Method for evaluating 5'capping efficiency

Abstract

The present invention relates to a method for analyzing 5' capping efficiency of prepared mRNA, wherein the method enables efficiency management in the production of mRNA. In addition, the 5' capping efficiency evaluation method of the present invention can produce high-quality mRNA by including a pretreatment procedure for effective 5' capping cleavage.

Description

METHOD FOR EVALUATING 5′CAPPING EFFICIENCY

[0001] The present invention relates to a method for evaluating mRNA 5' capping efficiency.

[0002]

[0003] RNA can encode all types of proteins, enabling the resolution of protein-targeting issues, the rapid derivation of candidates, and reduced concerns about impurities in the production process. Nevertheless, RNA is easily degraded in the body and has low durability, and thus, until recently, its development as a therapeutic has been considered limited.

[0004] However, since the development of mRNA vaccines for COVID-19, vaccines and therapeutics using mRNA have been actively researched, and attention has also been drawn to techniques for the efficient production of mRNA.

[0005]

[0006] A 5' cap structure of mRNA is one of the important factors to consider in mRNA production. The 5' cap refers to a structure where 7-methylguanosine (m7G) is linked to the 5'-end via a 5',5'-triphosphate bridge (ppp) during transcription or post-transcriptional modification in eukaryotes. This 5' cap is known to prevent mRNA degradation, increase translation efficiency, promote the migration of mRNA from the nucleus to the cytoplasm, and increase the efficiency of mRNA splicing.

[0007]

[0008] Therefore, it is essential to evaluate whether the 5' cap has been formed and to manage the 5' capping efficiency at an appropriate level, after mRNA synthesis, during the production and development of mRNA vaccines and therapeutics.

[0009]

[0010] To evaluate such 5' capping efficiency, a method using ribonuclease H (RNase H) and probes (US 2021-0108252 A1, etc.) and an ELISA method of capturing tagged biotin by streptavidin coating (WO 2014 / 152673 A1, etc.) have been known. However, these methods have the disadvantages of complex testing procedures and high analysis costs.

[0011]

[0012] As the demand for RNA-based vaccines and therapeutics is expected to increase, there is a need for a method for evaluating mRNA 5' capping efficiency that is more effective and facilitates quality control.

[0013]

[0014] An aspect of the present invention is to provide a method for evaluating mRNA 5' capping efficiency.

[0015] Another aspect of the present invention is to provide a kit for evaluating 5' capping efficiency.

[0016]

[0017] The 5' capping efficiency evaluation method of the present invention can produce high-quality mRNA by including a pretreatment procedure for effective 5' capping cleavage.

[0018]

[0019] FIG. 1 shows a site of mRNA that is cleaved by DNAzyme.

[0020] FIG. 2 shows the evaluation of cleavage efficiency depending on the type of catalytic motif (A) and the methylation modification (B) in order to select DNAzyme.

[0021] FIG. 3 evaluates the cleavage efficiency depending on the type of cations added to increase the cleavage efficiency (C) and the cleavage efficiency depending on the addition of spermine.

[0022] FIG. 4 shows the results of optimizing reaction conditions of main factors capable of maximizing cleavage efficiency through multivariate testing.

[0023] FIG. 5 evaluates 5' capping efficiency according to the method of the present invention.

[0024] FIG. 6 compares the cleavage efficiency depending on the reaction time after the mRNA cleavage reaction by DNAzyme was conducted from 10 minutes to 24 hours.

[0025] FIG. 7 compares the 5' capping efficiency depending on the reaction time after the mRNA cleavage reaction by DNAzyme was conducted from 10 minutes to 24 hours.

[0026] FIG. 8 confirms the cleavage efficiency depending on the binding arm length of DNAzyme.

[0027] FIG. 9 confirms the 5' capping efficiency depending on the binding arm length of DNAzyme.

[0028] FIG. 10 confirms the cleavage efficiency depending on the number of methylated bases of DNAzyme.

[0029] FIG. 11 confirms the 5' capping efficiency depending on the number of methylated bases of DNAzyme.

[0030]

[0031] In accordance with an aspect of the present invention, a method for evaluating mRNA 5' capping efficiency is provided.

[0032] In an embodiment, the method may include (a) providing a sample containing mRNA; and (b) bringing the sample into contact with a catalytic nucleic acid to cleave mRNA.

[0033] In another embodiment, in the cleaving of mRNA, mRNA may be cleaved by the catalytic nucleic acid in a reaction solution containing divalent cations and a buffer.

[0034] In any one of the previous embodiments, the reaction solution may contain divalent cations at a concentration of 12 to 30 mM.

[0035] In any one of the previous embodiments, the divalent ion may be Mg2+.

[0036] In any one of the previous embodiments, the buffer may have a pH of 7.0 to 7.5.

[0037] In any one of the previous embodiments, the catalytic nucleic acid may be DNAzyme.

[0038] In any one of the previous embodiments, the ratio of mRNA to DNAzyme reacting therewith may be 1:10 to 1:20.

[0039] In any one of the previous embodiments, the DNAzyme may include a binding arm binding to mRNA and a catalytic motif.

[0040] In any one of the previous embodiments, the DNAzyme may include two binding arms, which are located at both ends of the catalytic motif.

[0041] In any one of the previous embodiments, the binding arm may include 7 to 9 bases.

[0042] In any one of the previous embodiments, the binding arm may complementarily bind to mRNA.

[0043] In any one of the previous embodiments, the DNAzyme may include methylation of a base in the binding arm.

[0044] In any one of the previous embodiments, the binding arm may include 1 to 4 methylated bases.

[0045] In any one of the previous embodiments, the mRNA may have a cleavage site that is cleaved by the catalytic nucleic acid.

[0046] In any one of the previous embodiments, the DNAzyme or the catalytic motif may include an 8-17 motif.

[0047] In any one of the previous embodiments, the catalytic motif may be an 8-17 motif.

[0048] In any one of the previous embodiments, in the cleaving of mRNA, the sample may react with the catalytic nucleic acid for 2 to 30 hours.

[0049] In any one of the previous embodiments, the cleaving of mRNA may include (b-1) denaturing mRNA in the sample at 90 to 100°C for 5 to 15 minutes.

[0050] In any one of the previous embodiments, the cleaving of mRNA may include (b-2) binding mRNA to a catalytic enzyme at a temperature lower than the melting point of the catalytic enzyme by 5 to 10°C, for 20 to 40 minutes.

[0051] In any one of the previous embodiments, the cleaving of mRNA may include (b-3) cleaving mRNA by the catalytic enzyme at 35 to 40°C for 2 to 30 hours.

[0052] In any one of the previous embodiments, the method may further include (c) determining the ratio of 5'-capped mRNA and 5'-uncapped mRNA.

[0053] In any one of the previous embodiments, the method may further include separating the 5'-capped mRNA.

[0054] In any one of the previous embodiments, step (c) may be performed by any one or more methods of gel electrophoresis, capillary electrophoresis, mass spectrometry, and chromatography.

