Method for quantifying circular RNA

Abstract

The objective is to provide a novel method for accurately quantifying circular RNA. [Solution] A method for producing a reverse transcript from circular RNA, comprising a cleavage step of cleaving the circular RNA using a cleavage enzyme that cleaves the target cleavage site of the circular RNA, and a reverse transcription step of performing reverse transcription using the RNA cleaved in the cleavage step as a template, wherein the reverse transcription step uses a reverse transcription primer containing a base sequence complementary to the primer binding region, and the primer binding region does not include the target cleavage site.

Description

Technical Field

[0001] The present invention relates to a method for producing a reverse transcription product from circular RNA, a method for quantifying circular RNA, and the like.

Background Art

[0002] The demand for mRNA vaccines has increased with the worldwide spread of COVID-19. At present, with the convergence of COVID-19, mRNA vaccines are being developed not only for infectious disease vaccines but also for the treatment of various diseases including cancer treatment.

[0003] However, mRNA has the weakness of low stability and is rapidly degraded in the presence of degrading enzymes. Therefore, when administering mRNA to a living body as a drug, there is a problem of low blood retention.

[0004] As a method for improving the stability of mRNA, a method for circularizing mRNA has been proposed. Circular RNA obtained by circularizing mRNA is not degraded by exonucleases, which are intracellular degrading enzymes. Therefore, it is said to have higher stability compared to normal linear mRNA.

[0005] In the reaction for circularizing linear mRNA, since some mRNA remains linear without circularizing, it is common to obtain a mixture of circular mRNA and linear mRNA. As a conventional method, a method for quantifying the content of only circular RNA based on the degradation of linear RNA is known as described below.

[0006] Patent Document 1 discloses a touchdown PCR detection method for quantifying the expression level of circular RNA. The method involves extracting total RNA from HepG2 cells, a human hepatocellular carcinoma cell line, degrading linear RNA using RNase R enzyme, an exoribonuclease derived from Escherichia coli that has 3'-to-5' exonuclease activity, synthesizing cDNA by reverse transcription using the remaining circular RNA as a template, and performing relative quantification based on real-time fluorescence detection by subjecting the obtained cDNA to touchdown PCR.

[0007] Non-patent document 1 is a review article on methods for evaluating circular RNA. It describes a method for quantifying the content of circular RNA by preparing a circular RNA-containing sample from which linear mRNA has been removed by treatment with the Rnase R enzyme, and a circular RNA-containing sample from which circular RNA has not been treated with the Rnase R enzyme, and then performing qPCR using the cDNA obtained by subjecting these two samples to a reverse transcription reaction as a template. [Overview of the project] [Problems that the invention aims to solve]

[0008] The conventional method described above selectively quantifies circular RNA by degrading linear RNA contained in a sample containing circular RNA using the RNase R enzyme. However, when reverse transcription is performed using circular RNA as a template, the circular RNA sequence is repeated during reverse transcription. This can lead to the production of a reverse transcript containing two or more copies of the target sequence for amplification in subsequent amplification reactions, potentially resulting in an overquantification of the amount of circular RNA. Therefore, there is suspicion that the conventional method does not accurately quantify the amount of circular RNA.

[0009] The object of this invention is to provide a novel method for accurately quantifying circular RNA. [Means for solving the problem]

[0010] The present invention provides the following: A method for producing a reverse transcript from circular RNA, A cutting step, comprising cutting the circular RNA using a cutting enzyme that cuts the circular RNA at a target cleavage site, and Reverse transcription step: Using the RNA cut in the above-mentioned cutting step as a template, reverse transcription is performed. Includes, The reverse transcription step is performed using a reverse transcription primer containing a base sequence complementary to the primer binding region, wherein the primer binding region does not include the target cleavage site. [Effects of the Invention]

[0011] The present invention provides a novel method for accurately quantifying circular RNA. [Brief explanation of the drawing]

[0012] [Figure 1] This section outlines a method using cleavage products of circular RNA as a template for reverse transcription. Figure 1A shows that using circular RNA as a template without cleavage results in a reverse transcript containing repeated base sequences of the circular RNA. Figure 1B shows that using cleavage products of circular RNA as a template prevents the generation of a reverse transcript containing two or more copies of the target sequence. [Figure 2] The figure shows the cleavage product of circular RNA obtained by circularizing linear RNA, when it is cleaved at a different site than the linkage site used during circularization (indicated as "Cleavage product of circular RNA"), and the cleavage product of non-circular RNA, which was not circularized, when it is cleaved at the same site (indicated as "Cleavage product of non-circular RNA"). [Figure 3] This shows the first and second target regions that are amplified using reverse transcripts obtained by reverse transcription using circular RNA cleavage products and acyclic RNA cleavage products as templates for nucleic acid amplification. From circular RNA cleavage products, both the first and second target regions are amplified, while from acyclic RNA cleavage products, only the second target region is amplified. [Figure 4]This shows the results of verifying the effect of a cleavage reaction using RNase H to cleave circular RNA. The dashed line (RNase H treatment -) shows the qPCR measurement results when circular RNA was not cleaved with RNase H. The solid line (RNase H treatment +) shows the qPCR measurement results when circular RNA was cleaved with RNase H. The dotted line indicates the threshold used to calculate the Ct value. [Modes for carrying out the invention]

[0013] To solve the above problem, the inventors conceived of cleaving circular RNA with the aim of preventing the circular RNA sequence from being repeatedly reverse-transcribed. This is because if the reverse transcription reaction is performed using the cleaved circular RNA as a template, it is possible to prevent the same template from being repeatedly reverse-transcribed. Based on this idea, the inventors designed a primer that serves as the starting point for reverse transcription to hybridize with acyclic RNA obtained by cleaving circular RNA. Furthermore, the inventors positioned the reverse transcription primer so that the ligation site linked in the cyclization reaction is included within the region to be reverse-transcribed, thereby enabling the quantification of only circular RNA, distinguishing it from the linear RNA before cyclization. The inventors amplified and quantified the region corresponding to the region containing this ligation site in the reverse transcription product by quantitative PCR. As a result, they found that the quantitative value obtained when using uncleaved circular RNA as the reverse transcription template was 1.8 times higher than the quantitative value obtained when using a cleavage product obtained by cleaving circular RNA at a specific position as the reverse transcription template. These results support the finding that the amount of circular RNA quantified by conventional methods is 1.8 times higher than the actual amount. Therefore, the inventors have demonstrated that the accuracy of quantification can be significantly improved by cleaving the circular RNA before reverse transcription.

