Method for purifying circular RNA using thermostable nucleic acid exonuclease

Abstract

The present invention relates to a technique for purifying high-purity circular RNA by efficiently removing linear RNA impurities by using a 3'-exonulcease that can be activated at high temperatures. In particular, RNase R (gsRNR) derived from Geobacillus stearothermophilus, when excavated and purified and optimized to maintain stable and selective 3'-exonuclease activity at even high temperatures, is able to effectively decompose linear RNA, comprising a hairpin structure or ribozyme structure, which are difficult to remove with existing RNase R derived from E. coli. The enzyme according to the present invention can produce high-purity circular RNA without an auxiliary treatment such as poly A polymerase, and simplify purification processes and improve production yield, and thus can be effectively used for biopharmaceutical production, such as the development of RNA-based therapeutic agents and vaccines.

Description

Method for purifying circular RNA using a thermostable nuclease

[0001] The present invention relates to the purification of circular RNA, and more particularly, to a technique for purifying circular RNA by removing linear RNA impurities using a 3'-ribonuclease that is active at high temperatures.

[0002] Circular RNA (Circular RNA) has recently emerged as a promising protein expression platform and is being applied to the development of various biopharmaceuticals, including mRNA vaccines, gene therapies, and cell therapies. In particular, circular RNA eliminates the need for a 5' cap and 3' poly(A) tail, exhibits high structural stability, and has low immunostimulatory potential, enabling it to offer superior expression sustainability and safety compared to conventional linear mRNA. Due to these advantages, circular RNA is being actively researched as a next-generation RNA therapeutic and vaccine platform, and its potential applications are expanding to diverse fields such as anticancer immunotherapy, gene regulation, and intracellular translation control.

[0003] The global RNA-based therapeutics market was valued at approximately $11 billion in 2021 and is projected to reach approximately $35 billion by 2030, growing at a compound annual growth rate of over 14%. In particular, with the growth of the mRNA-based therapeutics and vaccine markets, demand for circular RNA platforms with superior stability and expression efficiency is rapidly increasing. Consequently, the development of purification technologies for the rapid and efficient production of high-purity circular RNA is becoming increasingly important.

[0004] The circular RNA manufacturing process typically involves the following steps: (1) linear RNA generation via in vitro transcription (IVT), (2) circularization using ligase or ribozyme, and (3) removal of linear RNA impurities. Among these steps, the removal of linear RNA impurities remaining after circularization is crucial for ensuring the purity and quality of the final circular RNA and directly impacts purification efficiency and production yield.

[0005] Currently, 3' exonucleases, such as E. coli-derived RNase R (ecRNR), are mainly used to remove linear RNA. However, these enzymes have the following limitations: (i) low processing efficiency for RNA substrates with double-stranded ends or complex structures, (ii) at high temperatures, most RNA double strands unwind, so complex-structured RNAs are also likely to be degraded by the enzyme, but the enzymes also tend to be inactivated or degraded at high temperatures, (iii) some exhibit non-specific endonuclease activity, which may damage the product RNA. In particular, ribozyme-based circularization methods are prone to generating linear byproducts with complex structures, and ecRNR cannot effectively remove them, requiring additional enzymatic treatment (PAP, XRN1, etc.), complicating the process.

[0006] Therefore, the discovery of novel endonucleases that are stable even at high temperatures and can effectively remove RNA with complex structures is essential. Such enzymes could significantly contribute to process simplification, shortened reaction times, improved purification efficiency, and reduced production costs, and could serve as a key technology for increasing the commercial viability of next-generation RNA-based pharmaceuticals.

[0007] The technical problem to be achieved by the present invention is to solve the problem of existing ecRNR being inactivated at high temperatures or exhibiting endonuclease activity, and to provide a high-temperature stable 3'-ribonuclease endonuclease capable of efficiently removing even RNA impurities having complex terminal structures, thereby providing an economical and effective circular RNA purification method.

[0008] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0009] For economical and effective removal of impurities in the production of circular RNA, the present invention provides RNase R (gsRNR) derived from Geobacillus stearothermophilus.

[0010] In the present invention, the gsRNR may include or consist of the amino acid sequence of sequence number 1.

[0011] As another embodiment of the present invention, the gsRNR may be fused with a tag for purification, and the tag may be a His tag, and the gsRNR fused with the His tag may include or consist of the amino acid sequence of SEQ ID NO: 2.