[0055] In accordance with another aspect of the present invention, a method for manufacturing a fragment containing an mRNA 5' cap is provided.

[0056] In an embodiment, the method may include (a) providing a sample containing mRNA; and (b) bringing the sample into contact with a catalytic nucleic acid to cleave mRNA.

[0057] In accordance with still another aspect of the present invention, a kit for analyzing mRNA 5' capping efficiency is provided.

[0058]

[0059] Hereinafter, detailed contents for implementing the present invention will be described below.

[0060] Each description and embodiment disclosed in the present application may also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present application fall within the scope of the present disclosure. Furthermore, the scope of the present invention is not limited by the specific description below.

[0061] Furthermore, a skilled person in the art will recognize, or be able to ascertain, by using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Furthermore, these equivalents are intended to be included in the present invention.

[0062]

[0063] In accordance with an aspect of the present invention, a method for evaluating mRNA 5' capping efficiency is provided. Specifically, the 5' capping efficiency evaluation method of the present invention comprises bringing mRNA into contact with a catalytic nucleic acid to cleave mRNA, wherein the mRNA 5' capping efficiency is evaluated by the ratio of 5' cap-containing fragments in the cleaved 5'-end fragments.

[0064]

[0065] In a specific aspect, the method of the present invention may include:

[0066] (a) providing a sample containing mRNA; and

[0067] (b) bringing the sample into contact with a catalytic nucleic acid to cleave mRNA.

[0068]

[0069] The present inventors attempted to develop a method for evaluating mRNA 5' capping efficiency that is more effective and facilitates quality control, and as a result, established a method for analyzing 5' capping efficiency by cleaving a portion of mRNA through a catalytic nucleic acid and determining whether a fragment of the 5'-end includes a 5' cap.

[0070] The 5' capping efficiency evaluation method according to the present invention can analyze 5' capping efficiency more easily and effectively by using a UV detector and confirm the specificity, precision, and robustness of a test method.

[0071]

[0072] As used herein, the term "mRNA", also called messenger RNA, refers to a genetic material that is transcribed from a DNA template and translated into a protein at the ribosome. In eukaryotic cells, precursor mRNA becomes mature mRNA via post-transcriptional modifications, including 5' capping, 3' polyadenylation, and RNA splicing.

[0073] For the purposes of this invention, the mRNA may indicate naturally synthesized mRNA in a subject or artificially synthesized mRNA. More specifically, the mRNA may be mRNA synthesized through in vitro transcription (IVT), but any mRNA that is produced by a known method and requires the evaluation of 5' capping efficiency is not limited to a specific production method. It may not be determined whether the mRNA has a 5' cap.

[0074]

[0075] It is important whether the mRNA of the present invention contains the AG sequence (a cleavage site by DNAzyme) within the 5'-untranslated region (UTR). The full length of the mRNA is not limited, and an appropriate length thereof may be determined considering a target protein that is ultimately intended by a person skilled in the art, a recognition site by a catalytic nucleic acid, and the like. In the present invention, the mRNA may further contain 5'-UTR, an open reading frame (ORF), 3'-UTR, a poly(A) tail, and the like.

[0076]

[0077] The providing of a sample containing mRNA (a) of the present invention may be preparing a sample containing mRNA to be analyzed, for analysis of 5' capping efficiency. Specifically, the providing may indicate preparing a sample containing synthesized mRNA, of which the presence or absence of the 5' cap has not been determined, but is not limited thereto.

[0078]

[0079] As used herein, the term "sample" refers to a specimen containing at least one mRNA to be analyzed for 5' capping efficiency. The sample may contain a collection of mRNAs synthesized through in vitro transcription or some thereof, where a mRNA with a 5' cap and mRNA without a 5' cap may be mixed. The ratio of mRNA with a 5' cap and mRNA without a 5' cap in the sample may be determined by the method for evaluating 5' capping efficiency of the present invention, but is not limited thereto.

[0080]

[0081] As used herein, the term "5' cap" refers to a structure of a 7-methylguanosine (m7G) cap formed on the 5'-end during transcription or post-transcriptional modification. The cap is attached to the first nucleotide at the 5'-end of mRNA through a 5',5'- triphosphate bridge (ppp) (m7GpppGpN structure). The presence or absence of the 5' cap in the produced mRNA greatly affects the functionality of mRNA, and thus it is essential to check the proportion of mRNA containing the 5' cap and maintain the proportion at an appropriate level during the production of mRNA.

[0082] The 5' cap of the present invention may have a naturally occurring, unmodified form, or may be a 5' cap analog that is artificially engineered to impart better stability to mRNA. The 5' cap analog may be prepared by the addition of a methyl group, but is not limited thereto. The 5' cap and its analogs are well known in the art, and may be appropriately selected by a person skilled in the art. A specific example of the 5' cap may be, but is not limited to, a 5' cap having a structure of chemical formula 1 below.

[0083]

[0084] [Chemical Formula 1]

[0085]

[0086]

[0087] The "5' capping efficiency" herein refers to the proportion of mRNA confirmed to be 5' capped among the total mRNA to be analyzed. In a specific embodiment, the 5' capping efficiency may refer to the proportion of 5' capped mRNA among the total mRNA in the sample, or may refer to the proportion of 5'-end truncated, 5' capped mRNA to 5'-end truncated mRNA in the sample. The 5' capped mRNA may be distinguished from mRNA without a 5' cap by chromatography, but is not limited thereto.

[0088]

[0089] In an embodiment, the 5' capping efficiency may be expressed by the following equation.

[0090]

[0091]

[0092] The "5' capping efficiency evaluation method" of the present invention indicates a method for determining the proportion of mRNA with a 5' cap among the total mRNA or mRNA in the sample during transcription or post-transcriptional modification. The 5' capping efficiency evaluation method of the present invention has the advantage of simple testing procedures and low analysis costs compared with existing 5' capping efficiency evaluation methods.

[0093]

[0094] In the 5' capping efficiency evaluation method of the present invention, any method for determining the proportion may be used in the method of the present invention without limitation to a particular method as long as the proportion can be determined by quantifying (i) total mRNA to be analyzed, (ii) mRNA with a 5' cap, or 5'-end fragments thereof, and / or (iii) mRNA without a 5' cap, or 5'-end fragments thereof.

[0095] The 5' capping efficiency may be determined using gel electrophoresis, capillary electrophoresis, mass spectrometry, or chromatography, but is not limited thereto. Specifically, the chromatography may be liquid chromatography, and more specifically, may be high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), or liquid chromatography-mass spectrometry (LC-MS), but is not limited thereto. The chromatography may use, as a column, an anion exchange column, a cation exchange column, a reverse-phase HPLC column, a hydrophobic interaction column, a size exclusion column, or a combination thereof. Additionally, an ion exchange resin, a mobile phase, and the like may be appropriately selected and used by a person skilled in the art.

