A heat-sensitive double-stranded specific nuclease mutant and its application

Abstract

The present invention relates to the field of biotechnology and specifically discloses a heat-sensitive double-stranded specific nuclease mutant and an application thereof. The amino acid sequence of the mutant is based on SEQ ID NO.1, except that the alanine at position 323 is mutated to the threonine A323T, and the proline at position 237 is mutated to the alanine P237A. The amino acid sequence of the mutant is shown in SEQ ID NO.5. The heat-sensitive double-stranded specific nuclease mutant is used to remove DNA residues in RNA samples before a reverse transcription reaction. The present invention significantly reduces the inactivation temperature of DSN by introducing two amino acid mutations, A323T and P237A, into a wild-type DSN, so that the DSN can be directly inactivated at a commonly used reverse transcription reaction temperature without the need for an additional heat inactivation step, thereby further simplifying the reverse transcription operation process.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a heat-sensitive double-stranded specific nuclease mutant and application thereof. Background Art

[0002] During RNA extraction, genomic DNA (gDNA) contamination is a significant concern, primarily due to DNA released during cell lysis. gDNA contamination can interfere with both quantitative and qualitative RNA analysis. In reverse transcription-fluorescence quantitative PCR, gDNA contamination can lead to false-positive results because primers can bind to both RNA and gDNA, resulting in the amplification of nonspecific products. To effectively remove gDNA contamination from RNA samples, deoxyribonuclease I (DNase I) treatment has been commonly used. DNase I specifically degrades both double-stranded and single-stranded DNA but is inactive against RNA. To ensure reliable experimental results, DNase I must be completely inactivated by adding ethylenediaminetetraacetic acid (EDTA) and heating to 65°C after the enzymatic digestion reaction to prevent nonspecific degradation of the DNA template and reverse transcription primers in subsequent experiments. However, this separate protocol for genomic DNA removal and reverse transcription not only increases the number of experimental steps and the potential for sample contamination, but also introduces the potential for inhibition of reverse transcriptase activity due to the EDTA chelator, potentially affecting the accuracy of experimental results.

[0003] Double-stranded nuclease (DSN) is a specialized class of endonucleases originally discovered from the hepatopancreas of marine crustaceans. DSN recognizes fully complementary double-stranded DNA or DNA / RNA hybrids and specifically hydrolyzes only the DNA strand within them, with little activity against single-stranded DNA or RNA. Furthermore, DSN can be inactivated by incubation in a certain concentration of the reducing agent dithiothreitol (DTT, already included in reverse transcription reagents) without the need for the addition of ethylenediaminetetraacetic acid (EDTA). Due to this characteristic, DSN is widely used to remove genomic DNA contamination from RNA samples.

[0004] However, known natural DSNs are not inactivated at the commonly used reverse transcription reaction temperature (50-55°C). Therefore, researchers have engineered some DSNs to be thermosensitive. The most representative example is a thermosensitive DSN derived by introducing a point mutation into the Arctic shrimp DSN (U.S. Patent No. 8551753B2). However, its inactivation temperature was measured at 58°C, still higher than the commonly used reverse transcription reaction temperature (50-55°C). In practical reverse transcription applications, after adding DSN to digest genomic DNA in RNA samples, an additional heat inactivation step is required to prevent uninactivated DSN from digesting the reverse transcribed cDNA product before the temperature can be lowered for reverse transcription. This increases the number of reverse transcription steps and introduces inconvenience. Therefore, screening for DSNs with improved thermosensitivity is of great application value. To this end, a thermosensitive double-stranded-specific nuclease mutant and its application are provided. Summary of the Invention

[0005] The purpose of the present invention is to address the defects of the prior art and provide a heat-sensitive double-stranded specific nuclease mutant and its application to solve the problems raised by the above background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a heat-sensitive double-stranded specific nuclease mutant, wherein the amino acid sequence of the mutant is based on SEQ ID NO.1, with the alanine at position 323 mutated to threonine A323T, and the proline at position 237 mutated to alanine P237A. The amino acid sequence of the mutant is shown in SEQ ID NO.5.

[0007] A use of the heat-sensitive double-stranded specific nuclease mutant as described above in removing residual DNA in RNA samples before reverse transcription reaction.

