A mutant ribonuclease R and its applications

Abstract

This invention provides a mutant ribonuclease R (RNase R) and its applications. Through creative selection and screening of mutations at the RNase R site in *E. coli*, this invention provides 10 mutant RNase R variants, obtaining mutants T495E, A246E, K457F, A246F, D281F, and D122L with significantly increased enzyme activity relative to the parental strain. Among these, mutant T495E shows the most significant increase in enzyme activity, with a relative increase of 37.48%. The other mutants show increases of 24.74%, 21.74%, 10.04%, 10.34%, and 6.75%, respectively. This invention also provides recombinant *E. coli* strains that highly express RNase R, enabling efficient production of RNase R mutants with promising industrial application prospects. The RNase R mutants with increased enzyme activity provided by this invention are suitable for large-scale industrial production of mRNA, particularly in the degradation of linear RNA or the production of circRNA.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and medicine, particularly to the field of ribonuclease R, and especially to mutated ribonuclease R and its applications. This application is a divisional application of application number 202211610204.8, entitled "A Mutated Ribonuclease R and Its Applications". Background Technology

[0002] Ribonuclease R (RNase R) is a magnesium-dependent 3'→5' exonuclease that digests virtually all linear RNA, but not lasso-like or circular RNA structures, or double-stranded RNA with 7 nucleotides shorter than the 3' overhang. Most cellular RNA is completely digested by RNase R, except for tRNA, 5S RNA, and proteins containing lasso. The 3' tail of the lasso is modified by RNase R into a branching nucleotide containing a 2',5'-phosphodiester bond.

[0003] Circular RNA (circRNA) is a novel class of circular non-coding RNA that differs from linear RNA. It has a long half-life and exhibits species conservation and tissue specificity. Its unique circular structure makes it difficult for RNases to degrade, resulting in strong intracellular stability. Therefore, it holds immense potential and research value in areas such as novel biomarkers and the study of biological mechanisms.

[0004] The concept of "circRNA" first appeared in 1971 and was discovered in RNA viruses. However, for a long time, due to its low expression abundance, it was considered "noise" in transcription and had no practical function. Until 2012, with the help of high-throughput sequencing technology, Salzman et al. (Salzman J, Gawad C, Wang PL, et al. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse celltypes. Plos One, 2012, 7(2): 1-12) found that leukemia cells, HeLa cell lines, and normal human primary blood cells all exhibit the phenomenon of forming non-linear circular transcripts through exon rearrangement. About 80 circular RNAs were reported for the first time. Subsequently, a large number of researchers entered this field and continuously produced research results on circular RNAs. Currently, the most studied circular RNAs are those formed by exons located in the cytoplasm. They contain a large number of miRNA binding sites and can act as miRNA sponges, binding and blocking the regulatory effects of miRNAs, thereby enhancing the expression of target genes.

[0005] With the deepening research on circular RNA, its preparation has attracted increasing attention. In the preparation process, to eliminate interference from other RNAs, RNase R must be used to remove other RNAs from the reaction system to obtain relatively pure circRNA. However, the quality of existing RNase Rs varies considerably, and validation processes have revealed that some RNase Rs have a significant degradative effect on circRNA. As research on circular RNA continues to gain momentum, the demand for RNase R in the market and research fields will further increase in the future.

[0006] CN114438054A (publication date: May 6, 2022) discloses a mutant RNase R named RNaseR_△M8, whose amino acid sequence is shown in SEQ ID NO. 5, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO. 6. The preparation process of the mutant RNase R_△M8 provided by this invention includes vector construction, vector transformation, protein induction expression, bacterial collection, protein purification, and activity assay. Compared with wild-type RNase R, the mutant RNase R provided by this invention has a higher protein expression level and can tolerate 150 mM NaCl.

