CN118126982A - Light-operated DNA polymerase, nucleic acid fragment, recombinant vector, recombinant cell and application - Google Patents
Light-operated DNA polymerase, nucleic acid fragment, recombinant vector, recombinant cell and application Download PDFInfo
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- CN118126982A CN118126982A CN202410572684.6A CN202410572684A CN118126982A CN 118126982 A CN118126982 A CN 118126982A CN 202410572684 A CN202410572684 A CN 202410572684A CN 118126982 A CN118126982 A CN 118126982A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1264—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07031—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention discloses light-operated DNA polymerase, nucleic acid fragments, recombinant vectors, recombinant cells and application, and relates to the technical field of biology. The light-operated DNA polymerase comprises a terminal deoxynucleotide transferase and cpLOV < 2 >, and the amino acid sequence of the terminal deoxynucleotide transferase is shown as SEQ ID NO:1, said cpLOV2 is inserted between the phenylalanine residue 202 and serine residue 203 of said terminal deoxynucleotidyl transferase. The light-operated DNA polymerase can show remarkably differentiated enzyme activity under different illumination conditions, the activity difference reaches hundreds of times under blue light illumination and darkness conditions, reversible optical regulation of catalytic activity is realized, and the light-operated DNA polymerase has the characteristics of high catalytic efficiency, high stability and strong controllability, and has wide application prospect.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to light-operated DNA polymerase, nucleic acid fragments, recombinant vectors, recombinant cells and application.
Background
DNA artificial synthesis technology is an important basis for modern genetic technology. The key methods for DNA artificial synthesis include: column chemical oligonucleotide synthesis, chip chemical oligonucleotide synthesis, oligonucleotide purification, oligonucleotide assembly, gene synthesis error correction and clone screening, large fragment gene synthesis assembly, and new generation enzymatic synthesis of DNA. Traditional methods for DNA synthesis mostly rely on phosphoramidites to complete chemical reactions. In recent years, the third generation of DNA artificial synthesis based on the principle of enzymatic synthesis is gradually rising, and the method becomes a DNA artificial synthesis method with wide prospects. Among them, the enzymatic synthesis technique using terminal deoxynucleotidyl transferase (TdT) as a core is a very promising strategy for DNA synthesis.
The novel enzymatic synthesis of nucleotides is in vitro oligonucleotide fragment synthesis using a template independent TdT. TdT was first discovered by Bollum and suggested that this enzyme can be used in the synthesis of single stranded oligonucleotides. Subsequent Schott and Schrade studies found that TdT has little bias on four nucleotides, high coupling efficiency, and continuous synthesis and extension of single stranded DNA can produce homopolymers up to 8000 nt. To achieve TdT-catalyzed controlled DNA synthesis, the activity of TdT needs to be reversibly controlled. TdT catalytic activity control mechanism constructed in 2018 by Keasing team utilizes reversible covalent bond connection of TdT and single nucleotide to prevent further extension of DNA chain synthesized by TdT, and when the reversible covalent bond is broken, the DNA chain can enter into new nucleotide addition cycle. The average coupling efficiency of the method can reach 97.7%, and only 2-3 min is needed for a single cycle. DNA synthesis based on innovative TdT has received general attention in academia and industry. Because of the increasingly remarkable DNA synthesis application of TdT, the development of enzyme engineering design aiming at TdT to realize flexible regulation of TdT enzyme activity has important significance.
The existing light-operated DNA polymerase (such as Chinese patent CN 116286722A) with TdT activity regulated and controlled by cpLOV is an advanced biological enzyme synthesis mode which does not depend on deprotection and is controlled by light, has the advantages of high catalytic efficiency, high stability and strong controllability, and is more beneficial to the application of DNA synthesis. Although the traditional artificial ultraviolet or chemical reagent deprotection step is replaced preliminarily, the reaction step is reduced, but the catalytic activity difference of the DNA polymerase under dark and blue light irradiation conditions is not obvious enough, only about 10 times, and the DNA polymerase has a revolutionary significance if the catalytic difference under dark and blue light irradiation conditions can be further improved.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
Based on the defects of the prior art, the invention aims to provide light-operated DNA polymerase, nucleic acid fragments, recombinant vectors, recombinant cells and application, and aims to solve the problem that the catalytic activity difference of the existing light-operated DNA polymerase with TdT activity regulated and controlled by optics by cpLOV under dark and blue light irradiation conditions is not obvious enough.
