Ribonuclease r variants and uses thereof

Abstract

This disclosure provides several ribonuclease R variants and their preparation methods. Compared to the parent enzyme, the variants described in this disclosure exhibit increased protease activity per unit and show promising prospects for industrial applications. Furthermore, this disclosure also provides the application of ribonuclease R variants in linear RNA degradation.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to variants of ribonuclease R and their applications. Background of the Invention

[0002] Circular RNA (circRNA) is a covalently closed single-stranded RNA that lacks 5'-Cap and 3'-polyA structures, making it less susceptible to RNase degradation. Compared to linear mRNA, circRNA exhibits higher stability and lower immunogenicity, providing a novel approach for RNA vaccine development. With the recent surge in circRNA research, the underlying preparation process has also garnered significant attention. The circRNA production process includes template preparation, in vitro transcription, DNA template removal, circularization, and purification. After RNA circularization, uncirculated impurities remain, which can be removed through optimization of the subsequent purification process. In contrast to this complex process, another method involves degrading uncirculated impurities using RNase R, which is also a popular area for process optimization.

[0003] Ribonuclease R (RNase R) belongs to the RNase II superfamily and is a 3'→5' exonuclease that stepwise cleaves RNA into dinucleotides and trinucleotides along the 3'→5' direction. Except for circular RNA, lasso-structured RNA, and short double-stranded RNA molecules with fewer than 7 nucleotides overhang at the 3' end, RNase R can generally digest all linear RNA. Therefore, using RNase R digestion during circRNA preparation can achieve relative enrichment of circRNA.

[0004] The most common RNase R currently derived from E. coli is difficult to express under conventional expression conditions, resulting in low enzyme activity, which does not meet the requirements for economical production. Although several 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 with high specific activity that is suitable for economical use. Summary of the Invention

[0005] In view of this, the purpose of this disclosure is to provide an RNase R variant with high specific activity and promising prospects for industrial application. Furthermore, this disclosure also provides a method for preparing this variant and its application in the degradation of linear RNA and circRNA.

[0006] To achieve the above objectives, this disclosure provides the following technical solutions:

[0007] This disclosure provides an RNase R variant whose amino acid sequence has at least 95% sequence identity with SEQ ID NO: 1, and the variant contains at least one mutation selected from the following amino acid sites: T627, H565, wherein the mutation type is substitution or deletion.

[0008] In some embodiments, the amino acid at position 627 is substituted with S or K. In some embodiments, the amino acid at position 565 is substituted with K.

[0009] In some implementations, the RNase R variant contains a mutation at any of the following amino acid sites: T627K, T627S, or H565K.

[0010] In some embodiments, any variant described in this disclosure has an enhanced specific activity compared to wild-type RNase R with an amino acid sequence as shown in SEQ ID NO: 1.

[0011] This disclosure also provides RNase R variants with enhanced specific activity, the amino acid sequences of which have at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences 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] This disclosure also provides nucleic acids encoding any RNase R variant of this disclosure. Due to codon degeneracy or codon preference of the host cell expressing the polypeptide, the nucleic acid sequence can be any nucleic acid sequence encoding the variant without altering the amino acid sequence.

[0014] 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.

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

[0016] This disclosure also provides host cells for transforming or transfecting the vector. In some embodiments, the host cells are selected from prokaryotic cells, yeast cells, or eukaryotic cells. In some embodiments, the host cells are *Escherichia coli*. In some embodiments, the host cells are BL21(DE3).

[0017] This disclosure also provides a method for preparing the RNase R variant, comprising: culturing the host cells and recovering the RNase R variant. In some embodiments, the method comprises: ligating any one of the nucleic acid sequences in SEQ ID NO: 6-8 into a pET-30a(+) vector, transforming it into Escherichia coli for expression and purification to obtain the RNase R variant.

[0018] This disclosure also provides a composition comprising: at least one RNase R variant as described in this disclosure; or at least one nucleic acid encoding an RNase R variant as described in this disclosure; or at least one expression vector as described in this disclosure; or at least one host cell as described in this disclosure.

[0019] This disclosure also provides a kit comprising: at least one RNase R variant as described in this disclosure; or at least one nucleic acid encoding an RNase R variant as described in this disclosure; or at least one expression vector as described in this disclosure; or at least one host cell as described in this disclosure; or a composition as described in this disclosure.

[0020] This disclosure also provides the use of the RNase R variant in the degradation of linear RNA.

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

[0022] Beneficial effects

[0023] 1. The RNase R variant provided in this disclosure has a higher specific activity compared with the wild-type RNase R shown in SEQ ID NO: 1. The increased specific activity can reduce the amount of reaction feed, thereby reducing the enzyme residue rate. Furthermore, the high specific activity RNase R variant 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. Brief description of the attached diagram

[0024] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids;

[0025] Figure 2 The expression of wild-type RNase R and its variants;

[0026] Figure 3 The specific activity values ​​are for wild-type RNase R and its variants.

