Ribonuclease r variants and uses thereof

Abstract

The present disclosure provides a plurality of RNase R variants and a preparation method thereof, wherein the expression amount of the variant RNase R is significantly increased relative to the parent enzyme, and the enzyme activity of the variant RNase R can be increased by 3 times, and the variant RNase R has a good industrial application prospect. In addition, the present disclosure also provides the application of the RNase R variant in the degradation of linear RNA and the preparation of circRNA.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biotechnology, in particular to a mutated ribonuclease R (RNase R) and its application. BACKGROUND

[0002] Circular RNA (circRNA) is a covalently closed single-stranded RNA, which does not contain 5'-Cap and 3'-polyA structure, and is not easily degraded by RNase. It has higher stability than linear mRNA and lower immunogenicity, which provides a new idea for the development of RNA vaccine. With the popularity of circRNA research in recent years, the underlying preparation process has also gradually attracted the attention of researchers. The production process of circRNA includes template preparation, in vitro transcription, DNA template removal, circularization and purification. After RNA circularization, there will be un-circularized impurities left, which can be removed by optimization of the later purification process. Compared with this complex process, there is also a method of degrading un-circularized impurities by RNase R, which is also a popular direction of process optimization.

[0003] Ribonuclease R (RNase R) belongs to the 3'→5' exonuclease of the RNase II superfamily, which can gradually cut RNA into dinucleotides and trinucleotides from the 3'→5' direction. Except for circular RNA, lariat structure RNA and short double-stranded RNA molecules with less than 7 nucleotides of 3' overhang, RNase R can digest all linear RNA. Therefore, in the process of circRNA preparation, the use of RNase R digestion can achieve the relative enrichment of circRNA.

[0004] The most common RNase R currently comes from Escherichia coli. The enzyme is difficult to express under conventional expression conditions, and has low enzyme activity, which does not meet the needs of economic production. Although various 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 a RNase R that meets the needs of economic use, has high expression level and high enzyme activity. SUMMARY

[0005] Therefore, the purpose of the present disclosure is to provide a RNase R variant with high expression and high enzyme activity, which has good industrial application prospects. In addition, the present disclosure also provides a preparation method of the variant and its application in degrading linear RNA and preparing circRNA.

[0006] To achieve the above-mentioned purpose, the present disclosure provides the following technical solutions:

[0007] The present disclosure provides RNase R variants having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1, and the variants comprise at least one mutation selected from the following amino acid positions: T627, S309, M692, the mutation type being substitution or deletion.

[0008] 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.

[0009] In some embodiments, the RNase R variants comprise any one of the following mutations selected from the following amino acid positions: T627I, S309G, M692V.

[0010] In some embodiments, any of the variants described in the present disclosure has improved enzymatic activity and / or increased expression level compared to RNase R shown in SEQ ID NO: 1.

[0011] The present disclosure also provides RNase R variants having improved enzymatic activity and / or increased expression level, which have an amino acid sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0012] 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.

[0013] The present disclosure also provides nucleic acids encoding any of the RNase R variants 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 the variants without changing the amino acid sequence.

[0014] In some embodiments, the nucleic acid sequence is shown in SEQ ID NO: 6. In some embodiments, the nucleic acid sequence is shown in SEQ ID NO: 7. In some embodiments, the nucleic acid sequence is shown in SEQ ID NO: 8.

[0015] The present disclosure also provides expression vectors or cloning vectors comprising the above-mentioned nucleic acids. In some embodiments, the backbone of the vector is pET-30a(+) vector.

[0016] 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 E. coli.

[0017] 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 one of the nucleic acid sequences in SEQ ID NO: 6-8 to a pET-30a(+) vector, transforming into E. coli for expression and purification, and obtaining the RNase R variant through column chromatography.

[0018] 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.

[0019] 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.

[0020] The present disclosure also provides the use of the RNase R variant in degrading linear RNA.

[0021] The present disclosure also provides the use of the RNase R variant in circRNA preparation.

[0022] Advantages

[0023] 1. The RNase R variant provided by the present disclosure has at least 1.5 times higher relative enzyme activity compared to the RNase R shown in SEQ ID NO: 1, and is suitable for degrading 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.

[0024] 2. The RNase R variant preparation method provided by the present disclosure can significantly improve the expression amount of the variant, and has good industrial application prospects. SUMMARY

[0025] Figure 1 It is a schematic diagram for construction of recombinant plasmid;

[0026] Figure 2 It is the expression of wild-type RNase R and its variants;

[0027] Figure 3 HPLC detection results of RNase R of WT group;

[0028] Figure 4 HPLC detection results of V1 variant;

[0029] Figure 5 HPLC detection results of V2 variant;

[0030] Figure 6 HPLC detection results of V3 variant;

[0031] Figure 7 Relative enzyme activity of wild type RNase R and its variants.

[0032] In the figure, the correspondence between the RNase R variant name and the amino acid is shown in Table 1.