[0096]

[0097] The 5' capping efficiency evaluation method of the present invention may use a combination of one or more chromatography methods, or chromatography in combination with mass spectrometry, but is not limited thereto. In an embodiment, the 5' capping efficiency may be determined by the method described in Example 2, but is not limited thereto.

[0098] In the 5' capping efficiency evaluation method of the present invention, 5'-end truncation is induced with high efficiency, thereby enabling effective analysis of 5' capping efficiency and the application thereof in the mRNA production process.

[0099]

[0100] The bringing of the sample into contact with a catalytic nucleic acid to cleave mRNA (b) of the present invention may be cleaving mRNA so as to determine the presence or absence of a 5' cap in order to analyze 5' capping efficiency. Specifically, the 5' capping efficiency evaluation method of the present invention may include preparing a 5'-end fragment through the cleaving step.

[0101]

[0102] As used herein, the term "catalytic nucleic acid" refers to a nucleic acid molecule having the activity to cleave a nucleic acid, wherein the catalytic nucleic acid has a length of 10-60 nt and can cleave the nucleic acid by recognizing a cleavage site of the nucleic acid. Examples of the catalytic nucleic acid may include ribozyme and DNAzyme. The mRNA of the present invention may have a site, which is recognized and cleaved by the catalytic nucleic acid. In the present invention, the catalytic nucleic acid may be DNAzyme, but is not limited thereto. The type, sequence, origin, and characteristics of the catalytic nucleic acid are well known to a person skilled in the art, and the design and production of the catalytic nucleic acid may be appropriately performed by a person skilled in the art considering mRNA to be analyzed.

[0103]

[0104] The DNAzyme of the present invention, which is one type of catalytic nucleic acid, may include binding arms complementarily binding to mRNA and a catalytic motif having mRNA cleavage activity. When the binding arms of the DNAzyme complementarily bind to mRNA, the AG base sequence in the mRNA, which is located therebetween, can be recognized and cleaved. In an embodiment, the DNAzyme may have the nucleic acid sequence of SEQ ID NO: 2, where the sequence of bases complementarily binding to the target mRNA is not limited while the base sequence of the catalytic motif recognizing and cleaving the AG base sequence in the target mRNA may be fixed.

[0105]

[0106] For the purpose of the present invention, the mRNA of the present invention may have a cleavage site, which is cleaved by DNAzyme. The cleavage site may correspond to positions 10-25 nt from the 5'-end of mRNA, and in an embodiment, the DNAzyme of the present invention may recognize and cleave the AG base sequence of mRNA. Specifically, the DNAzyme of the present invention may recognize and cleave the AG base sequence in the 5'-UTR in order to cleave fragments of about 10-25 nt to improve the separation degree between capped fragments and uncapped fragments.

[0107] However, a person skilled in the art can determine the cleavage site and DNAzyme as long as the 5' capping efficiency can be analyzed through fragments containing a 5' cap that have been manufactured.

[0108]

[0109] In a specific embodiment, the DNAzyme of the present invention includes a catalytic motif. The DNAzyme of the present invention may be naturally non-occurring, and may include modified forms for high cleavage activity and stability.

[0110] The DNAzyme may include a known catalytic motif, named an 8-17 motif, a 10-23 motif, a 10-12 motif, or the like. The DNAzyme of the present invention may include, as a catalytic motif, an 8-17 motif, a 10-23 motif, a 10-12 motif, or the like, and the sequence of each motif is known. Alternatively, the DNAzyme of the present invention may include a modified form of each of the motifs.

[0111]

[0112] The motifs included in the DNAzyme may include the following sequences, but are not limited thereto.

[0113]

[0114]

[0115]

[0116] The sequence portions in bold and italics may serve as catalytic motifs, and the other portions may serve as binding arms binding to mRNA. In a specific embodiment, the DNAzyme of the present invention may include the 8-17 motif of SEQ ID NO: 4 or the methylated 8-17 motif of SEQ ID NO: 2.

[0117] The binding arms are regions complementarily binding to mRNA to be analyzed for 5' capping efficiency, and the cleaving activity can be exhibited by the catalytic motif located between the binding arms. The catalytic motifs included in the DNAzyme of the present invention can recognize the AG sequence of mRNA to cleave mRNA, but are not limited thereto.

[0118]

[0119] Specifically, the DNAzyme of the present invention may include two binding arms, with a catalytic motif located between the respective binding arms. The sequences of the two binding arms included in the DNAzyme may be the same as or different from each other, and the sequences may be independently determined as long as the binding arms can bind to mRNA. The sequence of the binding arm may be any one of SEQ ID NOS: 7 to 18, but is not limited thereto.

[0120] In addition, the lengths of the two binding arms included in the DNAzyme may be the same as or different from each other. Each of the binding arms may contain 1 to 10 bases, 2 to 9 bases, 4 to 9 bases, or 7 to 9 bases, but is not limited thereto.

[0121] A binding arm with a short length may non-specifically bind to a region other than a target sequence to cause an undesired cleavage, resulting in a reduction in accuracy of 5' capping efficiency analysis, and even if such a binding arm binds to the target sequence, the binding arm may be easily detached again due to its weak binding affinity for mRNA, causing reduced cleavage efficiency. A binding arm with a long length may cause its target sequence to extend beyond the 5'-UTR to include even the ORF sequence in mRNA, and as a result, the binding arm needs to be changed for each mRNA sequence to be analyzed, causing poor versatility. The longer the binding arm, the higher the temperature required for mRNA binding, and such a high temperature may cause the denaturation of the mRNA itself to be analyzed. Therefore, it is important to determine an appropriate length of the binding arm usable for 5' capping efficiency analysis. In an embodiment of the present invention, as a result of analyzing the cleavage efficiency of mRNA by adjusting the length of the binding arm, the use of DNAzyme including binding arms each containing 7 to 9 bases at both ends thereof exhibited excellent cleaving efficiency, leading to effective analysis of 5' capping efficiency.

[0122]

[0123] The DNAzyme of the present invention may include the methylation of bases complementarily binding to mRNA, but is not limited thereto. Specifically, some bases of the binding arm included in the DNAzyme of the present invention may be methylated, and the inclusion of the methylation of bases complementarily binding to mRNA may increase the binding affinity for mRNA.

[0124] As used herein, the term "methylation" refers to the attachment of a methyl group to a base of the nucleic acid sequence. The position of the attachment of the methyl group is not particularly limited, and the methylated base of the present invention may result in a 2'-O-methylated nucleoside, but is not limited thereto. The methylation of bases in the binding arm may be carried out by a method known in the art.

[0125]

[0126] Specifically, some bases of the two binding arms included in the DNAzyme of the present invention may be methylated, and the numbers of methylated bases in the two binding arms may be the same as or different from each other, and the numbers of methylated bases may be independently determined as long as the binding arms can bind to mRNA. Specifically, the number of methylated bases in one binding arm may be 1 to 9, 1 to 6, or 1 to 4, and more specifically, 1, 2, 3, 4, 5, 6, 7, 8, or 9, but is not limited thereto. Alternatively, the number of methylated bases in the DNAzyme may be 1 to 18, 1 to 12, or 1 to 8, and more specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, but is not limited thereto.