[0007] Although mutant RNase R already exists in the prior art, there is still an urgent need in the field to provide different mutant RNase R with improved performance for industrial and scientific research, especially mutant RNase R with improved enzyme activity, which can yield circRNA with higher yield and purity. Summary of the Invention

[0008] To address the shortcomings of existing technologies in the RNase R field, one objective of this invention is to provide a mutant RNase R with improved performance, particularly a significant increase in enzyme activity compared to the parent. A further objective of this invention is to provide a recombinant strain, particularly recombinant *Escherichia coli*, that highly expresses the RNase R mutant, enabling efficient production of RNase R mutants with promising industrial application prospects. This invention also provides a recombinant nucleic acid encoding the mutant RNase R, comprising an expression cassette, vector, cells, strain, composition, and kit, as well as methods for preparing, purifying, and detecting the enzyme activity of the RNase R mutant, and its applications in degrading linear RNA and producing circRNA.

[0009] One aspect of the present invention provides a mutant ribonuclease R, characterized in that it contains a mutation based on the parental ribonuclease R, said mutation including T495E, A246E, K457F, A246F, D281F, or D122L, wherein the amino acid sequence of the parental ribonuclease R is shown in SEQ ID NO:1.

[0010] Further, the mutated ribonuclease R is a mutant T495E, A246E, K457F, A246F, D281F, or D122L. The amino acid sequence of mutant T495E is shown in SEQ ID NO:30, the amino acid sequence of mutant A246E is shown in SEQ ID NO:25, the amino acid sequence of mutant K457F is shown in SEQ ID NO:29, the amino acid sequence of mutant A246F is shown in SEQ ID NO:26, the amino acid sequence of mutant D281F is shown in SEQ ID NO:27, and the amino acid sequence of mutant D122L is shown in SEQ ID NO:23.

[0011] Furthermore, the DNA sequence of the parental ribonuclease R is shown in SEQ ID NO.2.

[0012] Another aspect of the present invention provides a recombinant nucleic acid, characterized in that it comprises a nucleic acid encoding the mutated ribonuclease R.

[0013] Another aspect of the present invention provides an expression cassette, characterized in that it comprises the recombinant nucleic acid.

[0014] Another aspect of the present invention provides a vector, characterized in that it comprises the recombinant nucleic acid or the expression cassette; preferably, the vector is an expression vector.

[0015] Another aspect of the present invention provides a cell characterized in that it comprises the recombinant nucleic acid or the expression cassette or the vector.

[0016] Another aspect of the present invention provides a recombinant bacterium, characterized in that it comprises the recombinant nucleic acid or the expression cassette or the vector; preferably, the bacterium is Escherichia coli; more preferably, the Escherichia coli is one of E. coli BL21(DE3), E. coli Origami B(DE3) or E. coli Rosetta Blue(DE3); most preferably, the Escherichia coli is E. coli BL21(DE3).

[0017] Another aspect of the present invention provides a composition characterized in that it comprises the mutated ribonuclease R or the recombinant nucleic acid or the expression cassette or the vector or the cell or the recombinant bacteria.

[0018] Another aspect of the present invention provides a kit characterized in that it comprises the mutated ribonuclease R or the recombinant nucleic acid or the expression cassette or the vector or the cell or the recombinant bacteria or the composition.

[0019] Another aspect of the present invention provides a method for preparing the mutant ribonuclease R, characterized by comprising the following steps:

[0020] (1) Construct a vector containing a nucleotide sequence encoding the mutated ribonuclease R;

[0021] (2) Transform the vector obtained in step (1) into the expression strain cells to obtain the expression strain;

[0022] (3) Expand the expression strain obtained in step (2) and induce protein expression;

[0023] (4) Collect the expression strains after expansion culture, and wash and lyse them;

[0024] (5) Perform protein purification;

[0025] (6) Perform enzyme activity detection.

[0026] Further, step (3) includes culturing the expression strain obtained in step (2) at 25-37°C until OD200. 600 The concentration of isopropyl-β-D-thiogalactoside (IPTG) is 0.6-1.0. Fermentation is induced at 16-37℃ for 8-16 hours to induce the expression of the mutant ribonuclease R.