[0014] Previous papers in this field have reported the RNase H Nicking method, which analyzes the size of cleavage products obtained by cleaving circular RNA with RNase H using agarose gel electrophoresis (Non-Patent Literature 2-3). This method confirms circularity based on the change in electrophoretic mobility between the circular RNA before cleavage and the linear RNA after cleavage. On the other hand, a method that uses the cleaved circular RNA as a template for reverse transcription, as in the present invention, has not been reported in the past.

[0015] 1. Method for preparing reverse transcript products 1-1. Overview A first aspect of the present invention is a method for producing a reverse transcript from circular RNA (hereinafter sometimes abbreviated as "production method"). The production method of the present invention is characterized by performing reverse transcription using circular RNA cleaved with a cleaving enzyme as a template, thereby preventing the circular RNA sequence from being repeated in the reverse transcription, and thus contributing to the highly accurate quantification of circular RNA. Furthermore, by placing a reverse transcription primer upstream of the ligation site in the direction of reverse transcription, the ligation site will be reverse transcribed, making it possible to amplify the region containing the ligation site using the reverse transcript as an amplification template, and making it possible to distinguish and quantify only the cleavage products derived from circular RNA from the cleavage products derived from non-cyclic RNA before cyclization.

[0016] 1-2. Definitions of Terms The terms frequently used in this specification are defined below.

[0017] In this specification, "RNA" or "RNA strand" includes any RNA, such as total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA. mRNA may be chemically synthesized mRNA or mRNA transcribed from a DNA sequence encoding mRNA.

[0018] As used herein, "circular RNA" refers to an RNA strand containing a sequence portion that forms at least one circular structure. Circular RNAs include both those in which the entire RNA strand forms a circular structure and those in which a part of the RNA strand is in a circular structure. However, when the entire RNA strand forms a single circular structure, it is preferred because it does not contain free 3'- and 5'-ends and thus has high resistance to degradation by exonucleases. In this specification, circular RNA is generally considered to form a circular structure by the ligation of the 5'-end and the 3'-end in the RNA strand. The ligation between the 5'-end and the 3'-end may be either a covalent bond or a non-covalent bond, but is the internucleoside bond unless otherwise specified. In addition, circular RNA may be either synthetic circular RNA or circular RNA derived from a living body or a natural source. The nucleotide sequence of circular RNA is not particularly limited, and may contain, for example, a nucleotide sequence encoding a protein. The proteins encoded by circular RNA are not limited, and examples include viral proteins for conferring immunity against viruses, therapeutic proteins introduced into a subject in gene therapy, and cancer antigens for targeting cancer cells in cancer immunotherapy. The length of circular RNA in this specification is not particularly limited, and may be, for example, 50 bases to 100,000 bases, 100 bases to 50,000 bases, 200 bases to 20,000 bases, 500 bases to 10,000 bases, 1,000 bases to 5,000 bases, or 2,000 bases to 3,000 bases. Note that circular RNA may be referred to as "circRNA" or "circular polynucleotide", etc., and these terms are used interchangeably with circular RNA in this specification.

[0019] As used herein, "non-circular RNA" refers to any RNA other than circular RNA. That is, non-circular RNA refers to any RNA that does not contain a sequence portion forming a circular structure. Non-circular RNAs include the following linear RNAs and RNA strands having a branched structure.

[0020] As used herein, "linear RNA" or "straight-chain RNA" means a linear RNA that does not have a branched structure among non-circular RNAs.

[0021] As used herein, "circularization" means circularizing the whole or a part of an RNA. For example, it includes circularizing the whole non-circular RNA such as the above-mentioned linear RNA by ligating the 5'-end and the 3'-end of the non-circular RNA via an internucleoside bond, or circularizing a part thereof.

[0022] As used herein, the "internucleoside bond" may be either a naturally occurring internucleoside bond (i.e., a phosphodiester bond) or an internucleoside bond having a substitution or any change from a phosphodiester bond (referred to herein as a "modified internucleoside bond"). Examples of the modified internucleoside bond include phosphorothioate bond, phosphorodithioate bond, phosphotriester bond, alkylphosphonate bond, alkylthiophosphonate bond, and phosphorodiamidate, etc.

[0023] As used herein, the "linking site" means the position where an RNA strand is linked when circularized. More specifically, it means at least two nucleotides linked by circularization.

[0024] As used herein, the "cleaving enzyme" means an enzyme having an activity of cleaving a nucleic acid strand. Unless otherwise specified, it refers to an RNA cleaving enzyme that cleaves an RNA strand. Specific examples of the RNA cleaving enzyme include ribonuclease H (RNase H), Cas13 protein, and Dicer protein.

[0025] In this specification, "nucleic acid primer" or "primer" refers to a nucleic acid molecule that specifically binds to a portion of a nucleic acid chain and provides a starting point for nucleic acid synthesis reactions by polymerase enzymes such as DNA polymerase and reverse transcriptase. In this specification, primers used for specific applications such as reverse transcription and nucleic acid amplification may be referred to as "reverse transcription primers" or "amplification primers" depending on the application. Nucleic acid primers are usually composed of single-stranded DNA and are designed to include a sequence complementary to at least a portion of the target sequence so that they can bind to the target sequence. The base length of the primer is not limited as long as it is long enough to bind to the target sequence to which the primer binds, and may be, for example, 12 bases or more, 15 bases or more, 18 bases or more, 20 bases or more, or 25 bases or more, and / or 100 bases or less, 80 bases or less, 60 bases or less, 40 bases or less, or 30 bases or less. In a preferred embodiment, the base length of the primer is within the range of the above base lengths as the upper and lower limits. Furthermore, it is preferable that the primer has several bases at its 3' end (2-15 bases, 3-12 bases, 4-10 bases, or 5-8 bases) that are perfectly complementary to the target sequence in order to function as a starting point for nucleic acid synthesis reactions.