The technical scheme of the invention is as follows:
In a first aspect of the present invention, there is provided an optically controlled DNA polymerase, wherein the optically controlled DNA polymerase comprises TdT and cpLOV < 2 >, and the amino acid sequence of TdT is as set forth in SEQ ID NO:1, said cpLOV2 is inserted between phenylalanine residue 202 and serine residue 203 of said TdT.
Alternatively, cpLOV2 consists of the core domain PAS and the jα helix.
Alternatively, the jα helix becomes a loose flexible structure under blue light irradiation conditions, and the loose flexible structure becomes a jα helix under dark conditions.
Optionally, the amino acid sequence of cpLOV is as shown in SEQ ID NO: 2.
Optionally, the amino sequence of the optically controlled DNA polymerase is as set forth in SEQ ID NO: 3.
In a second aspect of the invention, there is provided a nucleic acid fragment, wherein the nucleic acid fragment comprises a nucleotide sequence encoding the optically controlled DNA polymerase of the invention as described above.
In a third aspect of the present invention, there is provided a recombinant vector comprising a nucleic acid fragment according to the present invention as described above.
In a fourth aspect of the invention, there is provided a recombinant cell comprising a recombinant vector of the invention as described above.
In a fifth aspect, the present invention provides the use of an optically controlled DNA polymerase of the invention as described above, a DNA polymerase expressed from a nucleic acid fragment of the invention as described above, a DNA polymerase expressed from a recombinant vector of the invention as described above, or a DNA polymerase expressed from a recombinant cell of the invention as described above, for catalyzing a DNA synthesis reaction.
Alternatively, the optically controlled DNA polymerase catalyzes the repeated addition of deoxynucleotides to the 3 'hydroxyl end of single stranded DNA or the 3' hydroxyl end of double stranded DNA.
The beneficial effects are that: the light-operated DNA polymerase provided by the invention can show remarkably differentiated enzyme activity under different illumination conditions, the activity difference under blue light illumination and darkness conditions reaches hundreds of times (the activity under blue light condition is 106.14 times under darkness condition), reversible optical regulation of catalytic activity is realized, and the light-operated DNA polymerase has the characteristics of high catalytic efficiency, high stability and strong controllability, and has wide application prospect. The invention utilizes the optical control technology of the light-operated DNA polymerase activity, and improves the limitations of poor controllability, more synthetic reaction steps and dependence on the deprotection process when the traditional DNA polymerase synthesizes DNA. Compared with a biosynthesis method, the invention uses the optical control technology of the light-operated DNA polymerase activity to preliminarily replace the traditional artificial ultraviolet or chemical reagent deprotection step, reduces the reaction steps, improves the synthesis efficiency, reduces the synthesis cost and provides a new innovation strategy for the controllable synthesis of the DNA polymerase.
Drawings
In FIG. 1, a is a schematic structural diagram of the light-operated DNA polymerase, and b is a crystal structure diagram of the light-operated DNA polymerase.
FIG. 2 is a SDS-PAGE diagram of optically controlled DNA polymerase prepared in example 1 of the present invention.
FIG. 3 is a gel diagram of modified urea PAGE of the product of the catalytic reaction using optically controlled DNA polymerase in example 2 of the present invention.
FIG. 4 is a graph showing the catalytic rate of light-operated DNA polymerase in example 3 of the present invention.