[0027] Further, step (4) includes centrifuging the fermentation broth obtained in step (3) at 8000 rpm for 5 min and discarding the supernatant, then resuspending the cells in 50 mM PBS and centrifuging at 8000 rpm for 5 min and discarding the supernatant, adding PBS to resuspend the cells and mixing well; sonicating the cells on ice; placing the cell lysate in a pre-cooled centrifuge and centrifuging at 4°C and 8000 rpm for 10 min, collecting the precipitate, and redissolving the precipitate in a buffer solution containing 500 mM KCl.

[0028] Another aspect of the present invention provides a method for purifying the mutated ribonuclease R, characterized by comprising the following steps: purifying the crude enzyme solution containing the mutated ribonuclease R obtained by fermentation using a protein purification system via nickel column purification, equilibrating the nickel column with a pre-equilibrated buffer, eluting with an elution buffer of increasing concentration, collecting the target protein, and then performing electrophoretic analysis.

[0029] Another aspect of the present invention provides a method for detecting the enzyme activity of the mutated ribonuclease R, characterized by comprising the following steps: diluting the mutated ribonuclease (RNase R) through a series of gradient dilutions, degrading 10 μg of linear mRNA with the diluted RNase R at 37°C for 1 h, detecting by gel electrophoresis until no obvious band is detected by electrophoresis, and calculating the enzyme activity of the mutated ribonuclease R based on the amount of enzyme used when no obvious band is detected.

[0030] Another aspect of the present invention provides the application of the mutant ribonuclease R in the field of mRNA preparation.

[0031] Another aspect of the present invention provides the application of the mutant ribonuclease R in the field of degrading linear RNA or producing circRNA.

[0032] The mutant ribonuclease R and its applications provided by this invention have the following beneficial technical effects:

[0033] 1. This invention, through creative selection and screening of RNase R site mutations in *E. coli*, provides 10 mutated ribonucleases R, obtaining mutants T495E, A246E, K457F, A246F, D281F, and D122L with significantly increased enzyme activity relative to the parental strain. Among them, mutant T495E showed the most significant increase in enzyme activity, with a relative increase of 37.48%. Mutant A246E ranked second with a relative increase of 24.74%. Mutant K457F ranked third with a relative increase of 21.74%. Mutants A246F and D281F showed relative increases of approximately 10%. Mutant D122L showed a relative increase of less than 10%, at 6.75%.

[0034] 2. The method of the present invention for preparing mutant RNase R is simple and quick to operate, and the obtained enzyme has good stability.

[0035] 3. This invention provides a recombinant strain that highly expresses RNase R, particularly recombinant Escherichia coli, for the efficient production of RNase R mutants, and has good prospects for industrial application.

[0036] 4. The mutant ribonuclease R provided by this invention has a significantly increased enzyme activity compared to the parent, making it particularly suitable for large-scale industrial production of mRNA, especially in applications related to the degradation of linear RNA or the production of circRNA, resulting in higher yields and purity of circRNA. Attached Figure Description

[0037] Figure 1This is a spectrum of the pET-28a-RNase R vector of the present invention, wherein RNase R is Feature 15 in the figure;

[0038] Figure 2 The SDS-PAGE results show the expression levels of RNase R protein in whole cells (whole bacteria) and lysate supernatant (soluble) of recombinant E. coli BL21(DE3) at different final IPTG concentrations.

[0039] Figure 3 This is a nickel column chromatography program chart of the crude RNase R enzyme solution expressed by Escherichia coli in this invention;

[0040] Figure 4 This is the SDS-PAGE result of the protein content of the peak components collected by nickel column chromatography at different time periods in this invention;

[0041] Figure 5 The electrophoresis results show the linear EGFP mRNA residues after RNase R degradation at different gradient dilutions according to this invention. Detailed Implementation

[0042] Example 1: Construction of parental RNase R expression vector

[0043] Genome analysis of *E. coli* revealed the presence of the ribonuclease RNase R. The protein sequence encoded by this gene contains 814 amino acid residues and belongs to the RNR superfamily. The amino acid sequence of the RNase R protein is shown in SEQ ID NO.1. Codon optimization was performed according to *E. coli* expression guidelines, resulting in the DNA sequence shown in SEQ ID NO.2. The specific sequences are shown in Table 1. The DNA sequence was synthesized by our company (Tianlin Biotechnology Co., Ltd.), and the gene was successfully constructed into the pET-28a vector to obtain pET-28a-RNase R. Its plasmid expression map is shown below. Figure 1 As shown.