[0127]

[0128] The positions of methylation in the two binding arms included in the DNAzyme may be the same as or different from each other, and the positions of methylation may be independently determined as long as the binding arms can bind to mRNA. Additionally, the methylated bases may be consecutive or non-consecutive, but are not limited thereto. Specifically, the position of methylation may be a combination of 1 to 4 positions among the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth positions from the N-terminus of the binding arm, but is not limited thereto. As an example of methylation position, 1 to 4 bases from the edge of an end of the binding arm, to which the catalytic motif is not connected, may be consecutively methylated, but are not limited thereto.

[0129] In an embodiment of the present invention, as a result of analyzing the cleaving efficiency of mRNA according to the number of methylated bases, the use of DNAzyme including a binding arm containing 1 to 4 methylated bases exhibited excellent cleaving efficiency, leading to effective analysis of 5' capping efficiency.

[0130]

[0131] In a specific embodiment, the ratio of the mRNA of the present invention and DNAzyme reacting therewith may be 1:10-50, but is not limited thereto. Specifically, the molar ratio may be 1:10-50, 1:10-30, 1:10-25, or 1:10-20, but is not limited thereto. The mRNA reacting with the DNAzyme is mRNA that is contained in the sample and is not confirmed as to whether a 5' cap has been formed.

[0132]

[0133] As used herein, the term "5'-end truncation" indicates the preparation of a fragment containing a 5'-end site of mRNA by cleaving mRNA through a reaction with the catalytic nucleic acid, and the term "5'-end fragment" refers to a 5'-end fragment prepared through 5'-end truncation, wherein the fragment includes a 5'-end site among mRNA fragments cleaved by the catalytic nucleic acid. To solve the problem that the presence or absence of a 5' cap is difficult to determine in full-length mRNA, 5'-end truncation can be carried out to evaluate the 5' capping efficiency. For the purpose of the present invention, the 5'-end fragment may have a length of 10 to 25 nt, but the 5'-end fragment is not limited to a particular length as long as the length enables the analysis of the presence or absence of a 5' cap.

[0134]

[0135] Specifically, a 5'-end fragment of mRNA with a 5' cap may be a fragment containing a 5' cap, and a 5'-end fragment of mRNA without a 5' cap may be a fragment not containing a 5' cap. More specifically, the 5'-end fragment of the present invention may contain a 5' cap and 5'-UTR or a portion thereof, or may contain only 5'-UTR or a portion thereof.

[0136]

[0137] According to the 5' capping efficiency evaluation method of the present invention, the 5' capping efficiency can be evaluated by analyzing the proportion of 5'-end fragments with a 5' cap in the total 5'-end fragments. For effective evaluation of 5' capping efficiency, the occurrence of 5'-end truncation at a higher rate is advantageous, and thus it is necessary to induce a cleavage reaction by the catalytic nucleic acid in an environment with high 5'-end truncation efficiency.

[0138]

[0139] In a specific embodiment, the cleaving of mRNA of the present invention may be performed in a reaction solution containing cations and a buffer, but is not limited thereto. Specifically, the reaction solution may contain the buffer and cations in appropriate conditions so as to effectively induce mRNA cleavage activity by the catalytic nucleic acid.

[0140]

[0141] More specifically, the cations in the reaction solution may be Mg2+. In a specific embodiment, the reaction solution of the present invention may contain Mg2+.

[0142]

[0143] The reaction solution may contain the cations at a concentration of about 1 to 50 mM, about 5 to 50 mM, about 10 to 50 mM, about 10 to 40 mM, about 12 to 40 mM, or about 12 to 30 mM, but is not limited thereto.

[0144]

[0145] In another specific embodiment, the reaction solution of the present invention may contain Mg2+at a concentration of about 12 to 30 mM, but is not limited thereto.

[0146]

[0147] The term "about" as used herein refers to a range encompassing ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and the like, and includes all of the values in the range equivalent or similar to those stated after this term, but is not limited thereto.

[0148]

[0149] The reaction solution of the present invention may contain a buffer (buffer solution). The buffer may include any buffering agent known in the art, including alkaline salts (sodium or potassium phosphate or hydrogen or dihydrogen salts thereof), citrate salts (e.g., sodium citrate / citric acid), acetate salts (e.g., sodium acetate / acetic acid), or histidine or salts thereof, as well as mixtures thereof, but is not limited thereto. Specifically, the reaction solution of the present invention may contain an acetate buffer or a citrate buffer, and more specifically, a buffer containing sodium acetate, but is not limited thereto.

[0150] The concentration of the buffering material contained in the reaction solution of the present invention may be about 1 mM to 200 mM, about 1 to 150 mM, 1 to 100 mM, 10 to 100 mM, or 50 to 100 mM, but is not limited thereto.

[0151]

[0152] The pH of the buffer contained in the reaction solution of the present invention may be 6 to 8, about 6.5 to 8, about 6.5 to 7.5, about 7 to 8, about 7 to 7.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, or about 7.9, but is not limited thereto.

[0153]

[0154] More specifically, the buffer contained in the reaction solution of the present invention may be a buffer containing 50 to 100 mM sodium acetate at pH 7.0 to 7.5, but the buffer is not limited to a specific buffering material and pH as long as the buffer allows for 5'-end truncation.

[0155]

[0156] An example of the present invention established that the use of a reaction solution containing a buffer at pH 7.0 to 7.5 and divalent cations at 12 to 30 mM exhibited excellent cleaving efficiency by DNAzyme, leading to effective analysis of 5' capping efficiency. It was also identified that the cations and buffer contained in the reaction solution of the present invention affected 5'-end truncation efficiency by an interaction therebetween, and thus it is necessary to prepare a reaction solution enabling the improvement of 5'-end truncation efficiency by controlling the cations and the buffer.

[0157]

[0158] For the purpose of the present invention, the 5' capping efficiency evaluation method of the present invention may further include preparing a reaction solution containing a buffer and divalent cations. A 5'-end truncation reaction may be carried out in a mixture where the prepared reaction solution, a sample containing mRNA, and a catalytic nucleic acid are mixed.

[0159] As used herein, the term "reaction solution" may indicate a reaction solution containing a buffer and divalent cations, or may include a reaction solution allowing for a cleavage step by further containing a catalytic nucleic acid and mRNA (or a sample containing mRNA).

[0160]

[0161] As used herein, the term "5'-end cleaving efficiency" refers to the proportion of mRNA, of which the 5'-end is truncated by a catalytic nucleic acid (e.g., DNAzyme), in mRNA in the sample to be analyzed.

[0162] In the present invention, the 5'-end truncation efficiency may be expressed as follows, but is not limited thereto.

[0163]

[0164]

[0165] In the present invention, the 5'-end truncation efficiency may be analyzed without limitation to a particular method as long as the proportion of 5'-end truncated mRNA can be identified.