[0026] In this specification, "sample" refers to any sample containing circular RNA that is used in the preparation method, quantification method, etc., of the present invention. The sample is not particularly limited, and specific examples include preparations such as pharmaceuticals containing circular RNA (e.g., mRNA vaccines, and pharmaceuticals for gene therapy or cancer immunotherapy) and reagents, or nucleic acid-containing compositions as raw materials therefor, cells or tissues derived from any organism (e.g., animals such as humans, eukaryotes including plants, bacteria including Escherichia coli, and archaea), or extracts thereof.

[0027] In this specification, "complementary" means a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) via hydrogen bonding. Two complementary base sequences do not necessarily have to be perfectly complementary (i.e., 100% complementary); it is acceptable if the base sequences have at least 80%, preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity, particularly sufficient complementarity for the two base sequences to hybridize with sufficient specificity. The complementarity of base sequences can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two strands can anneal or hybridize, taking into account the degree of complementarity between the strands.

[0028] 1-3. Method The present invention's method includes a cleavage step and a reverse transcription step as essential steps, and may include other steps such as a cyclization step, an RNA extraction step, and / or an RNA degradation step as optional steps. The configuration of each step is described below.

[0029] (cutting process) The "cleavage process" is a process in which circular RNA is cleaved using a cleavage enzyme that cuts at a target cleavage site on the circular RNA. Through this process, the circular RNA is cleaved, and acyclic RNA is produced as the cleaved RNA (cleavage product).

[0030] In this specification, "target cleavage site" refers to a position on the RNA chain that is cleaved by the cleaving enzyme in this process, specifically, an internucleoside bond that is cleaved on the RNA chain (Figure 1B). The target cleavage site is not limited to any internucleoside bond contained in the RNA chain, and any position on the circular RNA can be cleaved. The number of target cleavage sites can be one or more, for example, 1 to 50, 2 to 40, 3 to 30, 4 to 20, or 5 to 10. In one embodiment, it is preferable to use a single cleaving enzyme to ensure that one non-cyclic RNA is reliably obtained as the cleavage product. In another embodiment, it is preferable to use multiple cleaving enzymes to ensure that the circular RNA is cleaved at one or more locations, i.e., to have two or more target cleavage sites. When there are two or more target cleavage sites, it is preferable that the target cleavage sites on the circular RNA are in the range of a few nucleotides to several hundred nucleotides in length. The target cleavage sites may be internucleoside bonds that are consecutive or non-consecutive on the circular RNA. For example, in the method using single-stranded DNA oligonucleotides together with ribonuclease H, as will be described later, consecutive nucleoside bonds within a region complementary to the single-stranded DNA oligonucleotide can serve as target cleavage sites.

[0031] The conditions for the cleavage reaction in this process should be set according to the type of cleavage enzyme used. For example, refer to Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press for appropriate settings. For example, the reaction can be carried out at a temperature of 32°C to 60°C for several seconds to several hours, such as 10 seconds to 3 hours, 20 seconds to 1 hour, 30 seconds to 10 minutes, 40 seconds to 5 minutes, or 1 minute to 2 minutes.

[0032] In one embodiment, the circular RNA cleaved in this process is formed by the circularization of an acyclic RNA such as linear RNA. In this embodiment, the sites where acyclic RNA is ligated during circularization are called ligation sites. Specific ligation sites include, for example, the sites where the 5' and 3' ends of the acyclic RNA before circularization are ligated in the circular RNA, and the sites where ligation occurs through spontaneous circularization in the PIE method described later. In this embodiment, it is preferable that the target cleavage site is at a different location from the ligation site. If the target cleavage site is a contiguous nucleoside linkage of two or more nucleosides, it is preferable that none of these target cleavage sites are included in the ligation site.

[0033] In a further embodiment, the circular RNA is cleaved such that the primer-binding region is located upstream of the ligation site in the direction of reverse transcription. That is, the reverse transcript obtained in the reverse transcription step described later will include the reverse-transcribed ligation site. For example, by setting the target cleavage site to be located on the 3' side of the primer-binding region and on the 5' side of the ligation site in the circular RNA, the ligation site will be included in the range from the base adjacent to the 5' side of the primer-binding region in the circular RNA, which serves as the template for reverse transcription in the reverse transcription step described later, to the target cleavage site where reverse transcription terminates. In other words, when reverse transcription is performed using a reverse transcription primer that binds to the primer-binding region, with the cleavage product obtained by cleaving the circular RNA at the target cleavage site as a template, the ligation site will be located downstream in the direction of reverse transcription (Figure 3A). By detecting the region containing the position corresponding to this ligation site in the reverse transcript using nucleic acid amplification or the like, it is possible to detect a unique sequence for the circular RNA (Figure 3A, regions amplified by primers A and B). On the other hand, the reverse transcript obtained by reverse transcription using non-cyclic RNA before circularization as a template does not include this ligation site (Figure 3B). Therefore, only circular RNA will be selectively detected.