[0044] Table 1 Parental RNase R sequence information

[0045]

[0046]

[0047] Example 2: Escherichia coli-induced expression of ribonuclease RNase R

[0048] The *E. coli* JM109 strain containing the synthesized pET-28a-RNase R from Example 1 was inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured. The plasmid was then extracted to obtain the recombinant plasmid pET-28a-RNase R. The recombinant expression vector pET-28a-RNase R was transformed into competent *E. coli* BL21(DE3) cells for positive clone selection.

[0049] The recombinant strain E. coli BL21(DE3) obtained through screening was induced to express its contents using IPTG. The recombinant bacteria were inoculated into LB medium and cultured at 37°C until OD500. 600 The OD value was 1.8-2.2. Subsequently, the cell culture medium was centrifuged at 8000g for 5 min to collect the bacterial cells. The cells were then resuspended in LB medium. 600 The concentration of IPTG was 0.9-1.1, and IPTG was added to the culture medium to a final concentration of 0.5 mM. Fermentation was induced at 25°C for 8 h. After centrifugation, the bacterial pellet was collected, sonicated, and analyzed by SDS-PAGE electrophoresis. The target protein, RNase R, had a molecular weight of approximately 92 kDa. The SDS-PAGE results of the expression levels of RNase R in whole cells (whole bacteria) and the supernatant (soluble) of the lysate from recombinant *E. coli* BL21(DE3) at different final IPTG concentrations are shown below. Figure 2 As shown, the lanes in the electrophoresis diagram, from left to right, are: Lane 1 (Marker), Lane 2 (BL21-pET28a+RNase-R-1-25℃-0% IPTG total cells), Lane 3 (BL21-pET28a+RNase-R-1-25℃-0.1% IPTG soluble), Lane 4 (BL21-pET28a+RNase-R-1-25℃-0.1% IPTG total cells), Lane 5 (BL21-pET28a+RNase-R-1-25℃-0.1% IPTG soluble), Lane 6 (BL21-pET28a+RNase-R-1-25℃-0.5% IPTG total cells), Lane 7 (BL21-pET28a+RNase-R-1-25℃-0.5% IPTG soluble), Lane 8 (BL21-pET28a+RNase-R-1-25℃-1.0%). Whole cell culture with IPTG, lane 9 BL21-pET28a+RNase-R-1, -25℃, -1.0% IPTG soluble. The -1 in RNase-R-1 in the lane represents the single clone strain number. The results showed that the target protein RNase R was well expressed under these conditions (final IPTG concentration of 0.5 mM, corresponding to lane 6, i.e., 0.5% IPTG), and batch fermentation can be used to prepare cell culture.

[0050] Example 3: Purification of RNase R using nickel column and desalting using gel column

[0051] When recombinant E. coli BL21(DE3) / pET28a-RNase R induces protein expression, the target protein is ultimately distributed intracellularly. Obtaining the target protein requires cell disruption. The fermentation broth is poured into a 50ml centrifuge tube, centrifuged at 8000rpm for 5min, and the supernatant is discarded. The cells are then resuspended in 50mM PBS and centrifuged again at 8000rpm for 5min, discarding the supernatant. PBS is added back to resuspend the cells, and the mixture can be pipetted to homogenize. During ultrasonic cell disruption, the cells should be placed on ice. The ultrasonic cell disruptor settings are: amplitude bar 6mm, time 60min, 50mL of cell suspension, ensuring the probe is close but not touching the bottom. The disrupted cell solution is placed in a pre-chilled centrifuge and centrifuged at 8000rpm for 10min at 4℃. The precipitate is collected and reconstituted in a 500mM KCl buffer. Subsequently, before purifying the crude enzyme solution sample, insoluble impurities are removed by filtering with a 0.45μm filter membrane (Sartorius).

