Ribonuclease R variant and application thereof

Abstract

The invention provides a plurality of RNase R variants and a preparation method thereof, compared with a parent enzyme, the expression quantity of the variants RNase R disclosed by the invention is remarkably increased, the enzyme activity of the variants RNase R can be improved by 3 times, and the RNase R has a good industrial application prospect. In addition, the invention also provides an application of the RNase R variant in degradation of linear RNA and preparation of circRNA.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a mutant ribonuclease R (RNase R) and applications thereof. Background of the Invention

[0003] Circular RNA (circRNA) is a covalently closed single-stranded RNA that does not contain 5'-Cap and 3'-polyA structures and is not easily degraded by RNase. Compared with linear mRNA, it has higher stability and lower immunogenicity, providing a new idea for the development of RNA vaccines. As circRNA research has become popular in recent years, the underlying preparation process has gradually attracted the attention of researchers. The production process of circRNA includes: template preparation, in vitro transcription, DNA template removal, cyclization and purification. After RNA cyclization, uncirculated impurities will remain, which can be removed by optimizing the subsequent purification process. Compared with this complex process flow, there is also a method of degrading uncirculated impurities by RNaseR, which is also a popular direction for process optimization.

[0004] Ribonuclease R (RNase R) belongs to the 3'→5' exoribonuclease of the RNase II superfamily, which can gradually cut RNA into dinucleotides and trinucleotides from the 3'→5' direction. RNase R can usually digest all linear RNAs except circular RNA, lasso RNA, and short double-stranded RNA molecules with less than 7 nucleotides protruding from the 3'. Therefore, the relative enrichment of circRNA can be achieved by using RNase R digestion during the preparation of circRNA.

[0005] The most common RNase R currently originates from Escherichia coli. Under conventional expression conditions, the enzyme is difficult to express and has low enzyme activity, which does not meet the requirements of economical production. Although a variety of RNase R variants with improved performance have been disclosed in the prior art (CN114438054A, CN116240199A, etc.), there is still a need in the art for an RNase R that meets economical use, has a high expression level and high enzyme activity. Summary of the invention

[0006] In view of this, the purpose of the present disclosure is to provide an RNase R variant, which has the characteristics of high expression and high enzyme activity and has good industrial application prospects. In addition, the present disclosure also provides a method for preparing the variant and its application in the preparation of degraded linear RNA and circRNA.

[0007] To achieve the above objectives, the present disclosure provides the following technical solutions:

[0008] The present disclosure provides an RNase R variant, the amino acid sequence of which has at least 95% sequence identity with SEQ ID NO: 1, and the variant comprises at least one mutation selected from the following amino acid sites: T627, S309, M692, and the mutation type is substitution or deletion.

[0009] In some embodiments, the amino acid T at position 627 is substituted with I. In some embodiments, the amino acid S at position 309 is substituted with G. In some embodiments, the amino acid M at position 692 is substituted with V.

[0010] In some embodiments, the RNase R variant comprises any one mutation selected from the following amino acid positions: T627I, S309G, M692V.

[0011] In some embodiments, any variant described in the present disclosure has improved enzymatic activity and / or increased expression level compared to the RNase R set forth in SEQ ID NO:1.

[0012] The present disclosure also provides RNase R variants with improved enzymatic activity and / or expression level, whose amino acid sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0013] In some embodiments, the amino acid sequence of the variant is shown in SEQ ID NO: 2. In some embodiments, the amino acid sequence of the variant is shown in SEQ ID NO: 3. In some embodiments, the amino acid sequence of the variant is shown in SEQ ID NO: 4.

[0014] The present disclosure also provides nucleic acids encoding any RNase R variant of the present disclosure. Due to the degeneracy of codons or the preference of host cells expressing polypeptides for codons, the nucleic acid sequence can be any nucleic acid sequence encoding a variant without changing the amino acid sequence.

[0015] In some embodiments, the nucleic acid sequence is as shown in SEQ ID NO: 6. In some embodiments, the nucleic acid sequence is as shown in SEQ ID NO: 7. In some embodiments, the nucleic acid sequence is as shown in SEQ ID NO: 8.