[0034] In one embodiment, the distance between the target cleavage site (e.g., the target cleavage site closest to the ligation site) and the ligation site in the circular RNA is at least 12 nucleotides long. This distance may be at least 12 nucleotides long, at least 15 nucleotides long, at least 18 nucleotides long, at least 20 nucleotides long, at least 25 nucleotides long, or at least 30 nucleotides long, so that one of the primer pair used to amplify the first target region described later binds to the corresponding area in the reverse transcript. The upper limit of this distance is not limited and may be, for example, 5,000 nucleotides or less, 4,000 nucleotides or less, 3,000 nucleotides or less, 2,000 nucleotides or less, 1,000 nucleotides or less, 500 nucleotides or less, 200 nucleotides or less, or 100 nucleotides or less.

[0035] In one embodiment, the cleavage enzyme used for cleaving the target cleavage site in this process is ribonuclease H, Cas13 protein, or Dicer protein. The cleavage method using these cleavage enzymes will be described in detail below.

[0036] (1) Ribonuclease H (RNase H) Ribonuclease H has the activity to specifically hydrolyze the phosphodiester bond of RNA hybridized with DNA. Therefore, when cleaving a target cleavage site with ribonuclease H in this process, a DNA strand capable of hybridizing with the region containing the target cleavage site in circular RNA (hereinafter referred to as the "cleavage target region") is required to provide cleavage activity at the target cleavage site. An example of DNA having a base sequence complementary to the base sequence of the cleavage target region is single-stranded DNA oligonucleotide. For example, by using one or more single-stranded DNA oligonucleotides consisting of base sequences complementary to the base sequence of a cleavage target region of approximately 12 to 1,000 bases, 15 to 500 bases, 20 to 200 bases, 30 to 100 bases, or 40 to 70 bases in length containing the target cleavage site, together with ribonuclease H, the target cleavage site in circular RNA can be cleaved. The number of single-stranded DNA oligonucleotides used for cleavage is not limited and may be one or more, for example, one, two, three, four, five, six, or seven or more.

[0037] (2) Cas13 protein In this specification, "Cas13 protein" refers to an enzyme that, upon binding to a short RNA called guide RNA, specifically recognizes and cleaves an RNA strand complementary to the guide RNA.

[0038] In this process, when the target cleavage site is cleaved with the Cas13 protein, a guide RNA having a nucleotide sequence complementary to the cleavage target region is required to provide cleavage activity at the target cleavage site. For example, by using the Cas13 protein together with a guide RNA having a nucleotide sequence complementary to the nucleotide sequence of the cleavage target region, which is approximately 12 to 30 nucleotides or 17 to 24 nucleotides long and includes the target cleavage site, the target cleavage site in circular RNA can be cleaved. The number of guide RNAs used for cleavage is not limited; it may be one or more, for example, one, two, three, four, five, six, or seven or more.

[0039] (3) Dicer protein In this specification, "Dicer protein" refers to an enzyme that has the activity to cleave double-stranded RNA into pieces of approximately 21 nucleotides in length.

[0040] In this process, when the target cleavage site is cleaved with the Dicer protein, RNA having a nucleotide sequence complementary to the cleavage target region is required to provide cleavage activity at the target cleavage site. For example, by using the DICER protein together with RNA having a nucleotide sequence complementary to the nucleotide sequence of the cleavage target region, which is 20 nucleotides or longer than 21 nucleotides and includes the target cleavage site, the target cleavage site in circular RNA can be cleaved. The number of RNA molecules used for cleavage is not limited; it may be one or more, for example, one, two, three, four, five, six, or seven or more.

[0041] The cleaving enzyme used in this process can be removed as needed before the reverse transcription process described later. For example, the cleaving enzyme may be removed using the same method as in the RNA extraction process described later.

[0042] Furthermore, if the sample containing circular RNA subjected to this process also contains acyclic RNA that was not cyclic in the cyclization process described later (Figure 2, "Acyclic RNA"), when the acyclic RNA is cleaved at the target cleavage site, two fragments may be produced: a fragment from the 5' end of the acyclic RNA to the target cleavage site, and a fragment from the target cleavage site to the 3' end of the acyclic RNA (Figure 2, "Cleavage Products of Acyclic RNA").

[0043] (Reverse transfer process) The "reverse transcription process" is a process in which reverse transcription is performed using the RNA that was cut in the cleavage process as a template. Through this process, the cleaved RNA is reverse transcribed to obtain cDNA, which is the reverse transcription product.

[0044] The reverse transcription primers used in this process contain a nucleotide sequence complementary to the primer binding region.

[0045] In this specification, "primer binding region" refers to the region on a nucleic acid strand to which a nucleic acid primer binds. Examples include the region on an RNA strand to which a reverse transcription primer binds, and the region on a DNA strand to which an amplification primer binds. Unless otherwise specified, it refers to the region to which a reverse transcription primer binds. By binding to the primer binding region, the reverse transcription primer can provide the starting point for the reverse transcription reaction in this process (Figures 1 and 3).

[0046] The primer-binding region is configured so as not to include the target cleavage site. This configuration allows the reverse transcription primer to hybridize to the acyclic RNA obtained by cleaving the circular RNA, providing a starting point for reverse transcription. The distance from the target cleavage site to the primer-binding region in the 3' direction is not limited. For example, it may be at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 500 nucleotides, at least 800 nucleotides, at least 1,000 nucleotides, at least 2,000 nucleotides, at least 3,000 nucleotides, at least 4,000 nucleotides, or at least 5,000 nucleotides, so that one or more target regions to be amplified by nucleic acid amplification in the quantitative method described later can be placed within the reverse transcript. Furthermore, the distance from the primer binding region to the target cleavage site in the 3' direction is not particularly limited, but may be, for example, 0 to 5,000 base pairs, 10 to 4,000 base pairs, 50 to 3,000 base pairs, 100 to 2,000 base pairs, or 500 to 1,000 base pairs.

[0047] The base length of the primer binding region is not limited as long as it is long enough to bind to the reverse transcription primer, and may be, for example, 12 bases or longer, 15 bases or longer, 18 bases or longer, 20 bases or longer, or 25 bases or longer, and / or 100 bases or shorter, 80 bases or shorter, 60 bases or shorter, 40 bases or shorter, or 30 bases or shorter.