[0052] Purification of crude RNase R enzyme solution from *E. coli*. The crude RNase R enzyme solution was purified using an AKTA protein purification system via a Ni column (Nanomicro, 77105-60042-531800). Equilibration: Using a Ni-NTA column pre-equilibrated with Buffer A (50mM NaH2PO4, 0.5M NaCl, pH 8.0), the column was washed with Buffer A (50mM NaH2PO4, 0.5M NaCl, pH 8.0) for 5 CV (column volume). After all baselines were equilibrated, sample loading was performed: Samples reconstituted with KCl buffer and membrane-treated were loaded, and flow-through samples were collected. Washing: The column was washed with equilibration buffer A (50mM NaH2PO4, 0.5M NaCl, pH 8.0) for 10 CV until baseline, pH, and conductivity stabilized. Elution: Elute with a 0-50% gradient using Buffer B (50mM NaH2PO4, 0.5M NaCl, 0.5M imidazole, pH 8.0), wash 30 CV, collect the sample peak, and retain the sample for analysis. Elution: Wash 5 CV with 100% Buffer B (50mM NaH2PO4, 0.5M NaCl, 0.5M imidazole, pH 8.0), monitor at 280 nm, collect the sample peak, and retain the sample for analysis. The nickel column chromatography program for E. coli expressing crude RNase R enzyme solution is shown below. Figure 3 As shown.

[0053] Purified samples were analyzed by SDS-PAGE. The sample was mixed with protein loading buffer (SDS, DTT, and bromophenol blue) in a specific ratio to ensure a final protein loading buffer concentration of at least 1X. The sample was then denatured at 98°C or in a boiling water bath for 10 minutes. Next, the electrophoresis tank and gel plates were installed (removing the sealing tape). Electrophoresis buffer was poured in, ensuring the inner tank was full and the liquid level in the outer tank was above the electrode wires. The combs of the precast gel were slowly removed. A syringe was used to draw buffer and insert it into each well to remove glycerol. The sample was then spotted, the tank lid was closed, and electrophoresis was performed at 80V for approximately 40 minutes, until the sample was compressed into a thin band. The voltage was increased to 120V, and electrophoresis continued until the indicator band approached or reached the bottom of the gel. The electrophoresed gel was then stained and destained using a protein staining instrument, with the program set to Stain 3 minutes; Destain 2 minutes; Destain 1 minute. The gel was transferred to a gel imaging system for imaging, image modification, lane setting and labeling, and corresponding analysis. SDS-PAGE results of the protein content of peak components collected at different time points using nickel column chromatography are shown below. Figure 4 As shown, the lanes in the electrophoresis image, from left to right, are: Lane 1 LC1A01 30 μl, Lane 2 RNase-R-2 30 μl, Lane 3 marker, Lane 4 XT1A07 30 μl, Lane 5 XT1A08 30 μl, Lane 6 XT1A09 30 μl, Lane 7 XT1A11 30 μl, Lane 8 XT1A12 30 μl, Lane 9 XT1B12 30 μl, and Lane 10 XT1B11 30 μl. The -2 in RNase-R-2 represents the monoclonal strain number. The results indicate that the eluted target protein RNase R can be purified in a single nickel column to obtain a large amount of purified product.

[0054] Imidazole was removed from the purified sample using a gel column (Nanomicro, 77105-60011-003400). The protein sample containing the target protein eluted from the nickel column was mixed and used as the sample for gel column loading. Imidazole was removed from the purified enzyme solution using the AKTA protein purification system. Equilibration: Using a gel column pre-equilibrated with Buffer A, the column was washed with Buffer A for 5 CV to reach baseline equilibrium. Loading: The sample purified from the nickel column was loaded, and the flow-through sample was collected. Collection was stopped when the conductivity increased. Subsequently, the gel column was flushed with Buffer A, and loading was repeated until all samples were completely free of imidazole. The collected sample was then concentrated by ultrafiltration to obtain the RNase R enzyme solution.