[0012] The present invention provides a method for purifying circular RNA, comprising the step of treating gsRNR in a solution containing circular RNA and linear RNA.

[0013] The above method may include the following steps:

[0014] (1) A step of preparing RNase R (gsRNR) derived from Geobacillus stearothermophilus;

[0015] (2) a step of inducing in vitro transcription of a circular RNA precursor expression vector; and

[0016] (3) A step of treating the gsRNR in a test tube after the completion of the transcription induction step.

[0017] As one embodiment of the present invention, the step (1) may include a step of culturing a bacterium transformed with a gsRNR expression vector, and may further include a step of purifying and obtaining gsRNR expressed by the bacterium.

[0018] As another embodiment of the present invention, the bacteria may be Escherichia coli.

[0019] As another embodiment of the present invention, the gsRNR may be fused with a His tag, and in this case, the gsRNR fused with the His tag may be expressed in a state in which a Hig tag is fused in the gsRNR expression vector.

[0020] As another embodiment of the present invention, the method may further include a step of increasing the temperature of the reaction mass to 45 to 70°C after the in vitro transcription induction step is completed.

[0021] As another embodiment of the present invention, the step (3) may be performed under a temperature condition of 45 to 70°C.

[0022] As another embodiment of the present invention, the method may further include a step of removing the circular RNA precursor expression vector by treating the circular RNA precursor with DNase in a test tube after (2), and the step may be performed before, after, or simultaneously with step (3).

[0023] The present invention provides a technology for purifying highly pure circular RNA by efficiently removing linear RNA with complex terminal structures using RNase R (gsRNR) derived from Geobacillus stearothermophilus that operates stably even at high temperatures. The enzyme of the present invention exhibits superior degradation efficiency for RNA substrates with double-stranded or tertiary structures at the 3'-end compared to existing RNase R derived from E. coli, and maintains activity even at high temperatures (60-65°C) while exhibiting almost no non-specific cleavage (endonuclease activity), resulting in high purification accuracy. Furthermore, purification is possible without auxiliary enzymes such as poly A polymerase, simplifying the process and reducing production costs. In particular, it is possible to increase the yield by about two times or more compared to existing methods for long circular RNAs. This technology is expected to have great practical and industrial value in the production process of biopharmaceuticals, such as the development of RNA-based therapeutics and vaccines.

[0024] Figure 1 shows the results of a comparative analysis of the sequence of ecRNR and the sequence of RNase R homologs of thermophilic bacteria discovered from genome sequence information. The red box indicates a region important for 3' exonuclease activity.

[0025] (1) gsRNR: WP_277392187.1 [Geobacillus stearothermophilus]

[0026] (2) tmRNR: AGL49648.1 [Thermotoga maritima MSB8]

[0027] (3) tcRNR: WP_038060459.1 [Thermodesulfobacterium commune]

[0028] (4) pgRNR: WP_043905984.1 [Parageobacillus genomosp. 1]

[0029] (5) ecRNR: E. coli RNase R

[0030] (6) ttRNR: WP_024119831.1 [Thermus thermophilus]

[0031] (7) taRNR: WP_053768358.1 [Thermus aquaticus]

[0032] Figure 2 is a diagram illustrating the expression and purification process of RNase R proteins from Escherichia coli and thermophilic bacteria, including the amylose affinity chromatography results of proteins tagged at the N-terminus with 7Lys-MBP. Each band visually represents a major purification step of the expressed protein (IN, input; Ub, unbound; W, wash).

[0033] Figure 3 is a diagram analyzing the 3'-terminal cleavage activity of pgRNR, tmRNR, and ecRNR at different temperatures (40°C, 50°C, and 60°C), and visualizing the enzyme activity by comparing the degradation patterns of circular and linear RNA substrates. The substrate used was 3DB-CVB3-RLuc (1828 nt) (without polyA tailing).

[0034] Figure 4 is a drawing analyzing the 3'-exonuclease activity of gsRNR, tcRNR, ttRNR, and taRNR at various temperatures, and is the result of comparing the high-temperature reaction characteristics and decomposition efficiency of each enzyme (M, size marker; IN, input).