[0048] The conditions for the reverse transcription reaction in this process can be any method known in the art. For example, it can be carried out using an enzyme such as reverse transcription polymerase, in accordance with the reverse transcription method described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press. The reaction conditions in this process are not particularly limited; for example, reverse transcription can be carried out at a temperature of 30°C to 45°C or 37°C to 42°C for 5 minutes to 3 hours or 30 minutes to 2 hours.

[0049] In embodiments where the circular RNA cleaved in the above-described cleavage step is formed by the cyclization of an acyclic RNA, the primer-binding region is preferably located upstream of the ligation site linked by cyclization in the direction of reverse transcription. More specifically, it is preferably located on the 5' side of the target cleavage site and the 3' side of the ligation site in the circular RNA. By positioning the primer-binding region within this range, a reverse transcript containing the position corresponding to the ligation site is reverse transcribed. In this case, it is preferable that the primer-binding region does not include the ligation site.

[0050] If the sample containing circular RNA subjected to the above-described cleavage step also contains acyclic RNA that was not cyclicized in the cyclization step described later (Figure 2, "acyclic RNA"), a second reverse transcript may be produced as a byproduct during the cleavage step. This second reverse transcript is reverse transcribed using the fragment containing the primer-binding region as a template from the fragment generated from the acyclic RNA, specifically the fragment from the 5' end of the acyclic RNA to the target cleavage site and the fragment from the target cleavage site to the 3' end of the acyclic RNA (Figure 3B). This second reverse transcript has either a nucleotide sequence complementary to the nucleotide sequence from the 5' end of the acyclic RNA towards the primer-binding region in the 3' direction, or a nucleotide sequence complementary to the nucleotide sequence from the target cleavage site towards the primer-binding region in the 3' direction. Figure 3B shows the former case.

[0051] (RNA extraction process) The "RNA extraction step" is a step in which RNA is extracted from any sample containing circular RNA before the cleavage step described above. This step is a selective step.

[0052] The method for extracting RNA from the sample in this process is not particularly limited. For example, RNA may be extracted using the general acid-phenol method, or RNA extraction reagents containing acid-phenol may be used. RNA extraction reagents and RNA extraction kits are commercially available from various life science manufacturers such as Qiagen, Takara Bio, Toyobo, Thermo Fisher Scientific, and Promega, and these can also be used. Furthermore, the RNA after extraction can be purified as needed.

[0053] (Circularization process) The "circularization process" is a step performed before the cleavage process described above, in which acyclic RNA is circularized to obtain circular RNA for use in the cleavage process (Figure 2).

[0054] The circularization of acyclic RNA in this process may be performed using either an enzyme-assisted method or an enzyme-assisted method, and the circularization can be carried out by referring to the relevant literature in the art as appropriate (Non-Patent Literature 4). In enzyme-assisted circularization, an RNA ligase such as T4 RNA ligase can be used as the enzyme that can catalyze the linkage between the 5' and 3' ends of the acyclic RNA. The acyclic RNA can be circularized by carrying out the RNA ligase-assisted circularization reaction for example at 30-42°C or 35-38°C for 30 seconds to 3 hours or 10 minutes to 1 hour, or by carrying out the reaction overnight at 10-20°C or 12-18°C. In the case of enzyme-assisted circularization, the Permuted Intron-Exon (PIE) method, which circularizes based on ribozyme-type self-splicing, can be used. The PIE method is used, for example, for Anabaena tRNA LeuThis method utilizes the fact that when a template obtained by permutation—which moves the 5' portion of a Group I tRNA intron in a precursor gene to the end of the tRNA exon and places the remaining 3' portion at the beginning of the exon—is transcribed in vitro, the transcribed RNA spontaneously becomes circular. Similar spontaneous circularization is known to occur in permutations of introns derived from the thymidine synthase (td) gene of the T4 bacteriophage. A PIE method based on permutations of Group II introns derived from yeast mitochondria is also known.

[0055] Furthermore, the method for producing the RNA according to this embodiment may optionally include an RNA synthesis step prior to this step, in which the acyclic RNA to be cyclicized in this step is synthesized using chemical synthesis or intracellular synthesis.

[0056] Furthermore, in the preparation method of this embodiment, the acyclic RNA may be degraded after this step and / or before the reverse transcription step described above, preferably before the cleavage step described above. The method of degrading the acyclic RNA is not particularly limited, and for example, an RNase R enzyme, which is an exoribonuclease exhibiting 3'-5' exonuclease activity, may be used. For example, an RNase R enzyme derived from E. coli may be used to degrade the acyclic RNA remaining after the cyclization step.

[0057] (RNA degradation process) The "RNA strand degradation step" is a process in which RNA, such as circular RNA, after the reverse transcription step described above is degraded using the RNase H enzyme. This step is selective. In this step, the heteroduplex formed by the hybridization of cDNA (the reverse transcription product) and RNA is recognized by the RNase H enzyme, and the RNA strand is cleaved, allowing only cDNA to be obtained.

[0058] 1-4. Effects According to the method of the present invention, by using cleaved circular RNA as a template for reverse transcription, a reverse transcript product can be obtained that does not contain repeating structures resulting from the reverse transcription of the circular RNA sequence. Furthermore, by positioning the primer binding region upstream of the ligation site linked in the cyclization reaction in the direction of reverse transcription, the ligation site is reverse transcribed, making it possible to amplify the region containing the ligation site using the reverse transcript product as a template. This makes it possible to distinguish and quantify only the cleavage products derived from circular RNA from the cleavage products derived from non-cyclic RNA before cyclization. Based on these effects, the quantification method described later makes it possible to quantify circular RNA with dramatically higher accuracy compared to conventional methods.