[0008] A method for preparing the above-mentioned heat-sensitive double-stranded specific nuclease mutant comprises the following specific steps:

[0009] Step 1: Vector construction: Use enzyme digestion-ligation or seamless cloning technology to clone the gene encoding the thermosensitive double-stranded specific nuclease mutant into a Pichia pastoris secretory expression vector to form a recombinant expression vector for the thermosensitive double-stranded specific nuclease mutant;

[0010] Step 2: Transformation and screening: The constructed recombinant expression vector is linearized using restriction enzymes. The linearized vector is then introduced into Pichia pastoris competent cells by electroporation. The transformed cells are plated on yeast culture plates containing antibiotics and cultured at 30°C for 2-3 days. Single colonies are picked for PCR verification to screen for positive clones containing the correct recombinant expression vector. The selected positive clones are sent to a sequencing company for sequencing of the inserted gene to ensure that the mutation site is correct and there are no other base mutations.

[0011] Step 3: Inducing expression: The positive Pichia pastoris clones obtained by screening are inoculated into a liquid culture medium containing an appropriate amount of antibiotics and cultured until the logarithmic growth phase. The cells are then collected by centrifugation, resuspended, and transferred to a new culture medium for culture. Methanol is regularly added to induce secretory expression of the heat-sensitive double-stranded specific nuclease mutant. During this period, the cell growth status and protein expression are regularly monitored. After the culture is completed, the supernatant is collected by centrifugation, which contains the secreted heat-sensitive double-stranded specific nuclease mutant.

[0012] Step 4: Protein purification: The collected culture supernatant containing the heat-sensitive double-stranded specific nuclease mutant is purified using a protein purification method to obtain a high-purity heat-sensitive double-stranded specific nuclease mutant protein.

[0013] As a preferred technical solution of the present invention, the protein purification method in step 4 includes affinity chromatography or ion exchange chromatography.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] Regarding protein thermosensitivity, the present invention introduces two amino acid mutations, A323T and P237A, into the wild-type DSN protein, altering its hydrophobic surface and internal secondary structure. This significantly lowers the inactivation temperature of DSN, enabling direct inactivation at the commonly used reverse transcription reaction temperature of 50°C. In terms of operational convenience and cost-effectiveness, the thermosensitive DSN mutant described in this invention can be used to remove residual genomic DNA from RNA samples before the reverse transcription reaction. This eliminates the need for the additional DSN heat inactivation step at elevated temperatures, further simplifying the reverse transcription process and saving time and costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : The results of RNA treatment with the thermosensitive DSN mutant of the present invention;

[0017] Figure 2 : Result diagram of the treatment of single-stranded DNA by the thermosensitive DSN mutant of the present invention;

[0018] Figure 3 : Schematic diagram of the heat inactivation effect of the heat-sensitive DSN mutant of the present invention;

[0019] Figure 4 : Diagram of the effect of the thermosensitive mutant of the present invention in the reverse transcription reaction;

[0020] A: DNA removal effect of the thermosensitive DSN mutant of the present invention in a simulated reverse transcription reaction system;

[0021] B: Effect of the thermosensitive DSN mutant of the present invention on the transcription product in the reverse transcription reaction. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0023] Example 1: Recombinant expression of DSN mutant protein

[0024] Using conventional molecular cloning techniques, the genes encoding the two DSN mutants (DNA sequences shown in SEQ ID NO. 4 (A323T) and SEQ ID NO. 6 (A323T / P237A)) were cloned into the pPIC9K vector (ThermoFisher, Catalog No. V17520), respectively, with a polyhistidine tag at the N-terminus. Recombinant expression plasmids for the two mutants were constructed and sent to a sequencing company for sequencing verification.