[0035] Figure 5 is a diagram showing the expression and purification process of gsRNR with a His-tag attached, and shows the purity and step-by-step purification status of the purified protein through high-temperature precipitation and nickel affinity chromatography (C, cell lysate; S, supernatant; P, precipitates; IN, input; Ub, unbound fraction; W, wash fraction; E, eluted fraction).

[0036] Figure 6 is a diagram comparing the time-dependent degradation activity of ecRNR and gsRNR for short RNAs with a hairpin structure, and shows the change in RNA band intensity according to the reaction time (1, 2, 4 hours) as a gel electrophoresis image.

[0037] Figure 7 is a diagram comparing the degradation efficiency of ecRNR and gsRNR for ribozyme-based RNA structures, and the reaction results over time (15, 30, and 60 minutes) for RNA substrates with complex terminal structures are presented as gel electrophoresis images. The substrate is a GroupI-CVB3-RLuc IVT reactant. A compares the reaction results over time at 37°C when MBP-tagged ecRNR is mixed with the substrate, and the reaction results over time at 65°C when 6His-tagged gsRNR is mixed with the substrate. The ecRNA shown in A was produced in-house by the present inventors. B shows the comparison results with ecRNR purchased from Company L.

[0038] The hypothesis underlying the present invention is as follows:

[0039] (1) The double-stranded and complex tertiary structure of RNA are unraveled at high temperatures, which exposes the 3' terminus and makes it susceptible to exonuclease cleavage. (2) The existing RNase R derived from E. coli (ecRNR) has optimal activity at 37°C, and its activity decreases sharply or endonuclease activity increases above 50°C, and it becomes inactive at high temperatures. On the other hand, RNase R analogs possessed by thermophilic bacteria can have stable 3'-terminal cleavage activity even at high temperatures. Accordingly, in order to discover RNase R analogs derived from thermophilic bacteria that are active even at high temperatures, we searched for RNase R analog sequences in various thermophilic bacterial genome databases, and secured a total of six candidate analog sequences with conserved 3'-exonuclease activity domains based on RNase R derived from E. coli.

[0040] Sequence analysis of the six candidate RNase R analogs identified confirmed that a domain important for 3' exonuclease activity was conserved. Subsequently, each of the candidate RNase R analogs was expressed and purified in E. coli to evaluate the physical availability and purification potential of the enzyme. As a result, gsRNR and pgRNR were selected as suitable RNase R analogs based on their expression levels and purity.

[0041] Next, the inventors of the present invention compared the high-temperature stability and functional activity of the candidate enzymes and confirmed that gsRNR exhibits high exonuclease activity even at high temperatures without endonuclease activity, confirming its suitability for circular RNA purification. In addition, the inventors of the present invention confirmed the suitability of gsRNR for the removal of RNA with a hairpin structure, which is simple in structure but difficult to remove, by comparing it with ecRNR, and as a result, confirmed that gsRNR exhibits degrading activity even for RNA with a stable structure. Through the above results, the degrading efficiency for RNA with complex terminal structures based on ribozymes, which are the most difficult to process in the actual circular RNA production process, was confirmed, and it was demonstrated that gsRNR can be used for high-purity circular RNA purification by effectively removing linear RNA at high temperatures compared to ecRNR.

[0042] Meanwhile, the inventors of the present invention introduced a His-tag system to overcome the instability of the MBP tag in high-temperature reactions.

[0043] In this specification, RNase R means ribonuclease R, which exhibits 3'-exonuclease activity that degrades RNA by base at the 3' end, and is also referred to as an enzyme because it exhibits enzymatic activity, and can also be referred to as RNR.

[0044] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0045]

[0046] [Example]

[0047] Example 1. Identification and sequence analysis of RNase R analogs derived from thermophilic bacteria.

[0048] The genome database of six thermophilic bacteria in Table 1 below was analyzed based on the sequence of RNase R from Escherichia coli (ecRNR; Protein ID: NP_418600.4).

[0049] Bacterial limit survival temperature Optimal survival temperature Aquifex pyrophilus 95°C 85°C Hermotoga maritima 90°C 80°C Hermodesulfobacterium commune 85°C 70°C Hermus thermophilus 82°C 65°C Hermus aquaticus 80°C 72°C Geobacillus stearothermophilus 65°C 60°C

[0050]

[0051] As a result of the analysis, six candidate RNase R analogs in Table 2 below were identified, and sequence similarity analysis was performed to confirm that the six candidate RNase R analogs in Table 6 below have conserved amino acid residues important for 3'-exonuclease activity (Fig. 1).