[0059] 2. Method for quantifying circular RNA 2-1. Overview A second aspect of the present invention is a method for quantifying circular RNA in a sample (hereinafter sometimes abbreviated as "quantification method"). The quantification method of the present invention is characterized by quantifying the reverse transcript product obtained by reverse transcription using circular RNA cleaved by a cleaving enzyme as a template, using a quantitative nucleic acid amplification method or the like. The quantification method of the present invention makes it possible to accurately quantify circular RNA by eliminating the influence of the reverse transcript product containing repeat sequences obtained by reverse transcription of the circular RNA sequence. Furthermore, by positioning a reverse transcription primer upstream of the ligation site in the direction of reverse transcription, the ligation site is reverse transcribed, and it becomes possible to amplify the region containing the ligation site using the reverse transcript product as an amplification template, making it possible to quantify only the cleavage product derived from circular RNA, distinguishing it from the cleavage product derived from acyclic RNA before cyclization. Furthermore, by amplifying the region that is commonly contained in both the cleavage product derived from circular RNA and the cleavage product derived from acyclic RNA before cyclization, it becomes possible to quantify the total amount of circular RNA and acyclic RNA, and thus quantify the relative abundance of circular RNA to the total amount of circular RNA and acyclic RNA.

[0060] 2-2. Method The quantitative method of the present invention includes a cleavage step, a reverse transcription step, and a quantitative step as essential steps, and includes an RNA extraction step, a cyclization step, and / or an RNA degradation step as optional steps. The configuration of the cleavage step, reverse transcription step, RNA extraction step, cyclization step, and RNA degradation step in this embodiment is the same as in the first embodiment, so a detailed explanation is omitted here, and only the configuration of the quantitative step will be described below.

[0061] (Quantitative process) The "quantification step" is a step in which the reverse transcript product obtained by the reverse transcription step is quantified. The quantification method used in this step is not particularly limited as long as it is a method that can quantify the reverse transcript product, which is DNA. This may include measuring the amount of DNA based on absorbance measurement using any measuring device such as nanodrops, measuring the DNA copy number using next-generation sequencing (NGS), PacBio sequencing, or nanopore sequencing, or using quantitative nucleic acid amplification methods.

[0062] In this specification, "nucleic acid amplification method" refers to a method of amplifying nucleic acids using nucleic acid polymerase with primers as needed. Examples include the PCR method, NASBA method, ICAN method (isothermal gene amplification method), LAMP® method, and RCA method. The nucleic acid amplification method used in the present invention is not limited, but the PCR method is preferred, and heat-resistant DNA polymerase is usually used. Each nucleic acid amplification method is known in the art, and the conditions described in the various protocols should be taken as a reference. In addition, nucleic acid amplification kits are commercially available from life science manufacturers and can also be used. In the amplification step, for example, several amplification cycles (e.g., 1-40 cycles, 2-30 cycles, 3-20 cycles, 4-15 cycles, or 5-10 cycles) can be performed using the PCR method.

[0063] Furthermore, in this specification, "quantitative nucleic acid amplification method" refers to a method that uses nucleic acid amplification for the quantification or semi-quantification of nucleic acids. Among quantitative nucleic acid amplification methods, a method that quantifies the degree of nucleic acid synthesis during nucleic acid amplification is called "real-time detection." For example, a real-time detection method based on the PCR method is called a real-time PCR method. Examples of real-time PCR methods include the intercalator method, which uses reagents that specifically bind to double-stranded DNA (e.g., SYBR Green, TB Green, Eva Green, etc.), and the method that uses fluorescently labeled probes (e.g., the TaqMan® probe method and the cycling probe method). For example, the TaqMan® probe method uses a probe modified with a quencher at the 5' end and a fluorescent dye at the 3' end. Normally, the quencher at the 5' end suppresses the fluorescent dye at the 3' end, but during the extension reaction, the probe is degraded by the 5'→3' exonuclease activity of Taq polymerase, thereby releasing the suppression by the quencher and causing it to emit fluorescence. The amount of fluorescence reflects the amount of amplification product. Since there is an inverse correlation between the number of cycles (Ct value) or time at which the amplification product reaches the threshold and the initial template amount, the initial template amount can be quantified by measuring the Ct value or time in real-time detection methods. By measuring the Ct value or time using several known amounts of template and creating a calibration curve, the absolute value of the initial template amount for an unknown sample can also be calculated. In this specification, semi-quantitative methods and semi-quantitative detections are included in quantitative nucleic acid amplification methods and real-time detections. An example of semi-quantitative real-time detection is a method of quantifying the amplification product at each amplification cycle number in PCR amplification using agarose gel electrophoresis, etc.

[0064] In a further embodiment, the quantitative nucleic acid amplification method in this step includes amplifying and quantifying a first target region contained in the reverse transcript.

[0065] In this specification, the "first target region" refers to the region in the reverse transcript that includes the ligation site where the 5' and 3' ends of the acyclic RNA are linked when the acyclic RNA is circularized to form a circular RNA (Figure 3A). Specifically, the first target region is located in the circular RNA within the region from the base adjacent to the 3' side of the target cleavage site to the primer binding region, and consists of a base sequence complementary to the region containing the ligation site. The length of the first target region is not particularly limited and may be, for example, 50 to 10,000 bases, 100 to 8,000 bases, 200 to 5,000 bases, 300 to 4,000 bases, 500 to 3,000 bases, or 1,000 to 2,000 bases. In the quantitative method of the present invention, by amplifying and quantifying the first target region, which is a region unique to the cleavage product of circular RNA, in a quantitative nucleic acid amplification method, it becomes possible to selectively quantify only the circular RNA in the sample.

[0066] For amplification of the first target region, a primer set capable of amplifying the first target region can be used. For example, a primer pair or primer set can be used that includes an amplification primer (Figure 3A, Primer B) containing a nucleotide sequence complementary to the primer-binding region for amplification located at the 3' end of the first target region, and an amplification primer (Figure 3A, Primer A) containing a nucleotide sequence complementary to the primer-binding region for amplification located at the 3' end of the complementary sequence of the first target region.

