described above, if the resonance energy transfer can be detected by the FRET technique, it is confirmed that the detection system of the first detectable label and the second detectable label are in infinite proximity, and it is further confirmed that the nucleotide analog of the present invention carrying the first detectable label undergoes a polymerization reaction by the candidate DNA polymerase, which is the target DNA polymerase. When the first detection marker is cy3 and the second detection marker is cy5, during the FRET detection, if resonance energy transfer occurs between cy3 and cy5, the fluorescence value of cy5 is increased, and the fluorescence value of cy3 is decreased, namely the fluorescence value increase rate of cy5 is greater than 0, or the fluorescence value decrease rate of cy3 is greater than 0, or the fluorescence value of cy5 is decreased, and the fluorescence value of cy3 is increased, namely the fluorescence value decrease rate of cy5 is greater than 0, or the fluorescence value increase rate of cy3 is greater than 0, the candidate DNA polymerase is proved to have the activity of catalyzing the polymerization reaction of the nucleotide analogue disclosed by the invention.
More particularly, in a final aspect of the invention, the invention provides an ex vivo method for rapid, efficient, high throughput screening of DNA polymerases capable of polymerization using MRT-modified nucleoside triphosphates. The method comprises the following steps: the method comprises the following steps of performing a screening reaction by using a Cy5 fluorescence-labeled template, dATP modified by Biotin Biotin-labeled MRT and streptavidin labeled by Cy3, and detecting the activity of a screening body by using a FRET (fluorescence resonance energy transfer) technology, wherein the principle of the method can be seen in figure 4, and the method mainly comprises the following steps:
1) looking up literature or related data to determine specific information and gene sequence of DNA polymerase to be screened, and searching corresponding crystal structure information in PDB protein database;
2) according to the crystal result information of the selected DNA polymerase, combining bioinformatics, utilizing related simulation software to properly adjust the crystal structure, and performing stability simulation on the adjusted result structure to establish a final structure, and utilizing the established structure to predict and simulate mutants to construct a simulated mutant library;
3) selecting a proper expression system according to the selected DNA polymerase and the gene sequence thereof, constructing an expression vector, carrying out a preliminary expression test, simultaneously testing whether the expression system is suitable for screening, adjusting the system according to an experimental result, and constructing the expression system suitable for screening;
4) selecting a proper experimental method for constructing the mutant according to the results of computer simulation and screening, and preparing a real-time experimental screening mutant library;
5) a small amount of mutant screening experiments are carried out by utilizing the constructed screening expression system to establish a complete high-throughput screening method, and the screening method is optimized, so that the method can meet various requirements of carrying out polymerization reaction DNA polymerase screening by utilizing MRT modified nucleoside triphosphates, and the requirements of simple operation and good stability are met;
6) inducing expression of a large number of screening bodies and preparing corresponding crude enzyme liquid (enzyme liquid without purification treatment);
7) screening the crude enzyme solution in vitro of the screening library according to the optimized screening method and experimental conditions, and detecting the polymerase activity of the screened body by utilizing an enzyme-labeling instrument with a fluorescence detection system
8) And (3) carrying out pair analysis on the detection result of the microplate reader, carrying out further enzyme activity analysis after the experimental effectiveness is determined, thus selecting an effective screening body, carrying out sequencing, determining the complete gene sequence of the screening body, and obtaining the specific information of the mutation point position so as to carry out the next experiment or deeper prediction and modification.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are conventional products which are commercially available, not indicated by the manufacturer, for example from NEB.
Examples
In the following examples, DNA polymerase polymerization activity was detected using a MRT-modified Biotin-labeled dATP (compound represented by formula (I)) (hereinafter referred to as 3 '-dATP-Biotin) and a Biotin-avidin system, wherein dATP-Biotin used for positive control means that Biotin is linked to the base position of dATP and MRT is not linked to 3' -OH.
1. Preparation of crude enzyme solution containing DNA polymerase screening Material
The preparation process refers to the preparation process of the crude enzyme solution in patents PCT/CN2017/070609 and CN201710287578.3, so as to obtain the crude enzyme solution containing the DNA polymerase to be screened (DNA polymerase screening body).
2. Activity detection of screening body
In this example, a reaction test is performed by using MRT modified Biotin labeled dATP, Cy3 fluorescently labeled streptavidin (streptavidin-Cy3), and Cy5 fluorescently labeled DNA template (template DNA-Cy5), and the relative reaction rate of the polymerase of the screening agent is detected by using a microplate reader, and the specific experimental method is as follows:
single-stranded primers with a 5' Cy5 fluorescent label:
S1A (5' -CGTGTATGCGTAATAGGATCCCGACTCACTATGGACG) and
S2A(5’-CGTGTATCGTCCATAGTGAGTCGGGATCCTATTACGC)
according to the following steps: 1, mixing the obtained product with the same molar concentration, naturally cooling the obtained product to room temperature for annealing after 10min at the temperature of 85 ℃, and storing the annealed product to-20 ℃ in a dark place to obtain the Cy5 fluorescence labeled template DNA-Cy 5.