[0052] 1WP_043905984.1 ribonuclease R [Parageobacillus genomosp. 1]pgRNR2WP_277392187.1 ribonuclease R [Geobacillus stearothermophilus]gsRNR3WP_038060459.1 ribonuclease R [Thermodesulfobacterium commune]tcRNR4AGL49648.1 3-to-5 exoribonuclease RNase R [Thermotoga maritimaMSB8]tmRNR5WP_024119831.1 ribonuclease R [Thermus thermophilus]ttRNR6WP_053768358.1 ribonuclease R [Thermus aquaticus]taRNR

[0053]

[0054] Example 2. Expression and purification of a thermophilic RNase R analogue.

[0055] Selected RNase R analogs were expressed in Escherichia coli with a 7Lys-MBP (7-lysine maltose-binding protein tag) tag attached to the N-terminus. Subsequently, they were purified by sequential cation exchange column chromatography and amylose affinity chromatography. As a result, it was confirmed that pgRNR and gsRNR had significantly higher expression levels and purification yields compared to other analogs, and that RNase R could be obtained with high purity (Fig. 2).

[0056]

[0057] Example 3. Evaluation of RNase R activity at high temperatures

[0058] The RNA substrate for the enzymatic reaction was prepared by mixing 1828 nt linear RNA containing the Renilla Luciferase gene and circular RNA whose both ends were joined using RNA ligase. The reaction was performed using 10 μg of the RNA substrate, 4 μg of the enzyme, 100 μL of reaction volume, 15 min of reaction time, and buffer conditions (1X RNase R Reaction Buffer: 20 mM Tris-HCl (pH 8.0), 100 mM KCl, and 0.1 mM MgCl2). The reaction was performed at different temperature conditions of 40, 50, or 60°C. The results are as follows (Figs. 3 and 4):

[0059] - ecRNR: Shows linear RNA cleavage activity at 37°C or 40°C. However, at 50°C, it also cleaves all circular RNAs, suggesting strong endonuclease activity at this temperature. No activity at 60°C.

[0060] - pgRNR and gsRNR: showed the best exonuclease activity at 60°C.

[0061] - tcRNR: Shows the best exonuclease activity at 60°C, but also exhibits endonuclease activity.

[0062] - tmRNR, taRNR, and ttRNR: exhibit strong endonuclease activity

[0063]

[0064] Example 4. Optimization of thermophilic RNase R expression and purification.

[0065] RNase R fused with 7Lys-MBP tag can affect the enzymatic activity of RNase by denaturing MBP even at high temperatures.

[0066] Accordingly, in order to induce effective enzyme reaction at high temperatures, the tag for RNase purification was replaced from the 7Lys-MBP tag to a histidine tag (Polyhistidine tag: His tag).

[0067] ProteinSequence MWSEQ NO.*86278.840000000051gsRNaseR-6HMDHVLAERILTFMRDEAYKPLTVEELEEAFGIEDADGFKEFVKTLVALEEEGLIVRTRSNRYGVPERMNLVRGKVIGHSKGFAFVTPEEPGLDDIFIPPSELKNAMHGDTVLVRVQADSSGARREGTIVRIVERGVKEVVGTYTESKYFGFVIPDDKRIVNDIFIPKHAANGAVEGHKVVVRLTSYPEGRMSAEGEVVQILGHKNDPGVDILSIIYKHGLPLQFPDDVIEHANRVPDVITEQDLEGRRDLRGEMIVTIDGEDAKDLDDAVTVTKLENGNYKLGVHIADVSHYVEEGSPIDREAYERGTSVYLVDRVIPMIPHRLSNGICSLNPKVDRLTLSCEMEITPQGEVVRHDIFQSVIRTTERMTYSDVNKILVDKDEALREKYAPLVPMFELMAELADILRTKRMKRGAIDFDFKEAKVLVDENGKPYDVVLRERSVAERLIEEFMLAANETVAEHFHWLNVPFMYRVHEDPKPEKLQRFLEFITNFGYVVKGTGNQIHPRALQQILEAVRGEPEEMVISTVMLRSMKQARYDAESLGHYGLSTEFYTHFTSPIRRYPDLIVHRLIRTYLINGQMDPETQRKWAEKLPEIAEHASNMERRAVEAERETDDLKKTEFMEDKIGMEFDGIISSVTNFGLFVELPNTIEGLVHVSYLTDDYYRYDERSYAMIGERTGKMYRIGDEITVRVINVNKDERIVDFEVVGMKGRRPPKAKAAPVVIEGKKQKNGKPKAEAKGKTGAGRAAKAKKKKKKKRSRHHHHHHSGR*87645.270000000062.