Detailed Description
The invention provides light-operated DNA polymerase, nucleic acid fragments, recombinant vectors, recombinant cells and application thereof, and the invention is further described in detail below for the purpose, technical scheme and effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
At present, in the existing bioenzyme synthesis technology, tdT has the advantages of high activity, high reaction efficiency and the like in the in-vitro oligonucleotide fragment synthesis reaction, but how to control the reaction initiation and termination of TdT and synthesize target fragments is still a difficult problem. This is because TdT has a high enzymatic activity and a low substrate specificity, which makes it impossible to directly synthesize natural deoxyribonucleotide substrates but to react with chemically modified artificial substrates. Essentially these substrates carry a reaction terminating group at the 3 'end (3' -OR, where R can be-CH 3-N3、-ONH2, etc.), allowing the reaction to terminate immediately after a single reaction has increased by one deoxyribonucleotide unit. Only the chemical groups of the last deoxyribonucleotide are removed by ultraviolet irradiation or chemical agent to continue the next round of reaction. The TdT mediated cyclic enzymatic synthesis method also has the advantages of increased cost of enzymatic DNA synthesis due to the need of artificial deprotection, longer synthesis period and very limited future application prospect. Therefore, the existing TdT-based DNA synthesis still needs multiple steps to be realized, and the automation degree of the enzymatic DNA synthesis is limited. In patent CN116286722a prior to the inventors, a DNA polymerase (denoted p 200-21) has been provided whose activity to intercalate TdT by cpLOV2 photoprotein can be controlled by a light signal, and whose difference in activity under dark and blue light irradiation can reach about 10-fold. However, compared with the application technology of DNA synthesis, the activity difference of the DNA polymerase still has the limitations of speed and accuracy in application, and needs to be further improved. Based on the activation characteristic of external physical signals to the polymerase and the reactive active site and the reversible inhibition characteristic of the reactive site after the reaction, the invention realizes the optical switch regulation of the DNA polymerase activity on a molecular level, fuses cpLOV photosensitive protein domain with TdT key active regulation site, realizes the reversible optical regulation of TdT reaction rate (or activity, reactivity, catalytic activity or catalytic rate) by utilizing the conformational change of cpLOV2 under the blue light irradiation and the dark condition, provides the light-operated DNA polymerase with higher activity and larger difference of the reactivity under the blue light irradiation and the dark condition, that is, the catalytic activity of TdT is regulated by controlling the change of cpLOV protein domain, thus realizing the reversible optical regulation of DNA polymerase conformation and its catalytic activity, and further eliminating the need of relying on protecting agent and deprotecting agent in DNA synthesis process. Specifically, the embodiment of the invention provides a full genetic coding light-operated DNA polymerase (a chimeric body of cpLOV and TdT, which is a light-operated protein formed by completely depending on amino acid arrangement and combination and protein space folding), wherein the light-operated DNA polymerase comprises TdT and cpLOV2, and the amino acid sequence of the TdT is shown as SEQ ID NO:1, said cpLOV2 is inserted between phenylalanine residue 202 and serine residue 203 of said TdT.
The light-operated DNA polymerase provided by the embodiment of the invention can show remarkably different enzyme activities under different illumination conditions, the activity difference under blue light illumination and darkness conditions is hundreds of times (the activity under blue light condition is 106.14 times under darkness condition) which is ten times of the activity difference of the DNA polymerase provided by the inventor before, the reversible optical regulation of the catalytic activity is realized, and the light-operated DNA polymerase has the characteristics of high catalytic efficiency, high stability and strong controllability (namely more sensitive light-operated performance) and has wide application prospect. The invention utilizes the optical control technology of the light-operated DNA polymerase activity, and improves the limitations of poor controllability, more synthetic reaction steps and dependence on the deprotection process when the traditional DNA polymerase synthesizes DNA. Compared with a biosynthesis method, the invention uses the optical control technology of the light-operated DNA polymerase activity to preliminarily replace the traditional artificial ultraviolet or chemical reagent deprotection step, reduces the reaction steps, improves the synthesis efficiency, reduces the synthesis cost, is more environment-friendly, and provides a new revolutionary strategy for the controllable synthesis of DNA polymerase.