[0016] The present disclosure also provides an expression vector or a cloning vector comprising the above nucleic acid. In some embodiments, the backbone of the vector is a pET-30a(+) vector.

[0017] The present disclosure also provides a host cell transformed or transfected with the vector. In some embodiments, the host cell is selected from a prokaryotic cell, a yeast cell or a eukaryotic cell. In some embodiments, the host cell is Escherichia coli.

[0018] The present disclosure also provides a method for preparing the RNase R variant, comprising: culturing the host cell and recovering the RNase R variant. In some embodiments, the method comprises: connecting any nucleic acid sequence in SEQ ID NO: 6-8 to a pET-30a(+) vector, transferring the nucleic acid sequence into Escherichia coli for expression and purification, and obtaining the RNase R variant by column chromatography.

[0019] The present disclosure also provides a composition, comprising: at least one RNase R variant as described in the present disclosure; or at least one nucleic acid encoding the RNase R variant as described in the present disclosure; or at least one expression vector as described in the present disclosure; or at least one host cell as described in the present disclosure.

[0020] The present disclosure also provides a kit, comprising: at least one RNase R variant as described in the present disclosure; or at least one nucleic acid encoding the RNase R variant as described in the present disclosure; or at least one expression vector as described in the present disclosure; or at least one host cell as described in the present disclosure; or a composition as described in the present disclosure.

[0021] The present disclosure also provides use of the RNase R variant in degrading linear RNA.

[0022] The present disclosure also provides the use of the RNase R variant in the preparation of circRNA.

[0023] Beneficial Effects

[0024] 1. The RNase R variant provided by the present disclosure has a relative enzyme activity at least 1.5 times higher than that of the RNase R shown in SEQ ID NO: 1; it is suitable for the degradation of linear RNA in the industrial circRNA preparation process to obtain high-purity and high-expression circRNA products, which is of great significance for the preparation of circRNA products;

[0025] 2. The method for preparing the RNase R variant provided in the present disclosure can significantly increase the expression level of the variant and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the construction of recombinant plasmid;

[0028] Figure 2 is the expression of wild-type RNase R and its variants;

[0029] Figure 3 This is the HPLC test result of RNase R in the WT group;

[0030] Figure 4 is the HPLC test result of V1 variant;

[0031] Figure 5 is the HPLC test result of V2 variant;

[0032] Figure 6 HPLC test results of V3 variant;

[0033] Figure 7 is the relative enzymatic activity of wild-type RNase R and its variants.

[0034] The correspondence between the RNase R variant names and amino acids in the figure is shown in Table 1.

[0035] Variant Name Amino acid sequence WT SEQ ID NO: 1 V1 SEQ ID NO: 2 V2 SEQ ID NO: 3 V3 SEQ ID NO: 4 DETAILED DESCRIPTION

[0036] The technical solution of the present disclosure is further described below in conjunction with specific embodiments. However, the following embodiments are merely examples of the present disclosure and do not represent or limit the scope of protection of the present disclosure. The scope of protection of the present disclosure shall be subject to the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the field, and the experimental methods and technical means used are conventional methods and means in the field.

[0037] Example 1 Preparation of RNase R variants and expression level determination

[0038] A His tag was added to the front end of the amino acid sequence SEQ ID NO: 1-4 of the wild-type RNase R and its variants, and PCR amplification was performed after DNA sequence synthesis (DNA sequence SEQ ID NO: 5-8), and then the NdeI and XhoI restriction sites of the expression vector pET-30a (+) were introduced to obtain a recombinant expression vector, which was transformed into E. coli BL21 (DE3), and after antibiotic resistance plate coating screening, a cloned strain was obtained. After successful recombination, the obtained strain was activated overnight in LB medium at 37°C and added to 200 mL of fermentation broth (LB medium) and cultured until the OD 600 value was 0.6-0.8. After adding IPTG with a final concentration of 0.5 mM and continuing to culture for 4-6 hours, the strain was collected by centrifugation at 12000 rpm and 5°C. The collected strain was washed with 0.2 M PBS buffer with a pH value of 7.0, and 15 mL of buffer was added to obtain a crude enzyme solution after ultrasonic disruption. The protein expression was verified by SDS-PAGE running. The test results showed that the expression levels of V1 and V3 variants were significantly higher than those of the WT group ( Figure 2).