[0055] Example 4: Enzyme activity detection of RNase R enzyme solution

[0056] RNase R enzyme activity assay. RNase R enzyme activity is defined as follows: in 20 mM Tris-HCl (pH 7.5), 100 mM KCl, and 0.5 mM MgCl₂... 2+ Under the given conditions, the amount of enzyme required to hydrolyze 1 μg of linear mRNA product at 37℃ is 1 activity unit. Using linear EGFP mRNA as a sample example (not limited to linear EGFP mRNA samples, any linear mRNA sample can be used), the prepared RNase R was serially diluted. The diluted RNase R was then used to degrade 10 μg of linear EGFP mRNA at 37℃. After 1 hour, the results were detected by gel electrophoresis. The electrophoresis results of the residual linear EGFP mRNA after degradation by RNase R at different serial dilutions are shown below. Figure 5 As shown, the lanes in the electrophoresis image from left to right are: Lane 1 DL 5000 Marker, Lane 2 Control (control, i.e., linear EGFP mRNA without RNase R), Lane 3 0.01 μL RNase R-mRNA, Lane 4 0.05 μL RNase R-mRNA, Lane 5 0.1 μL RNase R-mRNA, Lane 6 0.15 μL RNase R-mRNA, Lane 7 0.2 μL RNase R-mRNA, Lane 8 0.25 μL RNase R-mRNA, and Lane 9 0.3 μL RNase R-mRNA. The mRNA in lanes 3-9 refers to the linear EGFP mRNA sample. Adding 0.25 μL of enzyme solution and catalyzing at 37 °C for 1 h completely degraded 10 μg of mRNA until no obvious band was observed in the final gel electrophoresis. The enzyme activity of the prepared parent enzyme RNase R was 6.67 U / μL, based on the final amount of enzyme used.

[0057] Example 5: Construction of RNase R mutant and detection of enzyme activity. Enzyme activity detection of enzyme solution.

[0058] 5.1 Construction of Escherichia coli RNase R mutant

[0059] Using PCR technology, primers targeting the parental RNase R gene sequence (DNA nucleotide sequence as shown in SEQ ID NO:2) amplified in Example 1 were designed and synthesized to perform site-directed mutagenesis on the *E. coli* RNase R gene, targeting the parental D122L, K205L, A246E, A246F, D281F, D281Y, K457F, T495E, L610W, and V687N mutations (lowercase letters represent mutant bases). The primers used for PCR are shown in Table 2. The amino acid sequence of the RNase R mutant is shown in Table 3. The amino acid sequences of the RNase R mutant D122L are shown in SEQ ID NO:23, the amino acid sequence of the mutant K205L is shown in SEQ ID NO:24, the amino acid sequence of the mutant A246E is shown in SEQ ID NO:25, the amino acid sequence of the mutant A246F is shown in SEQ ID NO:26, the amino acid sequence of the mutant D281F is shown in SEQ ID NO:27, the amino acid sequence of the mutant D281Y is shown in SEQ ID NO:28, the amino acid sequence of the mutant K457F is shown in SEQ ID NO:29, the amino acid sequence of the mutant T495E is shown in SEQ ID NO:30, the amino acid sequence of the mutant L610W is shown in SEQ ID NO:31, and the amino acid sequence of the mutant V687N is shown in SEQ ID NO:32.

[0060] The PCR reaction system consisted of: 2 μL of forward primer (10 μM), 2 μL of reverse primer (10 μM), 1 μL of template DNA, 25 μL of NEBNext Ultra IIQ5 Master Mix (manufacturer NEB, 10135601), and double-distilled water to a final volume of 50 μL.