The enzyme activity was detected by using a BioTek microplate reader, and the reaction was carried out in 384 plates (Corning black, clear bottom384 plates) in a total volume of 50. mu.L.
The reaction system is as follows: 20 μ L of the crude enzyme solution from the screening, 0.25 μ M of 3' -dATP-Biotin, 2.5 μ M dTTP, 2.5 μ M dCTP, 2.5 μ M dGTP, 0.1 μ M template DNA-Cy5, enzyme reaction buffer 20mM Tris-HCl, 10mM (NH)4)2SO4、10mM KCl、30mM MgSO4、pH 8.8，25℃。
This example uses dATP (thermo Fisher scientific) (dATP-Biotin) and Taq DNA polymerase labeled with commercial Biotin as positive controls (positive control) (Taq DNA polymerase can perform normal polymerization reaction using dATP-Biotin), and 3 '-dATP-Biotin and Taq DNA polymerase as negative controls (negative control) (Taq DNA polymerase confirms that normal polymerization reaction using 3' -dATP-Biotin is not possible).
The reaction conditions are as follows: preparing a reaction solution according to the reaction system, adding crude enzyme solution containing screen polymerase or Taq DNA polymerase finally, reacting at 65 ℃ for 10min, placing the reaction solution on ice, adding 0.25 mu M streptavidin (streptavidin) -Cy3, and detecting once every 5min by using a BioTek enzyme-labeling instrument according to an enzyme reaction kinetics detection mode, wherein the detection conditions are as follows:
after the reaction is finished, a data table or an enzyme activity curve can be directly derived, and the reaction rate of the relative fluorescence value can be approximately calculated.
3. Data analysis
The enzyme activity curve detected by the microplate reader is shown in FIG. 5
(1) In the embodiment, the size of a target fluorescence value Cy5 is detected by using a FRET method, and in an enzyme activity curve diagram, the larger the rising amplitude and the larger the speed of a screened enzyme activity curve are, the stronger the polymerization activity of the selected enzyme activity curve in the process of modifying the dATP marked by the Biotin by using MRT is;
(2) in addition to observing the curve plot, a linear fit can be performed on the curve, the reaction rates between the screening entities are relatively compared, and the formula is: y is kx + b, wherein y is the fluorescence value; k is the relative speed of the rise of the fluorescence value; b is the intercept. The greater the k value in the same set of screeners, the better the polymerase activity relative to the screener
(3) From the results in FIG. 5, it can be seen that K159 was more active than B9 in polymerization using MRT-modified Biotin-labeled dATP. (wherein K159 and B9 are codes of two DNA polymerases having catalytic activity selected in the experiment)
In summary, in combination with the above specific examples, the method and the technical scheme for screening DNA polymerase described in the present application have the following advantages:
1) no pollution: the materials used in the invention can be purchased from commercial approaches, organic synthesis is not needed, and the use of radioactive substances is not involved; in addition, the expression system adopted by screening is a common escherichia coli prokaryotic expression system in molecular operation, does not need special treatment and has no pollution to the environment;
2) feasibility: the structure or sequence of the template substrate used for screening the modified or modified DNA polymerase has no special requirements, and the template substrate can be commercially synthesized, has small template dosage, simple and convenient design and is easy to realize;
3) simplicity: the method can adjust the experimental steps at any time according to different experimental requirements, can directly utilize the crude enzyme solution for experiment, has simple and convenient preparation of the crude enzyme solution, only needs to utilize lysozyme for cracking, does not need the steps of centrifugation and the like, has simple and easy operation, and does not need complicated operations such as purification and the like; in addition, the polymerization reaction is simpler than PCR reaction, no special instrument is needed, the reaction can be carried out at normal temperature, and different reaction temperatures can be set according to the properties of different enzymes
4) High flux: the method can directly utilize a micro 96-well or 384-well plate to directly carry out an expression test experiment, utilizes an enzyme-labeling instrument to carry out automatic detection, can simultaneously carry out a plurality of plate experiments according to the screening amount, has considerable experimental results, can be directly used and does not need to carry out additional processing on data.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen Huashengshengsciences institute
<120> nucleotide analogs and method for screening DNA polymerase
<130>PIDC3182995
<160>2
<170>PatentIn version 3.3
<210>1
<211>37
<212>DNA
<213>Artificial
<220>
<223> nucleotide sequence of Single-stranded nucleic acid
<400>1
cgtgtatgcg taataggatc ccgactcact atggacg 37
<210>2
<211>37
<212>DNA
<213>Artificial
<220>
<223> nucleotide sequence of Single-stranded nucleic acid
<400>2
cgtgtatcgt ccatagtgag tcgggatcct attacgc 37
Claims (10)
1. A nucleotide analog having the structure,
wherein the content of the first and second substances,
l represents a cleavable linking group;
label represents a first detectable Label;
p represents a triphosphate group;
base represents adenine, guanine, cytosine or thymine;
preferably, the cleavable linking group L has the following structure,
optionally, the first detectable label is a biotin label or a fluorescent label.