[0025] The plasmid, verified to be correct by sequencing, was linearized using the restriction enzyme SacI (Bristol-Myers Squibb, Cat. No. EG15565). 10 μg of the concentrated linearized recombinant plasmid was thoroughly mixed with 80 μL of Pichia pastoris GS115 competent cell suspension and transferred to a 0.2 cm electroporation cuvette on ice for 5 minutes. The electroporator operating parameters were set as follows: output voltage 1500 V, capacitance 25 μF, impedance 300 Ω, and pulse duration 2 ms. Immediately after electroporation, 1 mL of pre-chilled 1 M sorbitol solution was added. The cells were incubated on ice for 10 minutes before being transferred to a 30°C constant temperature incubator for 60 minutes. The revived bacterial suspension was evenly spread on auxotrophic (MD) selection medium and incubated at 30°C for 2-3 days. Transformant formation was observed until a single, well-developed colony emerged. After transformation, a single, well-developed colony was selected and plated onto a non-antibiotic YPD solid plate for streak culture. Single colonies were then picked with a sterilized toothpick and streaked onto G418 plates containing 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, and 4.0 mg / mL, respectively. Cultures were then placed in a 30°C incubator for 2-3 days until colonies emerged. Three to five highly resistant positive colonies were selected for colony PCR verification. The amplified products were purified and sent to a sequencing company for sequencing verification to confirm that the DSN encoding gene had been correctly integrated into the Pichia pastoris chromosome.

[0026] The positive clone strains were inoculated into 10 mL YPD medium containing antibiotics and cultured at 30°C and 220 rpm until the OD value of the culture medium reached 0. 600When the OD value reaches 2-4, transfer the bacterial solution to 25 mL BMGY medium according to 1% inoculation volume and continue to culture at the same temperature and speed conditions until the OD value reaches 2-4. 600 The value reached 2.3~3.0. After the culture was completed, the bacteria were collected by low-temperature centrifugation at 4000g and resuspended in 50mL BMMY induction medium containing 0.02% methanol. Pre-induction was performed at 30℃ for 2 hours, and then the methanol concentration was increased to 0.5% and the induction culture was continued. To maintain the induction intensity, 0.5% methanol was added every 24 hours. 24 hours, 48 ​​hours, and 72 hours after the induction treatment was completed, supernatant samples were taken for SDS-PAGE gel electrophoresis verification to determine the expression of the target protein and perform crude enzyme activity detection. Finally, the supernatant was collected by centrifugation.

[0027] The formula of MD plate medium is: 15 g agar powder + 800 ml ddH2O, autoclave at 121℃ for 20 min; when the temperature drops to 60℃, add 100 ml 10× YNB, 100 ml 10× glycerol, and 2 ml 500× biotin.

[0028] The formula of YPD medium is ( / L): 10 g Yeast Extract, 20 g PePtone, 20 g glucose, dilute to 1 L; add 1.5% agar powder to the solid; and sterilize by autoclaving at 115°C for 15 min.

[0029] The formula of BMGY medium is ( / L): 30 g Yeast Extract, 30 g Peptone, 60 mL 10× glycerol, 2 mL defoaming agent, dilute to 1.5 L, and sterilize at 120°C for 20 min. After sterilization, add 180 mL 10× YNB, 180 mL 1 M potassium phosphate, and 3.6 mL 500× biotin.

[0030] The above-mentioned biochemical reagents without indicating the source and product number were purchased from Aladdin and Sinopharm.

[0031] Example 2: Purification of DSN mutant protein

[0032] Load the collected fermentation supernatant onto a pre-equilibrated nickel column at 4°C at a flow rate of 1 mL / min. Elute impurities with 10 column volumes of Washing Buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), then elute the target protein with Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0), and collect the eluted peak.

[0033] The protein solution collected from nickel affinity chromatography was dialyzed into a low-salt buffer (20 mM Tris-HCl, pH 7.5) to remove imidazole and high-concentration salt ions. The dialyzed protein solution was then loaded onto an anion exchange chromatography column (Borgron BGL Q HP) pre-equilibrated with low-salt buffer. Unbound impurities were eluted with the low-salt buffer, followed by a linear elution using a 0-1 M NaCl gradient in 20 mM Tris-HCl (pH 7.5). The peak containing the target protein was collected by monitoring the absorbance of the eluate at 280 nm.

[0034] The eluted protein was dialyzed overnight against PBS buffer (pH 7.4) through a dialysis bag to remove impurities such as imidazole, thereby obtaining the purified DSN mutant. The purified protein was identified by SDS-PAGE gel electrophoresis.

[0035] Example 3: Verification of substrate specificity of DSN mutants

[0036] One unit (U) of DSN activity was defined as the amount of DSN required to completely degrade 1 μg of pUC19 plasmid DNA in a 50 μl reaction at 37°C for 10 min. Both the A323T and A323T / P237A mutants were diluted to 10 U / μL. Wild-type DSN (provided by Jiangsu Bristol-Myers Squibb Biotechnology Co., Ltd.) was diluted to the same concentration as a control.