In this example, the optically controlled DNA polymerase consists of TdT (a template independent terminal deoxynucleotidyl transferase) and a light sensitive protein cpLOV whose conformation is regulated by blue light. Wherein TdT may be added repeatedly to the 3' hydroxyl end of single-stranded or double-stranded DNA by catalytic deoxynucleotides. Enzymatic reactions of TdT require a short sequence containing at least 3 bases as primers. Another protein domain used in this example is the Light-oxygen-inductance domain (LOV) whose conformation is modulated by blue Light. This is a photosensitive protein domain that is widely found in algae, oats. In this example, the modified protein cpLOV2 after the optimal circular arrangement of LOV subunits (circular permutation) was used as a photoreceptor protein. cpLOV2 is composed of the core domain PAS and the J.alpha.helix (J.alpha.Helix). Under the irradiation of 420nm blue light, the noncovalent action between the core domain of cpLOV and the cofactor FMN is converted into covalent action, so that the J alpha helix at the N end of the ligand is uncoiled, namely a loose flexible structure is changed, and the loose flexible structure is changed into J alpha helix under dark conditions. Thus, this example uses conformational changes of proteins under blue light irradiation and darkness to alter the effector of jα attached to the N-terminus by embedding the photoreceptor protein cpLOV in TdT. When the light-operated DNA polymerase is under the blue light condition, the reactive center is in an open state; when the light-operated DNA polymerase is under dark condition, the catalytic activity of the catalytic active center of the DNA polymerase is inhibited, and the catalytic rate is reduced. The change of external physical illumination causes the conformational change of the photosensitive protein, and finally the conformational change is expressed as the controllability of the catalytic activity of the optically controlled DNA polymerase which is regulated and controlled by optical reversibility.
In addition, the active center of the optically controlled DNA polymerase in this example was in an "on" state under blue light conditions, and in an "off" state under dark conditions, as opposed to p 200-21.
Because of the complexity of combining the protein active site and the small molecular substrate, the embodiment combines a method of computer-aided protein design, establishes a molecular dynamics model, is based on an activity and conformation controllable protein design strategy, and obtains the light-controlled DNA polymerase with high catalytic efficiency, high stability and strong controllability through a high-flux enzyme screening platform. Specifically, the present example uses seamless cloning technology to amplify and ligate fragment vectors by constructing a plasmid gene sequence map of the protein. Transferring the plasmid vector into escherichia coli, amplifying the escherichia coli, inducing protein expression, and finally purifying target protein by utilizing an affinity screening mode.
In the invention, the amino acid sequence of TdT is shown in SEQ ID NO:1, it is specifically ZaTdT, i.e. TdT of avian origin. Generally speaking, embedding cpLOV2 in ZaTdT reduces the catalytic rate, but the catalytic rate of the DNA polymerase provided by the embodiment of the invention under the condition of blue light irradiation is relatively close to ZaTdT, which is more beneficial to DNA sequence synthesis.
In some embodiments, the cpLOV amino acid sequence is set forth in SEQ ID NO: 2.
In some embodiments, the optically controlled DNA polymerase has an amino sequence set forth in SEQ ID NO: 3.
The embodiment of the invention also provides a nucleic acid fragment, wherein the nucleic acid fragment comprises a nucleotide sequence for encoding the light-operated DNA polymerase.
The embodiment of the invention also provides a recombinant vector, wherein the recombinant vector contains the nucleic acid fragment disclosed in the embodiment of the invention. By way of example, the recombinant vector may be a recombinant plasmid containing the nucleic acid fragment of the embodiment of the present invention as described above.
The embodiment of the invention also provides a recombinant cell, wherein the recombinant cell contains the recombinant vector disclosed by the embodiment of the invention. By way of example, the recombinant cell may be a recombinant E.coli cell containing the recombinant vector of the embodiments of the invention as described above.
The embodiment of the invention also provides an application of the light-operated DNA polymerase disclosed in the embodiment of the invention, the DNA polymerase obtained by expressing the nucleic acid fragment disclosed in the embodiment of the invention, the DNA polymerase obtained by expressing the recombinant vector disclosed in the embodiment of the invention or the DNA polymerase obtained by expressing the recombinant cell disclosed in the embodiment of the invention in catalyzing DNA synthesis reaction.