[0067] In a further embodiment, the nucleic acid primers used to amplify the first target region (i.e., the first and / or second amplification primers described above) may consist of a nucleotide sequence that does not contain the sequence corresponding to the ligation site in the reverse transcript, or a complementary nucleotide sequence thereof. Specifically, the nucleic acid primers for amplifying the first target region may not contain one or both of the two bases that constitute the ligation site in the circular RNA, and may not contain one or both of the two bases that constitute the site corresponding to the ligation site in the nucleotide sequence complementary to the circular RNA.

[0068] In further embodiments, the quantitative nucleic acid amplification method of this step further includes amplifying and quantifying a second target region contained in the reverse transcript.

[0069] In this specification, the "second target region" is any region in the reverse transcript that does not include the region corresponding to the ligation site (Figure 3A), and it is preferable that it does not overlap with the first target region. For example, in circular RNA, it may be located in a region excluding the first target region, in a nucleotide sequence complementary to the region from the base adjacent to the 3' side of the target cleavage site to the primer binding region. The second target region may also be located in a nucleotide sequence complementary to the region in circular RNA from the base adjacent to the 3' side of the region complementary to the first target region to the base adjacent to the 5' side. Alternatively, the second target region may be located in a nucleotide sequence complementary to the region in circular RNA from the base adjacent to the 3' side of the region complementary to the first target region to the base adjacent to the 5' side of the target cleavage site. The length of the second target region is not particularly limited and may be, for example, 50 to 10,000 base pairs, 100 to 8,000 base pairs, 200 to 5,000 base pairs, 300 to 4,000 base pairs, 500 to 3,000 base pairs, or 1,000 to 2,000 base pairs. In the quantitative method of the present invention, by amplifying the second target region using quantitative nucleic acid amplification, a region common to both circular RNA and acyclic RNA is amplified, making it possible to quantify the total amount of RNA including both circular and acyclic RNA.

[0070] For amplification of the second target region, a primer set capable of amplifying the second target region can be used. For example, a primer pair or primer set can be used that includes an amplification primer (Figure 3A, primer D) containing a nucleotide sequence complementary to the primer-binding region for amplification located at the 3' end of the second target region, and an amplification primer (Figure 3A, primer C) containing a nucleotide sequence complementary to the primer-binding region for amplification located at the 3' end of the complementary sequence of the second target region.

[0071] As described above, if the sample containing circular RNA subjected to the cleavage step also contains acyclic RNA that was not cyclicized in the cyclization step (Figure 2, "Acyclic RNA"), a second reverse transcript is produced as a byproduct in the cleavage step. This second reverse transcript is reverse transcribed using the fragment containing the primer-binding region as a template from the fragment generated from the acyclic RNA, specifically the fragment from the 5' end of the acyclic RNA to the target cleavage site and the fragment from the target cleavage site to the 3' end of the acyclic RNA (Figure 3B). This second reverse transcript has either a nucleotide sequence complementary to the nucleotide sequence from the 5' end of the acyclic RNA towards the primer-binding region in the 3' direction, or a nucleotide sequence complementary to the nucleotide sequence from the target cleavage site towards the primer-binding region in the 3' direction (Figure 3B shows the former case). Since this second reverse transcript does not contain the first target region and only contains the second target region, only the second target region is amplified from the second reverse transcript. Figure 3B shows that amplification does not occur with the primer pair consisting of primer A and primer B, but the second target region is amplified with the primer pair consisting of primer C and primer D.

[0072] In one embodiment, the quantitative method of this embodiment quantifies the proportion of circular RNA in a sample. The proportion of circular RNA can be quantified, for example, as the proportion of circular RNA to total RNA, including circular and acyclic RNA, in the sample, or as the proportion of circular RNA to acyclic RNA.

[0073] The relative abundance of circular RNA in the sample can be calculated based on the quantitative results of the first and second target regions quantified in this step. The calculation method is not limited. As an example of a calculation method, the number of cycles (Ct value) when the amplified product reaches the detection threshold in the real-time detection method is measured, and the Ct value when the first target region is amplified is taken as "Ct1", and the Ct value when the second target region is amplified as "Ct2", and the relative abundance of circular RNA in the sample is calculated based on the values ​​of Ct1 and Ct2. -(Ct2-Ct1)It can be calculated as ×100(%). Note that there are no particular restrictions on how the detection threshold is set; any threshold can be used. For example, the maximum value (inflection point) of the derivative obtained by differentiating the amplification curve may be used as the threshold.

[0074] In another embodiment, the quantitative method of this embodiment quantifies the relative or absolute amount of circular RNA in a sample. For example, by measuring the Ct value using several known amounts of control DNA and creating a calibration curve, the absolute amount of circular RNA in the sample can be calculated. Alternatively, a known amount of control RNA can be mixed as an internal standard in the sample before the reverse transcription step in the quantitative method of the present invention, or a known amount of control DNA can be mixed as an internal standard in the sample containing the reverse transcript product to be quantified in the quantitative method of the present invention. The absolute amount of circular RNA in the sample can then be calculated by detecting the control reverse transcript product, which is the reverse transcript of a known amount of control RNA, or the known amount of control DNA, using a primer with a different label than the amplification primer. Furthermore, if different RNAs or DNAs that serve as a reference (e.g., mRNA of housekeeping genes such as actin genes and their reverse transcripts) are present in the same sample, the relative amount can also be quantified based on such RNAs or DNA.