[0037] First, verify whether the mutants possess ribonuclease (RNase) activity. Mix 1 μL of each of wild-type DSN, mutants A323T, and A323T / P237A with 500 ng of tomato RNA. Prepare the reaction solution according to Table 1 and incubate at 37°C for 30 min. Then, add 10 mM DTT and incubate at 80°C for 20 min to inactivate the reaction. Then, perform 2 μL of the product on agarose gel electrophoresis to analyze the reaction results.

[0038] Table 1: RNA substrate specific detection reaction system

[0039]

[0040] The results are as follows Figure 1 As shown, the DSN mutants A323T and A323T / P237A did not digest RNA like the wild type, and the RNA bands remained intact.

[0041] The mutants were then tested to see if they could digest single-stranded DNA.

[0042] Wild-type DSN, pre-inactivated wild-type DSN, mutant A323T, and mutant A323T / P237A were incubated with single-stranded DNA probes labeled with a FAM fluorophore and a BHQ quencher at each end. The reaction mixtures were prepared according to Table 2. T5 exonuclease (Bristol-Myers Squibb, Cat. No. EG24208) was used as a positive control. The cells were placed in a Macrostone SLAN-96S fluorescence quantitative PCR instrument and programmed at 37°C for 30 seconds for 30 cycles. Fluorescence signal changes were observed in real time.

[0043] Table 2: Single-stranded DNA substrate specific detection reaction system

[0044]

[0045] The results showed that the fluorescence intensity of the two mutants did not increase significantly when incubated with the single-stranded DNA probe, which was consistent with the wild type, indicating that the single-stranded DNA probe was not degraded and emitted fluorescence; while the fluorescence value using T5 DNA exonuclease as a control increased significantly ( Figure 2 ). This indicates that the mutants A323T and A323T / P237A, like the wild type, do not hydrolyze single-stranded DNA.

[0046] The above two experiments showed that the substrate specificity of mutants A323T and A323T was consistent with that of the wild type and they were inactive towards RNA and single-stranded DNA.

[0047] Example 4: Determination of inactivation temperature of DSN mutants

[0048] Both the A323T and A323T / P237A mutants were diluted to 10 U / μL. Wild-type DSN was diluted to the same concentration as a control. The reaction mixture was prepared according to Table 3 and incubated at 45°C, 50°C, 52°C, 55°C, 58°C, and 60°C for 10 min. Then, 1 μg of λ DNA was added and the reaction was incubated at 37°C for 10 min. The reaction was then incubated at 80°C for 20 min to inactivate the product. Afterwards, 2 μL of the product was subjected to agarose gel electrophoresis.

[0049] Table 3: DNA thermal stability detection system

[0050]

[0051] Gel electrophoresis showed that in the presence of DTT, the double mutant A323T / P237A was almost completely inactivated after incubation at 50°C for 10 minutes, and the substrate band remained basically intact; the single mutant A323T did not lose its activity until 60°C. However, the commercially available thermosensitive DSN could be inactivated at 58°C, while the wild-type DSN was not completely inactivated at 60°C ( Figure 3 ).

[0052] Example 5: Application of thermosensitive DSN mutants in a one-tube reverse transcription reaction

[0053] As shown in Example 4, while the inactivation temperature of the single mutant A323T is lower than that of the wild-type, it is still higher than that of commercially available thermosensitive DSNs, failing to meet the requirements. Therefore, only the double mutant A323T / P237A and commercially available thermosensitive DSNs were compared in a single-tube reverse transcription reaction.

[0054] First, the effectiveness of the mutants in removing DNA was investigated. Mutants A323T / P237A and commercially available thermosensitive DSN were added to a certain amount of tomato genomic DNA sample. All-in-One First-Strand Synthesis MasterMix Reverse Transcription Reagent (Bristol-Myers Squibb, Cat. No. EG20131) was also added, and a mock reverse transcription reaction was prepared according to Table 4. The genomic DNA was digested by incubation at 37°C for 2 minutes, followed by a simulated reverse transcription reaction at 50°C for 15 minutes. The simulated reverse transcription products were then used to prepare a quantitative PCR (qPCR) reaction according to Table 5. qPCR was used to detect the internal reference β-actin gene DNA. The qPCR program was set to a 95°C pre-denaturation for 30 seconds, followed by 45 cycles of amplification (95°C denaturation for 10 seconds, 58°C annealing for 10 seconds, and 72°C extension for 30 seconds).