In some embodiments, the optically controlled DNA polymerase catalyzes the repeated addition of deoxynucleotides to the 3' hydroxyl end of single stranded DNA or the 3' hydroxyl end of double stranded DNA, specifically, such as the 3' hydroxyl end of oligonucleotides, and the like.
The invention is further illustrated by the following specific examples.
Example 1
The preparation method of the light-operated DNA polymerase (TdT-cpLOV 2) comprises the following steps:
(1) TdT-cpLOV2 screening library construction
A TdT-cpLOV2 plasmid gene map embedded in cpLOV2 is constructed through Snapgene (the amino acid sequence of TdT is shown as SEQ ID NO:1, and the amino acid sequence of cpLOV2 is shown as SEQ ID NO: 2). PCR reaction with KOD DNA polymerase: the reaction started at 94 ℃ (5 min), followed by 30 cycles of 94 ℃ (20 s), annealing temperature 63 ℃ (1.5 min), and finally extension at 72 ℃ (5 min). The PCR product was digested with DpnI and transformed into competent E.coli BL21 (DE 3) cells, and after overnight, the monoclonal was picked up in a petri dish.
(2) Expression and purification of TdT-cpLOV2
The monoclonal mutation was selected and incubated in 200mL of LB medium containing 100. Mu.g/mL kanamycin for 3 hours to an absorbance (OD) value of 0.6. IPTG (isopropyl-. Beta. -d-thiogalactose) was added to a final concentration of 0.5mM. Induced at 230rpm and 16℃overnight. Cells were collected by centrifugation at 6000g for 10 min and then resuspended in 50mL lysis buffer (30 mM Tris-HCl buffer, 500mM NaCl,20mM imidazole). Cells were lysed by high pressure homogeniser and centrifuged at 6000g for 10 min at 4℃to remove cell debris. The clarified lysate is passed through a nickel affinity chromatography column under gravity. The non-target proteins were removed with 50mL of wash buffer (30 mM Tris-HCl buffer, 200mM NaCl,40mM imidazole) and then the target proteins were collected with 20mL of elution buffer (30 mM Tris-HCl buffer, 200mM NaCl,200mM imidazole). Concentrating and dialyzing the eluted protein by using an ultrafiltration centrifugal column with a molecular weight of 30kDa to obtain purified light-operated DNA polymerase, namely TdT-cpLOV2, the structure schematic diagram and the crystal structure of which are shown in figure 1, and the amino acid sequence of which is shown in SEQ ID NO:3, and the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) chart is shown in FIG. 2.
Example 2 testing of products after catalytic reactions Using optically controlled DNA polymerase
(1) Catalytic reaction using optically controlled DNA polymerase
Temporarily placing HEPES buffer solution (200mM NaCl,50mM HEPES,pH 7.2;HEPES is 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid) in an ice box for standby;
Mixing the oligonucleotide primer, deoxyribonucleotide, coCl 2 and HEPES buffer solution to obtain reaction preparation solution, and placing the reaction preparation solution in a metal bath at 30 ℃ for later use; the concentration of the oligonucleotide primer in the reaction preparation was 1. Mu.M, the concentration of deoxyribonucleotide was 0.1mM, and the concentration of CoCl 2 was 0.25mM.
The light-controlled DNA polymerase in example 1 (final concentration in the reaction preparation was 0.1. Mu.M) was added to 10. Mu.L of the reaction preparation, and after mixing uniformly with a pipette, the mixture was placed in a dark room and reacted at 37℃for 20 minutes, and then heated at 95℃for 20 seconds to stop the reaction, to obtain a first post-reaction solution.
Meanwhile, the light-operated DNA polymerase (final concentration in the reaction preparation solution is 0.1. Mu.M) in example 1 was added to 10. Mu.L of the reaction preparation solution, and after mixing uniformly with a pipette, it was placed in a blue light (420 nm) reactor to react at 37℃for 20 minutes, and then heated at 95℃for 20 seconds to stop the reaction, to obtain a second post-reaction solution.