[0061] PCR amplification conditions were as follows: pre-denaturation at 98℃ for 3 min; followed by 30 cycles (98℃ for 10 s, 55℃ for 15 s, 72℃ for 5 min); extension at 72℃ for 10 min. After verification of the PCR product's correctness, it was digested with DpnI and transformed into competent *E. coli* Tans10 cells. The competent cells were cultured overnight in LB solid medium (containing 50 mg / L kana), and clones were selected and cultured in LB liquid medium (containing 50 mg / L kana). Plasmids were extracted, and the mutant plasmids were transformed into competent *E. coli* BL21(DE3) cells expressing the host gene. All mutant plasmids were correctly sequenced. Recombinant strains were obtained and named D122L, K205L, A246E, A246F, D281F, D281Y, K457F, T495E, L610W, and V687N, respectively.

[0062] Table 2. Primer list for constructing RNase R mutants

[0063]

[0064]

[0065] Table 3 Amino acid sequences of RNase R mutants

[0066]

[0067]

[0068]

[0069]

[0070] 5.2 Expression and purification of Escherichia coli RNase R mutant

[0071] The RNase R mutant expression engineered strain constructed in step 5.1 was used to express the mutant RNase R according to Example 2 and to purify the RNase R mutant according to Example 3, finally obtaining the purified RNase R mutant enzyme.

[0072] 5.3 Enzyme activity detection of Escherichia coli RNase R mutant

[0073] The enzyme activity of the mutants was detected according to the method in Example 4. The degradation ability of wild-type parental E. coli RNase R (WT) and the mutants on mRNA was listed in Table 4. The results showed that the enzyme activities of the 10 RNase R mutants, D122L, K205L, A246E, A246F, D281F, D281Y, K457F, T495E, L610W, and V687N, were increased by 6.75%, -8.28%, 24.74%, 10.04%, 10.34%, -4.95%, 21.74%, 37.48%, -23.24%, and -8.1% respectively compared with the wild type (where negative values ​​represent decreases). Therefore, the T495E mutant showed the most significant increase in enzyme activity among the 10 RNase R mutants, with a relative increase of 37.48%. The second-highest relative enzyme activity was achieved by mutant A246E, with a 24.74% increase. The third-highest was mutant K457F, with a 21.74% increase. Mutants A246F and D281F showed increases of approximately 10%. Mutant D122L showed an increase of less than 10%, at 6.75%. Mutants K205L, D281Y, L610W, and V687N showed poor results, with varying degrees of decrease in relative enzyme activity. Specifically, for D281F and D281Y, different mutations at the same site had different effects; D281F showed an increase in relative enzyme activity, while D281Y showed a decrease. Thus, it can be seen that the present invention creatively selects and screens the RNase R site of Escherichia coli ribonuclease and the type of mutated amino acids to obtain mutants T495E, A246E, K457F, A246F, D281F, and D122L with significantly increased enzyme activity compared to the parental strain.

[0074] Table 4. Relative enzyme activities of wild-type Escherichia coli RNase R and multiple mutant enzymes.

[0075]

[0076] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A mutant ribonuclease R, characterized in that, The parental ribonuclease R was mutated to D122L, and the amino acid sequence of the parental ribonuclease R is shown in SEQ ID NO:

1.

2. The mutated ribonuclease R according to claim 1, characterized in that, The mutated ribonuclease R is mutant D122L, and the amino acid sequence of mutant D122L is shown in SEQ ID NO:

23.

3. The mutated ribonuclease R according to claim 1 or 2, characterized in that, The DNA sequence of the parental ribonuclease R is shown in SEQ ID NO.

2.

4. A recombinant nucleic acid, characterized in that, The nucleic acid comprising encoding the mutated ribonuclease R of any one of claims 1-3.

5. An expression box, characterized in that, It includes the recombinant nucleic acid as described in claim 4.

6. A carrier, characterized in that, It comprises the recombinant nucleic acid of claim 4 or the expression cassette of claim 5.