[0166] The 5'-end truncation efficiency may be determined using gel electrophoresis, capillary electrophoresis, mass spectrometry, or chromatography, but is not limited thereto. Specifically, the chromatography may be liquid chromatography and, more specifically, may be high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), or liquid chromatography-mass spectrometry (LC-MS). Examples of the column for the chromatography may include an anion exchange column, a cation exchange column, a reverse-phase HPLC column, a hydrophobic interaction column, a size exclusion column, or a combination thereof. Additionally, an ion exchange resin, a mobile phase, and the like may be appropriately selected and used by a person skilled in the art.

[0167] The 5'-end truncation efficiency of the present invention may be evaluated by using a combination of one or more chromatography methods, or chromatography in combination with mass spectrometry, but is not limited thereto. In an embodiment, the 5'-end truncation efficiency may be determined by the method described in Example 2, but is not limited thereto.

[0168]

[0169] For the purpose of the present invention, in the cleaving of mRNA of the 5'-capping efficiency evaluation method, the 5'-end truncation efficiency may be about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 99.9% or more, but is not limited thereto.

[0170]

[0171] The mRNA or catalytic nucleic acid of the present invention may be a derivative containing at least one chemical modification in sugar or base, but is not limited thereto.

[0172] In an embodiment, the derivative may have a chemical modification in at least one sugar moiety, and specifically, the -OH group at the 2' position of the pentose in a nucleotide may be substituted with a methyl group (OMe).

[0173] In another embodiment, the derivative may have a chemical modification of at least one base, but is not limited thereto. Specifically, the derivative may include an introduction of an amino group, a thiol group, or an alkyl group into a base, or may include a base in a modified form. Examples of the modification may include a chemical modification including pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-azauridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thiopseudouridine, 4-methoxypseudouridine, 4-thio-1-methylpseudouridine, 4-thiopseudouridine, 5-azauridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, or 2'-O-methyluridine, but are not limited thereto. In a specific embodiment, mRNA of the present invention may include N1-methylpseudouridine, and DNAzyme may include a methylated sugar modification. The N1-methylpseudouridine may be a modified form of uridine.

[0174]

[0175] The chemically modified mRNA or catalytic nucleic acid of the present invention may have two or more types of chemical modifications in a sugar, a linkage between nucleotides, or a base, but is not limited thereto.

[0176]

[0177] In a specific embodiment, the mRNA of the present invention is not limited to a particular sequence as long as the 5' capping efficiency can be analyzed by the 5' capping efficiency evaluation method of the present invention. Specifically, the mRNA of the present invention can be used in the 5' capping efficiency evaluation method, without limitation to a particular sequence, as long as the 5'-end is truncated by a catalytic nucleic acid (e.g., DNAzyme) and the ratio of 5'-capped fragments and 5'-uncapped fragments can be determined. In an embodiment, any mRNA can be used in the 5' capping efficiency evaluation method of the present invention without limitation as long as the mRNA has a complementary base sequence recognizable by DNAzyme of the present invention and has the AG sequence acted on by the catalytic motif of DNAzyme. In an embodiment, the mRNA of the present invention may include or consist (essentially) of the base sequence of SEQ ID NO: 1, but is not limited thereto.

[0178]

[0179] Although a nucleotide sequence (base sequence) is defined as a particular sequence number, such a nucleotide sequence does not exclude a mutation that may occur by the addition of a meaningless sequence upstream or downstream of the nucleotide sequence of the corresponding sequence number, or a mutation that may occur naturally, or a silent mutation thereof, as long as such a nucleotide sequence has functions and activity identical or corresponding to those of the nucleotide sequence of the corresponding sequence number, and it would be obvious that even a nucleotide sequence with such a sequence addition or mutation falls within the scope of the present invention.

[0180]

[0181] Specifically, the nucleotide sequence defined in the present invention may have or include a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, but less than 100% homology or identity to the nucleotide sequence of the corresponding sequence number, or may consist of or consist essentially of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, but less than 100% homology or identity to the nucleotide sequence of the corresponding sequence number, but is not limited thereto.

[0182]

[0183] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given nucleotide sequences, and may be expressed as a percentage. The terms homology and identity may often be used interchangeably with each other.

[0184] A method of determining sequence similarity or identity between two or more nucleotide sequences or amino acid sequences is known in the art.

[0185] For example, the similarity or identity may be determined by a known computer algorithm, such as the "FASTA" program, by using default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, this may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which is performed in the Needleman program of the European Molecular Biology Open Software Suite (EMBOSS) package (Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or versions thereafter) (including GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48: 1073). For example, the homology or identity may be determined using BLAST of the National Center for Biotechnology Information database, or ClustalW.

[0186]

[0187] In the method for evaluating mRNA 5' capping efficiency according to the present invention, the cleaving of mRNA may be performing denaturing, binding, and cleaving steps in a reaction solution containing a buffer, divalent cations, a nucleic acid, and mRNA (or a sample containing mRNA), but the cleaving step is not limited to a particular method as long as mRNA can be cleaved into two or more fragments. Specifically, an mRNA forming a secondary structure is unfolded into a primary structure during the denaturing step, and the catalytic nucleic acid binds to a cleavage site within the mRNA through the complementary base sequence during the binding step. Subsequently, during the cleaving step, the phosphodiester bond between A and G within the bound cleavage site by the catalytic motif of the catalytic nucleic acid is cleaved, resulting in the cleavage of the 5'-end of mRNA, but is not limited thereto.

[0188] The conditions for performing the denaturing, binding, and cleaving steps may be appropriately selected and determined by a person skilled in the art considering the reaction solution, experimental apparatuses, and the like. The denaturing, binding, and cleaving steps may be performed using the same apparatus or different apparatuses. Additionally, when one step is completed and then the next step is performed, the reaction may proceed by further adding a reaction solution containing a buffer and divalent cations, but is not limited thereto.

[0189]

[0190] In a specific embodiment, in the cleaving of mRNA, the sample may react with the catalytic nucleic acid for 2 to 30 hours, 2 to 25 hours, or 2 to 24 hours.

[0191]

[0192] In one example of the present invention, when the cleavage of mRNA was induced by DNAzyme, the increase rate in 5'-end truncation efficiency was reduced from the time when 2 hours had elapsed, and significant 5'-end truncation efficiency and 5' capping efficiency were obtained by a reaction time of 2 hours. It was therefore confirmed that an mRNA cleavage reaction for at least 2 hours enables the sufficient ensuring of cleaved mRNA, leading to the analysis of 5' capping efficiency.

[0193]

[0194] In another aspect embodiment, the cleaving of mRNA may include, but not limited thereto, at least one step of (b-1) to (b-3) below:

[0195] (b-1) denaturing mRNA in the sample at 90 to 100°C for 5 to 15 minutes;

[0196] (b-2) binding mRNA to a catalytic enzyme at a temperature lower than the melting point of the catalytic enzyme by 5 to 10°C, for 20 to 40 minutes; and

[0197] (b-3) cleaving mRNA by the catalytic enzyme at 35 to 40°C for 2 to 30 hours.