[0027] The correspondence between RNase R variant names and amino acids in the figure is shown in Table 1.

[0028] Table 1: Correspondence between RNase R variants and amino acids

[0029]

[0030]

[0031] Detailed Implementation

[0032] The technical solutions of this disclosure are further illustrated below with reference to specific embodiments. However, the following embodiments are merely examples of this disclosure and do not represent or limit the scope of protection of this disclosure. The scope of protection of this disclosure is determined by the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the art, and the experimental methods and techniques used are conventional methods and techniques in the art.

[0033] In this embodiment of the disclosure, the unit enzyme activity (U) is defined as the enzyme required to convert 1 μg poly(A) into acid-soluble nucleotides in 10 minutes under standard reaction conditions at 37°C.

[0034] Example 1: Preparation and expression level determination of RNase R variant

[0035] A His tag was added to the front of the amino acid sequences of wild-type RNase R and its variants (SEQ ID NO: 1-4). After DNA sequence synthesis, PCR amplification was performed (DNA sequence SEQ ID NO: 5-8). The DNA was then introduced into the NdeI and XhoI restriction sites of the expression vector pET-30a(+) to obtain a recombinant expression vector, which was transformed into E. coli BL21(DE3). After screening by antibiotic resistance plate plating, cloned strains were obtained. After successful recombination, the obtained strain was activated overnight at 37°C in LB medium and then added to 200 mL of fermentation broth (LB medium). The strain was cultured until the OD600 value reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mM, and the strain was cultured for another 4–6 hours. The strain was then collected by centrifugation at 12,000 rpm and 5°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0), and 15 mL of buffer was added for ultrasonic disruption to obtain crude enzyme solution. The crude enzyme solution was purified using an AKTA protein purifier (Bio-lab 100) and its expression was analyzed by SDS-PAGE.

[0036] The test results are as follows Figure 2 As shown, all of the above mutants can achieve normal expression.

[0037] Example 2: RNase R enzyme activity assay

[0038] Enzyme activity was estimated based on the concentration of purified enzyme protein, and the purified enzyme from Example 1 and RNase R standard (epicentre, catalog number RNRO7250) were serially diluted 1.5-fold from 0.15 U / μL to 0.000771 U / μL, for a total of 14 gradients. The enzyme activity reaction mix was prepared according to the reaction system in Table 3, and the diluted purified enzyme (4 μL) was added. The mixture was vortexed, centrifuged, and reacted at 37°C for 20 min, then at 70°C for 10 min.

[0039] Prepare the fluorescent dye mixture according to Table 4. Add 180 μL of the fluorescent dye mixture to the reacted liquid and mix well. Pour the mixture into a black ELISA plate and detect the fluorescence value using an ELISA reader. The enzyme activity of the sample to be tested is EC50. 标准品 / EC50 待测样品 ×Estimated enzyme activity. Specific activity = enzyme activity / protein content.

[0040] Table 2.10×RNase R ReactionBuffer Ratio

[0041] Components 50ml (reference value) 1 M Tris-HCl (pH 8.0, 25°C) 10ml 2MKCl 25ml 2MMgCl2 200μl DEPC water 14.8ml

[0042] Table 3. Reaction system ratio

[0043]

[0044] Table 4. Reaction system composition (Vazyme, catalog number EQ212)

[0045] Components Volume (μl) EqualbitRNABRReagent 2.5 EqualbitRNABRBuffer 200

[0046] Test results are shown Figure 3 All mutants effectively increased specific activity, and the H565K variant significantly increased specific activity from 26.33 U / μg of wild type to 83.50 U / μg, an increase of >300%.

Claims

1. A RNase R variant, characterized in that, The amino acid sequence of the variant has at least 95% identity with the sequence shown in SEQ ID NO: 1, and contains at least one mutation selected from the following amino acid sites: T627S, T627K, H565K.

2. The variant according to claim 1, characterized in that, The amino acid sequence is shown in any of SEQ ID NO: 2-4.

3. The nucleic acid encoding the variant of claim 1 or 2.

4. The nucleic acid of claim 3, wherein, The nucleotide sequence of the nucleic acid is shown in any of SEQ ID NO: 6-8.

5. An expression vector, characterized in that, It contains the nucleic acid as described in claim 3 or 4.

6. A host cell characterized in that, The host cell was transformed with the expression vector of claim 5.

7. The 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 as described in claim 1 or 2; or 2) At least one nucleic acid as described in claim 3 or 4; or 3) At least one expression vector as described in claim 5; or 4) At least one host cell as described in claim 6.

9. Reagent kit, containing: 1) At least one RNase R variant as described in claim 1 or 2; or 2) At least one nucleic acid as described in claim 3 or 4; or 3) At least one expression vector as described in claim 5; or 4) At least one host cell as described in claim 6; or 5) The composition as described in claim 8.

10. The use of the RNase R variant as described in claim 1 or 2 in the degradation of linear RNA.

11. The use of the RNase R variant as described in claim 1 or 2 in the preparation of circRNA.

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