[0055] Table 4: Reaction system for digestion of genomic DNA

[0056]

[0057] Table 5: Fluorescence quantitative PCR reaction system

[0058]

[0059] The results showed that after simulating the reverse transcription reaction process, the double mutation A323T / P237A and the commercially available thermosensitive DSN had no Ct value detected by fluorescence quantitative PCR ( Figure 4 A), indicating that both can efficiently digest genomic DNA.

[0060] The effects of the two mutants on a single-tube reverse transcription reaction were then compared. A323T / P237A and A323T proteins were added to a certain amount of tomato total RNA sample, along with reverse transcription reagents. The reverse transcription reaction mixture was prepared according to Table 6. The reaction mixture was incubated at 37°C for 2 minutes, followed by a 15-minute incubation at 50°C. The reverse transcription product was also prepared into a qPCR reaction mixture according to Table 4. β-actin gene expression in the reverse transcription product was detected by qPCR. The qPCR reaction procedure was the same as above.

[0061] Table 6: Reverse transcription reaction system

[0062]

[0063] It can be seen that compared with the control without DSN, the mutant A323T / P237A of the present invention is inactivated during the reverse transcription reaction at 50°C, with almost no effect on the reverse transcription reaction; while the commercially available thermosensitive DSN cannot be inactivated at 50°C and will degrade a portion of the cDNA product in the RNA / cDNA hybrid chain during reverse transcription, resulting in a 2-cycle lag in the Ct value of the reverse transcription product qPCR ( Figure 4 B).

[0064] The above embodiments merely illustrate the implementation methods of the present invention. Although the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A heat-sensitive double-stranded specific nuclease mutant, characterized in that: The amino acid sequence of the mutant is based on SEQ ID NO.1, with the alanine at position 323 mutated to threonine A323T and the proline at position 237 mutated to alanine P237A. The amino acid sequence of the mutant is shown in SEQ ID NO.

5.

2. A use of the heat-sensitive double-stranded specific nuclease mutant according to claim 1 for removing residual DNA in an RNA sample before a reverse transcription reaction.

3. A method for preparing the heat-sensitive double-stranded specific nuclease mutant according to claim 1, characterized in that: The specific steps are as follows: Step 1: Vector construction: Use enzyme digestion-ligation or seamless cloning technology to clone the gene encoding the thermosensitive double-stranded specific nuclease mutant into a Pichia pastoris secretory expression vector to form a recombinant expression vector for the thermosensitive double-stranded specific nuclease mutant; Step 2: Transformation and screening: The constructed recombinant expression vector is linearized using restriction enzymes. The linearized vector is then introduced into Pichia pastoris competent cells by electroporation. The transformed cells are plated on yeast culture plates containing antibiotics and cultured at 30°C for 2-3 days. Single colonies are picked for PCR verification to screen for positive clones containing the correct recombinant expression vector. The selected positive clones are sent to a sequencing company for sequencing of the inserted gene to ensure that the mutation site is correct and there are no other base mutations. Step 3: Inducing expression: The positive Pichia pastoris clones obtained by screening are inoculated into a liquid culture medium containing an appropriate amount of antibiotics and cultured until the logarithmic growth phase. The cells are then collected by centrifugation, resuspended, and transferred to a new culture medium for culture. Methanol is regularly added to induce secretory expression of the heat-sensitive double-stranded specific nuclease mutant. During this period, the cell growth status and protein expression are regularly monitored. After the culture is completed, the supernatant is collected by centrifugation, which contains the secreted heat-sensitive double-stranded specific nuclease mutant. Step 4: Protein purification: The collected culture supernatant containing the heat-sensitive double-stranded specific nuclease mutant is purified using a protein purification method to obtain a high-purity heat-sensitive double-stranded specific nuclease mutant protein.

4. The method for preparing a heat-sensitive double-stranded specific nuclease mutant according to claim 3, characterized in that: The protein purification method in step 4 includes affinity chromatography or ion exchange chromatography.

Images