[0068]

[0069] 4-1. 히스티딘 태그 gsRNA 발현 및 정제

[0070] A histidine-tagged gsRNA construct was transformed into Escherichia coli for overexpression, and the E. coli was heated to remove impurities. The supernatant was then collected by centrifugation and subjected to cobalt or nickel affinity chromatography to obtain gsRNA (Fig. 5).

[0071]

[0072] 4-2. Comparison of exonuclease activity for RNA with non-exposed 3' ends

[0073] In order to confirm the degrading activity of the above-obtained RNase R on short double-stranded RNA, small hairpin RNA with a double helix length of 10 bp and a short loop of 5 nt was synthesized and used as a substrate. The reaction was performed using 20 units* each of ecRNR and gsRNR for 50 ng RNA substrate. The reaction was performed at 37°C for ecRNR and at 65°C for gsRNR. *1 unit: The amount of RNase R that can completely degrade 1 ug linear RNA into nucleotides within 10 minutes under standard reaction conditions (reaction volume 50 μL; 1X RNase R Reaction Buffer: 20 mM Tris-HCl (pH 8.0), 100 mM KCl, and 0.1 mM MgCl2). After 1, 2, or 4 hours of reaction, gel electrophoresis was performed to measure the decrease in RNA band intensity. It was confirmed that ecRNR removed only about 32% of RNA during 4 hours of reaction, while gsRNR removed about 90% (Fig. 6).

[0074]

[0075] 4-3. Comparison of exonuclease activity for RNAs with complex 3'-terminal structures

[0076] Group I intron ribozyme is used in the PIE system for producing circular RNA. During the process of producing circular RNA using the PIE system, the remaining RNA, excluding the circular RNA generated by the ribozyme after in vitro transcription (IVT), must be removed. The linear RNA, which is the target for removal, contains parts of the group I intron ribozyme at both ends, forming a complex secondary structure, making removal difficult. The present inventors confirmed whether the secured RNase R was effective in removing linear RNA that was not circularized after the IVT reaction. The substrate was 10 μg of the Group I-CVB3-RLuc IVT reaction product, and 10 units of each RNR enzyme were used. ecRNR was reacted at 37°C and gsRNR at 65°C for 15, 30, and 60 minutes, respectively. As a result, gsRNR removed most of the linear RNA in 15 minutes (Fig. 7A, lane 6), whereas ecRNR showed that a significant amount of linear RNA remained even after 60 minutes (Fig. 7A, lane 5).

[0077]

[0078] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0079] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. (1) A step of preparing RNase R (gsRNR) derived from Geobacillus stearothermophilus; (2) a step of inducing in vitro transcription of a circular RNA precursor expression vector; and (3) A method for purifying circular RNA, comprising a step of treating the gsRNR in a test tube after the completion of the transcription induction step.

2. In paragraph 1, A method for purifying circular RNA, wherein the step (1) is achieved by culturing bacteria transformed with a gsRNR expression vector and obtaining gsRNR.

3. In paragraph 1, The above gsRNR is a circular RNA purification method in which a His tag is fused.

4. In paragraph 1, A method for purifying circular RNA, wherein the above step (3) is performed under temperature conditions of 45 to 70°C. 5.(a) a step of preparing a solution containing circular RNA and linear RNA; and (b) A method for purifying circular RNA, comprising the step of treating the solution with RNase R (gsRNR) derived from Geobacillus stearothermophilus.

6. In paragraph 5, A method for purifying circular RNA, wherein the step (a) is achieved by performing in vitro transcription of a circular RNA precursor expression vector.

7. In paragraph 6, A method for purifying circular RNA, wherein the method further comprises a step of increasing the temperature of the reaction product to 45 to 70°C after completion of the in vitro transcription induction step.

8. In paragraph 5, A circular RNA purification method, wherein the above gsRNR processing step is performed under temperature conditions of 45 to 70°C.

Images