[0198] In another specific embodiment, the denaturing and binding steps may be performed in a PCR machine, and the cleaving step may be performed in a heating block, but are not limited thereto.

[0199]

[0200] The method for evaluating mRNA 5' capping efficiency may further include (c) quantifying 5' capped mRNA and 5' uncapped mRNA and / or determining the ratio of the 5' capped mRNA and the 5' uncapped mRNA, but is not limited thereto.

[0201]

[0202] In a specific embodiment, step (c) may be performed by any one or more methods of gel electrophoresis, capillary electrophoresis, mass spectrometry, and chromatography, but is not limited thereto. Specifically, any one or more methods of gel electrophoresis, capillary electrophoresis, mass spectrometry, and chromatography may be used to quantify the total mRNA, 5' capped mRNA, and 5' uncapped mRNA and determine the ratio thereof, but are not limited thereto.

[0203]

[0204] In another specific embodiment, the method for evaluating mRNA 5' capping efficiency of the present invention may further include separating 5'-end fragments before the determining of the ratio of capped mRNA and uncapped mRNA (c), but is not limited thereto. The 5'-end fragments may be separated and used for the analysis of 5' capping efficiency. The separating may be appropriately performed through a known method in the art by using a difference in fragment size, or the like.

[0205]

[0206] In another specific embodiment, the method for evaluating mRNA 5' capping efficiency of the present invention may further include removing a phosphate group from 5' uncapped mRNA before the determining of the ratio of the capped mRNA and the uncapped mRNA (c), but is not limited thereto. The 5' uncapped mRNA may exist in four forms, that is, 5'-triphosphate, diphosphate, monophosphate, and hydroxyl. When all the 5' uncapped mRNA is induced into a 5'-hydroxyl form by treating the 5' uncapped mRNA with phosphatase for clear separation from the peaks of the 5' capped mRNA fragments, the 5' capping efficiency can be evaluated more accurately. The phosphatase is not limited to a specific type or sequence as long as the phosphatase is a known enzyme capable of removing the phosphate group.

[0207]

[0208] The description may be equally applied to all aspects of the inventions described herein.

[0209]

[0210] In accordance with still another aspect of the present invention, a kit for evaluating mRNA 5' capping efficiency is provided.

[0211] The kit may be a kit for performing the method of the present invention.

[0212]

[0213] In a specific embodiment, the kit may include a reaction solution containing (i) a buffer and (ii) divalent cations, but is not limited thereto.

[0214] In another specific embodiment, the kit may further include (iii) a catalytic nucleic acid (e.g., DNAzyme), but is not limited thereto.

[0215] The reaction solution and the catalytic nucleic acid are as described above.

[0216] The kit is for use in the production of mRNA and the evaluation of 5' capping efficiency, and may further include analytical reagents and instruction manuals usable for laboratories.

[0217]

[0218] In accordance with still another aspect of the present invention, a method for producing 5'-end fragments is provided.

[0219] In a specific embodiment, the method for producing 5'-end fragments may include:

[0220] (a) providing a sample containing mRNA; and

[0221] (b) bringing the sample into contact with a catalytic nucleic acid to cleave mRNA.

[0222] In another specific embodiment, the method may further include obtaining 5'-end fragments of the cleaved mRNA.

[0223] "mRNA", "5' cap", "sample", "5'-end truncation fragment", and "cleavage" are as described above.

[0224]

[0225] The overlapping description is omitted considering the complexity of the specification, and it would be obvious that the description provided in an aspect is applied to other aspects. Terms not otherwise defined herein have the meanings commonly used in the technical field to which the present disclosure pertains.

[0226]

[0227] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present disclosure, and the scope of the present disclosure is not limited thereto.

[0228]

[0229] Example 1: Cleavage efficiency of DNAzyme depending on buffer and cation conditions

[0230]

[0231] Example 1-1. Selection of DNAzyme

[0232]

[0233] DNAzyme was composed of binding arms consisting of base sequences complementary to a target RNA region and a catalytic motif recognizing a particular base sequence to cause hydrolysis. To select a catalytic motif with high target site cleavage efficiency among several types of catalytic motifs, deoxyribozymes having binding arms complementary to the target RNA region while having three different catalytic motifs, the 8-17 motif and the 10-23 motif (Theranostic DNAzymes, 2017), and the 10-12 opt motif (A Novel Small RNA-Cleaving Deoxyribozyme with a Short Binding Arm, 2019), were synthesized. As a result of comparing the cleavage efficiency% depending on the type of catalytic motif, the 8-17 motif showed an efficiency of 40.5%, which was the highest compared with 33.1% for the 10-23 motif and 7.4% for the 10-12 opt motif.

[0234]

[0235] In addition, to investigate whether increasing binding affinity for mRNA could increase the cleavage efficiency by DNAzyme, binding arms of a deoxyribozyme were 2'-O-methylated to examine the cleavage efficiency. To this end, the 2'-O-methylated binding arms were introduced to the 8-17 catalytic motif confirmed to show the highest cleavage efficiency% (four bases at each edge of the 7-base-long binding arm on either side being 2'-O-methylated). As a result, the 2'-O-methylation of the binding arm increased the cleavage efficiency% by 1.3 times (39.9% -> 53.2%) (FIG. 2).

[0236]

[0237] Example 1-2. Selection of metal ions in DNAzyme reaction buffer

[0238]

[0239] To determine the type of divalent metal ion added to a reaction buffer for increasing cleavage efficiency, divalent ions, such as Mg2+, Mn2+, and Zn2+, were added to examine the cleavage efficiency.

[0240] As a result, the addition of 10 mM MgCl2increased the cleavage efficiency% by 1.7 times (39.8% -> 66.9%) compared with control (1 mM MgCl2), while the addition of 10 mM MnCl2and the addition of 10 mM ZnCl2did not show a cleavage reaction. Additionally, the reaction was not observed by even the addition of 500 μM spermine, which was known to stabilize a deoxyribozymes / RNA hybrid (FIG. 3).

[0241]

[0242] Example 1-3. Establishment of DNAzyme reaction optimal conditions through DoE

[0243]

[0244] As a result of screening main factors affecting the reaction through multivariate testing, it was identified that the pH of a reaction buffer and the concentration of MgCl2involved in divalent metal ions acted as main factors. As a result of optimizing the treatment conditions for the two factors, the addition of 17 mM MgCl2in the reaction buffer at pH 7.5 showed a maximum cleavage efficiency, which was improved by 2.3 times compared with the existing cleavage efficiency (36.7% -> 85.5%) (FIG. 4).

[0245]

[0246] The interaction effect between the pH of the reaction buffer and the concentration of Mg2+was confirmed through the multivariate testing, and on the basis of these results, the cleavage efficiency was examined under the conditions as shown in Table 2 to check the conditions allowing high cleavage efficiency.