2. A method of screening for a DNA polymerase comprising:
subjecting a reaction mixture comprising a nucleic acid molecule, a nucleotide analog, and a candidate DNA polymerase to first conditions suitable for a DNA polymerization reaction to occur;
detecting the first detectable label in the reaction mixture; and
determining a target DNA polymerase based on the detection result,
wherein the content of the first and second substances,
the nucleic acid molecule has a double-stranded region and a 5 '-single-stranded region, the 5' -single-stranded region having double-stranded region contiguous nucleotides;
the nucleotide analog is the nucleotide analog of claim 1, and the base of the nucleotide analog matches the base of the adjacent nucleotide of the double-stranded region.
3. The method according to claim 2, wherein the nucleic acid molecule is linear, and has a 5' -single-stranded region at each end;
optionally, both 5' -single stranded regions have the same contiguous nucleotides of the double stranded region;
optionally, the 5' -single stranded region carries a second detectable label.
4. The method of claim 2, wherein the nucleic acid molecule is linear, having one and only one 5' -single stranded region at one end;
optionally, the 5 ' -single stranded region and/or the 5 ' end of the nucleic acid strand in which the non-5 ' -single stranded region is located carries a second detectable label.
5. The method of claim 2, wherein said nucleic acid molecule is of the circular form, said nucleic acid molecule having a 5' -single stranded region;
optionally, the non-5 '-single stranded region is located on the nucleic acid strand at the 5' end of which carries a second detectable label.
6. The method of any one of claims 3 to 5, wherein the detection system for the first detectable label and the second detectable label are configured to allow resonance energy transfer;
optionally, the second detectable label and the detection system of the first detectable label are separated by no more than 30 nt;
preferably, the interval between the detection systems of the second detectable label and the first detectable label is 15-30 nt.
7. The method of claim 6, wherein the first detectable label is biotin, the detection system of the first detectable label comprises streptavidin carrying a first fluorescent label, the second detectable label is a second fluorescent label, and the first fluorescent label and the second fluorescent label are suitable for resonance energy coupling transfer;
optionally, the first fluorescent marker is one of cy5 and cy3 and the second fluorescent marker is the other of cy5 and cy 3;
preferably, the first fluorescent label is cy3 and the second fluorescent label is cy 5.
8. The method according to claim 3, wherein the nucleic acid molecule is obtained by:
carrying out annealing reaction on the single-stranded nucleic acid with the cy5 at the 5' end, wherein the annealing reaction is carried out at 85 ℃ for 10 min;
optionally, the single-stranded nucleic acid has a nucleotide sequence shown as SEQ ID NO 1-2;
preferably, the molar ratio of the single-stranded nucleic acids having the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 is 1: 1.
9. The method of claim 7, wherein the DNA polymerization is performed by:
1) the reaction mixture containing the first reaction system is placed at 65 ℃ for reaction for 10min,
2) contacting the reaction mixture obtained in step 1) with cy3 fluorescently labeled streptavidin to obtain a second reaction system, an
3) Reacting the reaction mixture containing the second reaction system at 40 ℃ for 3 hours;
wherein, based on 50 μ L of the first reaction system, the first reaction system comprises the following components:
20. mu.L of a candidate DNA polymerase solution, 0.25. mu.M of the nucleotide analog represented by the formula (I), 2.5. mu.M of dTTP, 2.5. mu.M of dCTP, 2.5. mu.M of dGTP, 0.1. mu.M of a nucleic acid molecule and an enzyme reaction buffer,
wherein the enzyme reaction buffer comprises 20mM Tris-HCl, 10mM (NH)4)2SO4、10mM KCl、30mM MgSO4The pH of the enzyme reaction buffer solution is 8.8;
optionally, the detecting is performed by:
detecting once every 5min in the process of the step 3) of the DNA polymerization reaction by using a BioTeK enzyme-labeling instrument under the detection conditions that the excitation wavelength is 530nm and the emission wavelength is 676nm to obtain a curve of the change of the fluorescence value of cy5 along with time;
preferably, further comprising: placing a reaction mixture containing the nucleic acid molecule, dTTP, dCTP, dGTP, dATP with biotin-linked bases, and Taq DNA polymerase under second conditions suitable for DNA polymerization to occur to obtain a positive control reaction system;
placing a mixture containing the nucleic acid molecule, dTTP, dCTP, dGTP, the nucleotide analogue represented by the formula (I) and Taq DNA polymerase under a third condition suitable for DNA polymerization to obtain a negative control reaction system;
the first, second, and third conditions are the same.
10. The method of claim 9, wherein a rate of change of the fluorescence value of cy5 that is greater than 0 is indicative that the candidate DNA polymerase is a target DNA polymerase.
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