[0039] After the crude enzyme solution was purified by AKTA protein purifier, the protein concentration was detected by HPLC. The detection method was as follows:

[0040] Neutral hydrophilic silica high-efficiency size exclusion column (Nano BioCore SEC-150, 5 μm, 7.8 × 300 mm);

[0041] Detection wavelength: 280nm;

[0042] Mobile phase: 150 mM phosphate buffer (pH 6.8);

[0043] Flow rate: 1.0mL / min

[0044] Injection volume: 20 μL.

[0045] Test results see Figure 3-Figure 6 , further confirming that the expression levels of V1 and V3 variant proteins far exceeded those of the WT group.

[0046] Example 2 RNase R enzyme activity assay

[0047] The enzyme activity was estimated based on the concentration of the purified enzyme protein, and the purified enzyme of Example 1 and the RNase R standard (Vazyme, catalog number JE2303KA) were diluted in a gradient from 0.15 U / μL to 0.000771 U / μL, 1.5-fold serial dilution, for a total of 14 gradients. The enzyme activity reaction Mix was prepared according to the reaction system in Table 3, the diluted purified enzyme was added, vortexed and centrifuged, and the reaction was carried out at 37°C for 20 min and at 70°C for 10 min.

[0048] Table 2. 10×RNase R Reaction Buffer Ratio

[0049] Components 50ml(reference value) 1M Tris-HCl (pH 8.0, 25°C) 10ml 2M KCl 25ml 2MMgCl2 200 μl DEPC water 14.8ml

[0050] Table 3. Reaction system ratio

[0051]

[0052] Prepare the fluorescent dye mixture according to Table 4, add 180 μL of the fluorescent dye mixture to the liquid after the reaction, mix well, add to the black ELISA plate, and use an ELISA instrument to detect the fluorescence value. The enzyme activity of the sample to be tested = EC50 标准品 / EC50 待测样品 ×Estimated enzyme activity.

[0053] Table 4. Reaction system ratio (Vazyme, product number EQ212)

[0054] Components Volume (μl) EqualbitRNABRReagent 2.5 EqualbitRNABRBuffer 200

[0055] Test results see Figure 7 The enzyme activities of V1-V3 variants were significantly improved, among which the relative enzyme activity of V1 variant was about 3 times that of the WT group.

Claims

1. An RNase R variant, the amino acid sequence of which is at least 95% identical to the sequence shown in SEQ ID NO: 1, and comprises at least one mutation selected from the following amino acid positions: T627I, S309G, M692V.

2. The variant according to claim 1, whose amino acid sequence is shown in any one of SEQ ID NOs: 2-4.

3. A nucleic acid encoding a variant according to claim 1 or 2. The nucleic acid according to claim 3 , wherein the nucleotide sequence is shown in any one of SEQ ID NOs: 6-8.

5. An expression vector comprising the nucleic acid according to claim 3 or 4.

6. A host cell transformed with the expression vector according to claim 5.

7. A method for preparing the variant according to claim 1 or 2, comprising culturing the host cell according to claim 6 and recovering the RNase R variant.

8. A composition comprising: 1) at least one RNase R variant according to claim 1 or 2; or 2) at least one nucleic acid according to claim 3 or 4; or 3) at least one expression vector according to claim 5; or 4) At least one host cell according to claim 6.

9. A kit comprising: 1) at least one RNase R variant according to claim 1 or 2; or 2) at least one nucleic acid according to claim 3 or 4; or 3) at least one expression vector according to claim 5; or 4) at least one host cell according to claim 6; or 5) The composition according to claim 8.

10. Use of the RNase R variant according to claim 1 or 2 in degrading linear RNA.

11. Use of the RNase R variant as claimed in claim 1 or 2 in the preparation of circRNA.

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