(2) Modified urea polyacrylamide gel electrophoresis (modified urea PAGE) test of reaction products
Mixing 5 mu L of the first reaction solution and 5 mu L of the 2X loading buffer solution to obtain a first sample to be tested (corresponding to a dark condition); mixing 5 mu L of second reaction solution and 5 mu L of 2X loading buffer solution to obtain a second sample to be detected (corresponding to blue light irradiation conditions);
Preparing 10mL of 20% urea denatured glue, pouring the urea denatured glue between glass plates of a glue making frame, inserting a sample comb, installing an electrophoresis system, adding TBE electrophoresis buffer solution to pull out the sample comb, adding a Marker (molecular weight standard protein) and a first sample to be tested into a sample hole, carrying out electrophoresis at a constant voltage of 200V for 60min, observing a result under a gel imager, and testing a second sample to be tested by the same method. As shown in FIG. 3, it is clear that the length of the ssDNA sequence of the product is 10-35 nt under the dark condition, and the length of the ssDNA sequence of the product is at least 75nt under the blue light irradiation condition, which shows the difference of the catalytic rates of the optically controlled DNA polymerase under different conditions (blue light irradiation and darkness), namely, the difference of the reaction rates of the optically controlled DNA polymerase under the condition of blue light irradiation or not.
Example 3 catalytic Rate test of catalytic reactions Using optically controlled DNA polymerase
Temporarily placing HEPES buffer (200mM NaCl,50mM HEPES,pH 7.2) in an ice bin for later use;
Mixing the oligonucleotide primer, deoxyribonucleotide, coCl 2 and HEPES buffer solution to obtain reaction preparation solution, and placing the reaction preparation solution in a metal bath at 30 ℃ for later use; the concentration of the oligonucleotide primer in the reaction preparation was 1. Mu.M, the concentration of deoxyribonucleotide was 0.1mM, and the concentration of CoCl 2 was 0.25mM.
Preparing two white 96-well ELISA plates, placing one white 96-well ELISA plate on a darkroom shaking table, placing the other white 96-well ELISA plate on a blue light (420 nm) reactor, sequentially adding 90 mu L of reaction preparation solution and 10 mu L of light-operated DNA polymerase (prepared in example 1) into the two ELISA plates by using a multi-channel pipette, and immediately blowing and sucking uniformly for a plurality of times (at least 8 times) by using the multi-channel pipette; the reaction was shaken at 200rpm for 20min. Immediately after the reaction, the mixture was placed on a metal plate at 95℃and heated for 20 seconds to stop the reaction, thereby obtaining a reaction solution.
Detecting the reaction condition of light-controlled DNA polymerase catalyzing DNA synthesis, and comparing the reaction rate (or catalyzing rate) of the light-controlled DNA polymerase under the two conditions of blue light irradiation and darkness. Specifically, light-operated DNA polymerase catalytic rate testing was performed using a pyrophosphoric acid fluorescent probe kit. mu.L of the post-reaction solution and 50. Mu.L of the pyrophosphate fluorescent probe solution were added to a white 96-well ELISA plate using a multi-well pipette. After incubation for 10 minutes at room temperature, the light intensity values at the wavelength of 316nm and the wavelength of 456nm are read by a Flex Station3 multifunctional enzyme-labeled instrument in a light-emitting mode, the average light-emitting value in the time period is continuously monitored for 2 minutes, and the ratio of the fluorescence intensity of the reacted solution obtained by the reaction under the condition of blue light irradiation to the fluorescence intensity of the reacted solution obtained by the reaction under the dark condition is calculated.
As can be seen from fig. 4, the catalytic rate of the optically controlled DNA polymerase is significantly different under blue light irradiation and darkness, and the difference in the reaction rate (i.e., catalytic rate or catalytic activity) can be 106.14 times.