[0033] 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

[0034] The technical solutions of the present disclosure will be further described below in combination with specific examples. However, the following examples are only examples of the present disclosure and do not represent or limit the protection scope of the present disclosure. The protection scope of the present disclosure is subject to the claims. In the following examples, if not specifically stated, the reagents and consumables used are purchased from ordinary suppliers in the art, and the experimental methods and technical means used are conventional methods and means in the art.

[0035] Example 1 Preparation and expression determination of RNase R variants

[0036] His tags were added to the front of the amino acid sequences of wild type RNase R and its variants SEQ ID NO: 1-4, and after DNA sequence synthesis, PCR amplification (DNA sequences SEQ ID NO: 5-8) was performed, then the Ndel and Xhol enzyme digestion sites of the expression vector pET-30a(+) were introduced to obtain a recombinant expression vector, which was transformed into E. coli BL21(DE3). After screening by antibiotic-resistant plate coating, a clonal strain was obtained. After verifying the success of recombination, the obtained strain was activated overnight at 37°C in LB medium, and then 200 mL of fermentation broth (LB medium) was added. The culture was incubated to an OD 600 value of 0.6-0.8, and then 0.5 mM IPTG was added to continue incubation for 4-6 h. The strain was collected by centrifugation at 12000 rpm and 5°C, and the collected strain was washed with 0.2M PBS buffer with a pH value of 7.0. After ultrasonic crushing with 15 mL of buffer, a crude enzyme solution was obtained. SDS-PAGE was used to verify the protein expression amount, and the detection results showed that the expression amount of V1 and V3 variants was significantly higher than that of WT group (P<0.05). Figure 2).

[0037] After the above-mentioned crude enzyme solution was purified by AKTA protein purification instrument, HPLC was used for protein concentration detection, and the detection method was as follows:

[0038] Neutral hydrophilic silica gel high-performance size exclusion column (Nano BioCore SEC-150, 5 μm, 7.8 x 300 mm);

[0039] Detection wavelength: 280 nm;

[0040] Mobile phase: 150 mM phosphate buffer (pH 6.8);

[0041] Flow rate: 1.0 mL / min

[0042] Injection volume: 20 μL.

[0043] The detection results are shown in Figures 3-6 , which further proves that the expression amounts of V1 and V3 variant proteins are much higher than that of the WT group.

[0044] Example 2: RNase R enzyme activity determination

[0045] According to the enzyme protein concentration after purification, the enzyme activity was estimated, and the purified enzyme of Example 1 and RNase R standard (Vazyme, item number JE2303KA) were gradient diluted from 0.15 U / μL to 0.000771 U / μL, 1.5 times continuous dilution, 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, reacted at 37°C for 20 min, and reacted at 70°C for 10 min.

[0046] Table 2. 10 x RNase R Reaction Buffer ratio

[0047] Component 50 ml (reference value) 1 M Tris-HCl (pH 8.0, 25°C) 10ml 2 M KCl 25ml 2 M MgCl2 200 μl DEPC water 14.8ml

[0048] Table 3. Reaction system ratio

[0049]

[0050] The fluorescence dye mixture was prepared according to Table 4, 180 μL of fluorescence dye mixture was added to the reacted liquid, mixed well, added to a black enzyme plate, and the fluorescence value was detected by an enzyme marker. The enzyme activity of the sample to be tested = EC50 标准品 / EC50 待测样品 x estimated enzyme activity.

[0051] Table 4. Reaction system ratio (Vazyme, item number EQ212)

[0052] Component Volume (μl) Equalbit RNA BR Reagent 2.5 Equalbit RNA BR Buffer 200

[0053] The results of the detection are shown in Table 2 Figure 7 The relative enzyme activity of the V1-V3 variants was significantly improved, and the relative enzyme activity of the V1 variant was about 3 times that of the WT group.

Claims

1. A RNase R variant, whose amino acid sequence is shown in any one of SEQ ID NO: 2-4.

2. A nucleic acid encoding the variant of claim 1.

3. The nucleic acid of claim 2, whose nucleotide sequence is shown in any one of SEQ ID NO: 6-8.

4. An expression vector comprising the nucleic acid of claim 2 or 3.

5. A host cell transformed with the expression vector of claim 4.

6. A method for preparing the variant of claim 1, comprising culturing the host cell of claim 5, and recovering the RNase R variant.

7. A composition comprising: 1) at least one RNase R variant of claim 1; or 2) at least one nucleic acid of claim 2 or 3; or 3) at least one expression vector of claim 4; or 4) at least one host cell of claim 5.

8. A kit comprising: 1) at least one RNase R variant of claim 1; or 2) at least one nucleic acid of claim 2 or 3; or 3) at least one expression vector of claim 4; or 4) at least one host cell of claim 5; or 5) the composition of claim 7.

9. Use of the RNase R variant of claim 1 in degrading linear RNA.

10. Use of the RNase R variant of claim 1 in circRNA preparation.

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