[0247]

[0248]

[0249]

[0250] As a result, as confirmed in Table 2 above, the cleavage efficiency was high when Mg2+was contained in a concentration range of 12 to 30 mM in the reaction solution and the pH of the reaction buffer ranged from 7 to 7.5.

[0251]

[0252] Example 2: Evaluation of mRNA 5' capping efficiency using DNAzyme

[0253]

[0254] Example 2-1. Preparation of mRNA and DNAzyme

[0255]

[0256] mRNA (SEQ ID NO: 1) used in the evaluation of mRNA 5' capping efficiency was as shown in Table 3.

[0257]

[0258]

[0259]

[0260]

[0261] The bold text in the mRNA of SEQ ID NO: 1 corresponds to the 5' cap (TriLink CleanCap, cat#. N-7413), the structure of which is as shown in Chemical Formula 1 below. The underlined portions of SEQ ID NO: 1 correspond to the 5'-UTR, and Ψ represents N1-methylpseudouridine.

[0262]

[0263] [Chemical Formula 1]

[0264]

[0265]

[0266] The DNAzyme used to cleave the mRNA was as follows.

[0267]

[0268]

[0269]

[0270] The bold text in the DNAzyme of SEQ ID NO: 2 indicates a catalytic motif, and the other sequence portions correspond to binding arms.

[0271]

[0272] The DNAzyme was used to cleave the 5'-UTR of the mRNA, thereby preparing a 5' fragment containing a 5' cap, which was then used to evaluate the 5' capping efficiency.

[0273]

[0274] The cleavage site by DNAzyme is as shown in FIG. 1, and the cleaved fragment is as follows.

[0275]

[0276]

[0277]

[0278] Example 2-2. Preparation of sample for evaluating mRNA 5' capping efficiency

[0279]

[0280] A sample for evaluating mRNA 5' capping efficiency was prepared considering the conditions for increasing cleavage reaction efficiency, determined in Example 1.

[0281]

[0282] Specifically, 500 μL of mRNA (1.0 mg / mL), which was not confirmed as to whether the 5' cap of Chemical Formula 1 was included, and 100 mM sodium acetate (pH 7.5) were placed in a 30-kDa Centricon, and subjected to centrifugation at 12000 rpm for 3 minutes at 10°C. This procedure was repeated three times. Thereafter, 100 mM sodium acetate (pH 7.5) was adjusted to a final volume of 500 μL, and then transferred to an E-tube.

[0283]

[0284] To 500 μL of the prepared reaction solution, 38 μL of DNAzyme (400 pmol / μL), 60 μL of 10x reaction buffer (500 mM Tris, 170 mM MgCl2, pH 7.5), and 2 μL of distilled water were added, thereby preparing a sample for efficiency evaluation. Specifically, mRNA and DNAzyme were contained at a ratio (molar ratio) of 1:20 in the sample.

[0285]

[0286] Example 2-3. Denaturation, binding, and cleavage reactions

[0287]

[0288] The sample (600 μL) prepared in Example 2-1 was dispensed into a 96-well microplate, with 100 μL per well. The 96-well microplate was placed in a real-time PCR machine, followed by denaturation at 95°C for 10 minutes, binding at 65°C for 30 minutes, and cooling down at 37°C for 30 minutes.

[0289]

[0290] Thereafter, 600 μL of the sample was transferred to an E-tube, and then 70 μL of 10Х reaction buffer (500 mM Tris, 170 mM MgCl₂, pH 7.5) and 30 μL of distilled water were added, and placed in a heating block, followed by incubation at 37°C for 27 hours. As a result, the cleavage efficiency was 85.5% (AVG±3SD, 5 runs: 72.2-98.7%).

[0291]

[0292] The cleavage efficiency was determined as follows.

[0293] - Theoretical ratio = deoxyribozyme extinction coefficient 268500 Х molar ratio 20 / capped fragment extinction coefficient 123400 = 43.52

[0294] - Cleaved mRNA ratio = theoretical ratio 43.52 Х capped fragment peak area [A] / deoxyribozyme peak area [B]

[0295] - Normalized full-length mRNA peak area = analyzed full length mRNA peak area [C] Х (1 - cleaved mRNA ratio)

[0296] - Cleavage efficiency% =

[0297]

[0298]

[0299] Example 2-4. Removal of phosphate from uncapped mRNA 5'-end

[0300]

[0301] To remove phosphate from the 5'-end of a small amount of uncapped mRNA that might exist in the sample, 23 μL of calf intestinal phosphatase (CIP), 10 μL of T4 polynucleotide kinase (T4 PNK), 90 μL of 10x T4 PNK buffer, and 77 μL of distilled water were added to 700 μL of the sample of Example 2-2, and the mixture was placed in a heating block, followed by a reaction at 37°C for 30 minutes.

[0302]

[0303] Example 2-5. Buffer exchange and concentration

[0304]

[0305] The sample of Example 2-3 (900 μL) and mobile phase A were placed in a 3-kDa Centricon, followed by centrifugation at 14000 rpm for 10 minutes at 10°C (repeated a total of 3 times). Specifically, mobile phase A (1% HFIP 0.1% DIEA 2 μM EDTA in Water) was prepared by adding 1 mL of N,N-diisopropylethylamine (DIPEA), 10 mL of hexafluoroisopropanol (HFIP), and 4 μL of 0.5 M EDTA disodium salt solution to 900 mL of distilled water filtered through a 0.22-㎛ filter, followed by filling with distilled water filtered through a 0.22-μm filter to make a total volume of 1 L.

[0306]

[0307] The mobile phase A was adjusted to a final volume of 200 μL, and then transferred into an LC vial.

[0308]

[0309] Example 2-6. Ultra performance liquid chromatography (UPLC) analysis

[0310]

[0311] Ultra-high performance liquid chromatography was performed under the conditions as shown in Table 6, and the results are shown in FIG. 5. Mobile phase B (0.075% HFIP, 0.0375% DIEA, 2 μM EDTA in 65 / 35 ACN / Water) was prepared by adding 375 μL of N,N-diisopropylethylamine (DIPEA), 750 μL of hexafluoroisopropanol (HFIP), and 4 μL of 0.5 M EDTA disodium salt solution to 344 mL of distilled water filtered through a 0.22-㎛ filter, followed by filling with acetonitrile to make a total volume of 1 L.

[0312]

[0313]

[0314]

[0315] As a result, the method for evaluating mRNA 5' capping efficiency according to the present invention enables effective analysis of 5' capping efficiency by including cleaving mRNA with high efficiency through a catalytic nucleic acid.

[0316]

[0317] On the basis of the results of the example, the following tests were conducted to establish more optimized conditions for the analysis of 5' capping efficiency.

[0318]

[0319] Example 3: Evaluation of cleavage efficiency depending on reaction time of mRNA and DNAzyme

[0320]

[0321] To establish the minimum time required for the mRNA cleavage process by DNAzyme in the analysis of 5' capping efficiency, the cleavage efficiency depending on the cleavage reaction time was investigated.