7. The carrier according to claim 6, characterized in that, The carrier is an expression carrier.

8. A cell, characterized in that, It comprises the recombinant nucleic acid of claim 4, or the expression cassette of claim 5, or the vector of claim 6 or 7.

9. A recombinant bacterium, characterized in that, It comprises the recombinant nucleic acid of claim 4, or the expression cassette of claim 5, or the vector of claim 6 or 7.

10. The recombinant bacteria according to claim 9, characterized in that, The bacteria in question is Escherichia coli.

11. The recombinant bacteria according to claim 10, characterized in that, The Escherichia coli is one of E. coli BL21 (DE3), E. coli Origami B (DE3), or E. coli Rosetta Blue (DE3).

12. The recombinant bacteria according to claim 11, characterized in that, The Escherichia coli mentioned is E. coli BL21(DE3).

13. A composition, characterized in that, It comprises the mutated ribonuclease R of any one of claims 1-3, or the recombinant nucleic acid of claim 4, or the expression cassette of claim 5, or the vector of claim 6 or 7, or the cell of claim 8, or the recombinant bacteria of any one of claims 9-12.

14. A reagent kit, characterized in that, The composition comprises the mutated ribonuclease R of any one of claims 1-3, or the recombinant nucleic acid of claim 4, or the expression cassette of claim 5, or the vector of claim 6 or 7, or the cell of claim 8, or the recombinant bacteria of any one of claims 9-12, or the composition of claim 13.

15. A method for preparing the mutated ribonuclease R according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Construct a vector containing a nucleotide sequence encoding the mutated ribonuclease R; (2) Transform the vector obtained in step (1) into the expression strain cells to obtain the expression strain; (3) Expand the expression strain obtained in step (2) and induce protein expression; (4) Collect the expression strains after expansion culture, and wash and lyse them; (5) Perform protein purification; (6) Perform enzyme activity detection.

16. The method according to claim 15, characterized in that, The step (3) comprises culturing the expression strain prepared in step (2) at 25-37°C to OD 600 is 0.6-1.0, and isopropyl-β-D-thiogalactoside (IPTG) is added to the medium to a final concentration of 0.1-1.5 mM, and the fermentation is induced at 16-37°C for 8-16 h to induce expression of the mutated ribonuclease R.

17. The method according to claim 15 or 16, characterized in that, Step (4) involves centrifuging the fermentation broth obtained in step (3) at 8000 rpm for 5 min and discarding the supernatant, then resuspending the cells in 50 mM PBS and centrifuging at 8000 rpm for 5 min and discarding the supernatant, adding PBS to resuspend the cells and mixing well; sonicating the cells on ice; placing the cell lysate in a pre-cooled centrifuge and centrifuging at 4°C and 8000 rpm for 10 min, collecting the precipitate, and redissolving the precipitate in a 500 mM KCl buffer solution.

18. A method for purifying the mutated ribonuclease R according to any one of claims 1-3, characterized in that, The process includes the following steps: the crude enzyme solution containing the mutated ribonuclease R obtained from fermentation is purified by nickel column purification using a protein purification system. The crude enzyme solution is eluted with an elution buffer of increasing concentration using a nickel column that has been pre-equilibrated with equilibration buffer. The target protein is collected and then analyzed by electrophoresis.

19. A method for detecting the enzyme activity of the mutated ribonuclease R according to any one of claims 1-3, characterized in that, The procedure includes the following steps: the mutant ribonuclease R is serially diluted, and the diluted mutant ribonuclease R is degraded at 37°C for 10 μg of linear mRNA. After 1 hour, the degradation is detected by gel electrophoresis until no obvious band is detected. The enzyme activity of the mutant ribonuclease R is calculated based on the amount of enzyme used when no obvious band is detected.

20. The application of the mutated ribonuclease R according to any one of claims 1-3 in the field of mRNA preparation.

21. The use of the mutated ribonuclease R according to any one of claims 1-3 in the field of degrading linear RNA or producing circRNA.

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