In summary, the invention provides the light-operated DNA polymerase, the nucleic acid fragment, the recombinant vector, the recombinant cell and the application, wherein the light-operated DNA polymerase can show remarkably different enzyme activities under different illumination conditions, the activity difference under blue light illumination and darkness conditions reaches hundreds of times (the activity under blue light condition is 106.14 times under darkness condition), the reversible optical regulation of the catalytic activity is realized, and the light-operated DNA polymerase has the characteristics of high catalytic efficiency, high stability and strong controllability. The invention utilizes the optical control technology of the light-operated DNA polymerase activity, and improves the limitations of poor controllability, more synthetic reaction steps and dependence on the deprotection process when the traditional DNA polymerase synthesizes DNA. Compared with a biosynthesis method, the invention uses the optical control technology of the light-operated DNA polymerase activity to preliminarily replace the traditional artificial ultraviolet or chemical reagent deprotection step, reduces the reaction steps, improves the synthesis efficiency, reduces the synthesis cost and provides a new innovation strategy for the controllable synthesis of the DNA polymerase.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (10)
1. A light-operated DNA polymerase, comprising a terminal deoxynucleotidyl transferase and cpLOV a2, wherein the amino acid sequence of the terminal deoxynucleotidyl transferase is shown in SEQ ID NO:1, said cpLOV2 is inserted between the phenylalanine residue 202 and serine residue 203 of said terminal deoxynucleotidyl transferase.
2. The light-operated DNA polymerase of claim 1, wherein cpLOV2 consists of the core domain PAS and the jα helix.
3. The light-operated DNA polymerase of claim 2, wherein the ja helix becomes a loose flexible structure under blue light irradiation conditions and becomes a ja helix under dark conditions.
4. The light-operated DNA polymerase of claim 1, wherein the cpLOV amino acid sequence is set forth in SEQ ID NO: 2.
5. The light-operated DNA polymerase of claim 1, wherein the amino sequence of the light-operated DNA polymerase is as set forth in SEQ ID NO: 3.
6. A nucleic acid fragment comprising a nucleotide sequence encoding the optically controlled DNA polymerase of any one of claims 1-5.
7. A recombinant vector comprising the nucleic acid fragment of claim 6.
8. A recombinant cell comprising the recombinant vector of claim 7.
9. Use of an optically controlled DNA polymerase according to any one of claims 1 to 5, a DNA polymerase expressed from the nucleic acid fragment according to claim 6, a DNA polymerase expressed from the recombinant vector according to claim 7 or a DNA polymerase expressed from the recombinant cell according to claim 8 for catalyzing a DNA synthesis reaction.
10. The use of claim 9, wherein the optically controlled DNA polymerase catalyzes the repeated addition of deoxynucleotides to the 3 'hydroxyl end of single stranded DNA or the 3' hydroxyl end of double stranded DNA.
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| CN111269325A (en) * | 2020-02-18 | 2020-06-12 | 中山大学 | Fusion protein of light-regulated BK channel and preparation method and application thereof |
| US20200263152A1 (en) * | 2017-05-22 | 2020-08-20 | The Charles Stark Draper Laboratory, Inc. | Modified template-independent dna polymerase |
| CN112746063A (en) * | 2019-10-29 | 2021-05-04 | 中国科学院天津工业生物技术研究所 | New function and application of nucleoside transferase |
| CN116240213A (en) * | 2022-08-10 | 2023-06-09 | 中山大学 | Light-responsive nucleic acid aptamer, light-activated DNA polymerase and application |
| CN116286722A (en) * | 2023-04-24 | 2023-06-23 | 中国科学院深圳先进技术研究院 | Light-operated DNA synthetase with complete genetic code, nucleic acid and application |
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| US20200263152A1 (en) * | 2017-05-22 | 2020-08-20 | The Charles Stark Draper Laboratory, Inc. | Modified template-independent dna polymerase |
| CN112746063A (en) * | 2019-10-29 | 2021-05-04 | 中国科学院天津工业生物技术研究所 | New function and application of nucleoside transferase |
| CN111269325A (en) * | 2020-02-18 | 2020-06-12 | 中山大学 | Fusion protein of light-regulated BK channel and preparation method and application thereof |
| CN116240213A (en) * | 2022-08-10 | 2023-06-09 | 中山大学 | Light-responsive nucleic acid aptamer, light-activated DNA polymerase and application |
| CN116286722A (en) * | 2023-04-24 | 2023-06-23 | 中国科学院深圳先进技术研究院 | Light-operated DNA synthetase with complete genetic code, nucleic acid and application |
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