[0322]

[0323] Specifically, mRNA and a sample containing the mRNA were prepared by the same method as in Example 2, and DNAzyme for the analysis of mRNA 5' capping efficiency was prepared, followed by denaturation, binding, and cleavage reactions.

[0324]

[0325] Specifically, DNAzyme and the sample were reacted for up to 24 hours, and the cleavage efficiency was measured at 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours. The results are shown in Table 7 and FIGS. 6 and 7.

[0326]

[0327]

[0328]

[0329] As a result, the 5' capping efficiency within the reference range was confirmed from 2 hours, and it was therefore identified that the minimum reaction time for the analysis of 5' capping efficiency was 2 hours. Specifically, the reference range refers to a range in which 99% of the 5' capping efficiency results are expected to be distributed when the test is conducted under standard conditions (conditions of Example 2).

[0330]

[0331] Example 4: Evaluation of cleavage efficiency depending on length of binding arms

[0332]

[0333] To increase mRNA cleavage efficiency, the optimal binding arm length was investigated by adjusting the length of binding arms included in DNAzyme.

[0334]

[0335] To this end, DNAzyme in which binding arms having 4 to 9 bases were connected to both ends of the 8-17 catalytic motif was prepared. Table 8 below shows the sequences of binding arms located at the 5'-end and 3'-end of the catalytic motif.

[0336]

[0337]

[0338]

[0339]

[0340] The mRNA cleaving efficiency was investigated using DNAzyme by the same method as in Example 2. As for the reaction time, the cleavage reaction was carried out for 2 hours according to the time confirmed in Example 3, and the 5' capping efficiency was analyzed.

[0341]

[0342] As a result, as shown in Table 9 and FIGS. 8 and 9, the longer the binding arm length, the higher the cleavage efficiency. Specifically, the use of DNAzyme having binding arms of 7 to 9 bases achieved significant cleavage efficiency, showing 5' capping efficiency within the reference range, and thus binding arms of 7 to 9 bases were suitable for the analysis of 5' capping efficiency. Specifically, the reference range refers to a range in which 99% of the 5' capping efficiency results are expected to be distributed when the test is conducted under standard conditions (conditions of Example 2).

[0343]

[0344]

[0345]

[0346] Example 5: Evaluation of cleavage efficiency depending on number of methylated bases in binding arms

[0347]

[0348] Furthermore, the number of methylated bases in binding arms was determined as optimal conditions for the analysis of 5' capping efficiency.

[0349]

[0350] To this end, DNAzyme containing binding arms of 7 bases on both ends in Example 4 was prepared, and then the cleavage reaction was carried out by the same method as in Example 2 to analyze the 5' capping efficiency.

[0351]

[0352] The binding arms used in the present example were obtained by changing the number of methylated bases in the same sequences as in the binding arms of SEQ ID NO: 10 (5'-end) and SEQ ID NO: 16 (3'-end) in Example 4. Specifically, in each binding arm, 1 to 7 bases were sequentially methylated from the edge of the end unconnected to the catalytic motif (Table 10).

[0353]

[0354]

[0355]

[0356]

[0357] As a result, as shown in Table 11 and FIGS. 10 and 11, the use of DNAzyme with 1 to 4 methylated bases showed significant values in cleavage efficiency and 5' capping efficiency, but DNAzyme containing binding arms with 5 methylated bases showed lower 5' capping efficiency compared with the reference range. Specifically, the reference range refers to a range in which 99% of the 5' capping efficiency results are expected to be distributed when the is was conducted under standard conditions (conditions of Example 2). Furthermore, DNAzyme containing binding arms with 6 and 7 methylated bases showed low cleavage efficiency.

[0358]

[0359]

[0360]

[0361] Therefore, the use of binding arms with 1 to 4 methylated bases was appropriate in the analysis of 5' capping efficiency.

[0362]

[0363] The above examples confirmed that the method for evaluating mRNA 5' capping efficiency according to the present invention enables effective analysis of 5' capping efficiency, and determined the optimal conditions with respect to the reaction time, binding arm length, and number of methylated bases for the analysis of 5' capping efficiency.

[0364]

[0365] While the present invention has been described with reference to the particular illustrative embodiments, a person skilled in the art to which the present invention pertains can understand that the present invention may be embodied in other specific forms without departing from the technical spirit or essential characteristics thereof. In this regard, the embodiments described above should be understood to be illustrative rather than restrictive in every respect. The scope of the invention should be construed that the meaning and scope of the appended claims rather than the detailed description and all changes or variations derived from the equivalent concepts fall within the scope of the present invention.

Claims

1.A method for evaluating mRNA 5' capping efficiency, the method comprising:(a) providing a sample containing mRNA; and(b) bringing the sample into contact with a catalytic nucleic acid to cleave mRNA.2.The method of claim 1, wherein in the cleaving of mRNA, the contacting between the sample and the catalytic nucleic acid is performed in a reaction solution containing Mg2+and a buffer.3.The method of claim 2, wherein the reaction solution contains Mg2+at a concentration of 12 to 30 mM.4.The method of claim 2, wherein the buffer has a pH of 7.0 to 7.5.5.The method of claim 1, wherein the catalytic nucleic acid is DNAzyme.6.The method of claim 5, wherein the ratio of mRNA to DNAzyme in the sample is 1:10 to 1:20.7.The method of claim 5, wherein the DNAzyme comprises a binding arm binding to mRNA and a catalytic motif.8.The method of claim 7, wherein the DNAzyme comprises two binding arms, which are located at both ends of the catalytic motif.9.The method of claim 8, wherein the binding arms each include 7 to 9 bases.10.The method of claim 7, wherein the binding arm complementarily binds to mRNA.11.The method of claim 7, wherein the DNAzyme includes methylation of a base in the binding arm.12.The method of claim 11, wherein the binding arm includes 1 to 4 methylated bases.13.The method of claim 1, wherein the mRNA has a cleavage site, which is cleaved by the catalytic nucleic acid.14.The method of claim 7, wherein the catalytic motif is an 8-17 motif.15.The method of claim 1, wherein in the cleaving of mRNA, the sample reacts with the catalytic nucleic acid for 2 to 30 hours.16.The method of claim 1, wherein the cleaving of mRNA comprises at least one step of (b-1) to (b-3) below:(b-1) denaturing mRNA in the sample at 90 to 100°C for 5 to 15 minutes;(b-2) binding mRNA to a catalytic enzyme at a temperature lower than the melting point of the catalytic enzyme by 5 to 10°C, for 20 to 40 minutes; and(b-3) cleaving mRNA by the catalytic enzyme at 35 to 40°C for 2 to 30 hours.17.The method of claim 1, further comprising (c) determining the ratio of 5'-capped mRNA and 5'-uncapped mRNA.18.The method of claim 11, wherein step (c) is performed by any one or more methods of gel electrophoresis, capillary electrophoresis, mass spectrometry, and chromatography.

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