[0075] 2-3. Effects According to the quantitative method of the present invention, by reverse transcribing circular RNA after cleavage, a reverse transcript product can be obtained that does not contain repeating structures resulting from the reverse transcription of the circular RNA sequence. By using this reverse transcript as the target for quantification, the amount and abundance ratio of circular RNA can be quantified with higher accuracy compared to conventional methods. Furthermore, by positioning the reverse transcription primer used in the reverse transcription step upstream of the linkage site linked in the cyclization reaction in the direction of reverse transcription, the linkage site is reverse transcribed, making it possible to quantify only the cleavage product derived from circular RNA, distinguishing it from the cleavage product derived from acyclic RNA before cyclization. Moreover, by amplifying the region that is common to both the cleavage product derived from circular RNA and the cleavage product derived from acyclic RNA before cyclization, the total amount of circular RNA and acyclic RNA can be quantified, and therefore the abundance ratio of circular RNA to the total amount of circular RNA and acyclic RNA can also be quantified.

[0076] The present invention also provides a method for producing a formulation containing quantified circular RNA, comprising: a cleavage step of cleaving the circular RNA in the formulation using a cleavage enzyme that cleaves the target cleavage site of the circular RNA; a reverse transcription step of performing reverse transcription using the RNA cleaved in the cleavage step as a template; and a quantification step of quantifying the reverse transcript product obtained by the reverse transcription step, wherein the reverse transcription step uses a reverse transcription primer having a base sequence complementary to the primer binding region, and the primer binding region does not include the target cleavage site. This production method may optionally include, before the cleavage step, an acyclic RNA synthesis step of synthesizing acyclic RNA, and / or a cyclization step of cyclizing the acyclic RNA to obtain circular RNA. [Examples]

[0077] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0078] <Example 1: Quantification of circular RNA> (the purpose) Conventional methods for quantifying circular RNA involve reverse transcription using circular RNA as a template, amplifying the target sequence within the reverse transcript, and then quantifying the resulting amplified product. Because circular RNA is circular, it lacks the 3' end of the RNA strand, unlike linear RNA. Therefore, in the reverse transcription reaction of circular RNA, there is no stopping position, and it is thought that a reverse transcript containing repeated base sequences of the circular RNA is produced. When the target sequence is amplified using such a reverse transcript, a reverse transcript containing two or more copies of the target sequence may be produced, potentially leading to an overestimation of the amount of circular RNA being quantified.

[0079] In this example, the objective is to improve the quantitative performance of the conventional method by performing a reverse transcription reaction using a cleavage product obtained by cleaving circular RNA as a template, in order to prevent the generation of reverse transcripts containing two or more copies of the target sequence.

[0080] (Methods and Results) (1) Synthesis and cyclization of linear RNA by in vitro transcription, and cleavage of cyclic RNA Using a pMXs-IRES-GFP Retroviral Vector (cell biolab, RTV-013) consisting of the nucleotide sequence shown in SEQ ID NO: 4 as a template for amplification, PCR was performed using a primer pair containing a ligation region (a forward primer and a reverse primer, consisting of the nucleotide sequences shown in SEQ ID NO: 1 and 2, respectively). The amplification product obtained by PCR, consisting of the nucleotide sequence shown in SEQ ID NO: 5, was used as a template for transcription, and in vitro transcription was performed using the HiScribe® T7 High Yield RNA Synthesis Kit (NEB, E2040S) to synthesize linear RNA consisting of the nucleotide sequence shown in SEQ ID NO: 6. Subsequently, ligation was performed between the 5' and 3' ends of the synthesized linear RNA using T4 RNA Ligase 1 (NEB, MS204S) and DNA splint (SEQ ID NO: 3) to create a circular RNA consisting of the nucleotide sequence shown in SEQ ID NO: 7, with a length of 1449 nt.

[0081] To cleave the above circular RNA with the RNase H enzyme, three 25 nt DNA oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 8-10 were prepared to form heteroduplexes with the circular RNA. The nucleotide sequences of these three DNA oligonucleotides were designed to hybridize to mutually contiguous regions in the circular RNA.

[0082] The RNA sample containing the above circular RNA (4 μg) and each of the above DNA oligonucleotides were mixed in a 1:5 ratio (molar ratio), and the resulting mixture was heat-treated at 65°C for 5 minutes. After standing at room temperature for 5 minutes, 10 μL of 10×RNase H reaction buffer (NEB, B0297SVIAL) and 2 μL of RNase H (NEB, M0297SVIAL) were added to the sample, and NFW (Invitrogen, 10977015) was added to adjust the volume to 100 μL. This was incubated at 37°C for 30 minutes to carry out the cleavage reaction with RNase H. The RNA sample after the reaction was purified using the Monarch RNA Cleanup Kit (NEB, T2030S).

[0083] (2) Agarose gel electrophoresis and extraction from the gel The RNA sample purified in (1) above was subjected to electrophoresis using a 2% agarose gel. After electrophoresis at 50V for approximately 60 minutes, the band with the target molecular weight was excised from the gel. RNA was extracted from the excised gel using the Monarch RNA Cleanup Kit (NEB, T2030S). The concentration of the extracted RNA was measured using a Nanodrop 2000 (Thermo Scientific). Note that the purification step using agarose gel electrophoresis is not essential; even if the RNA obtained in (1) above was column purified with the Monarch RNA Cleanup Kit, the amount of RNA could be quantified using the same method as in (3) and subsequent methods below.

[0084] (3) Reverse transcription The reverse transcription primer used for reverse transcription was designed to hybridize to the region of the circular RNA excluding the cleavage site in the aforementioned cleavage reaction, and to ensure that the ligation site linked by the cyclization reaction in (1) above is included within the region to be reverse transcribed. Specifically, a 25-mer primer was designed consisting of a nucleotide sequence (SEQ ID NO: 11) complementary to the sequence from position 896 to 920 from the 5' end of the linear RNA before cyclization, and binding to a position 5 nt away from the RNase H cleavage site in the circular RNA. The RNA sample extracted in (2) above (10 pg to 500 ng), the reverse transcription primer (2 pmol), and 10 mM dNTP mix (1 μL) were mixed, heat-treated at 65°C for 5 minutes, and then cooled on ice for 1 minute. A mixture of 4 μL of 5×First-Stand buffer (Invitrogen, 18080044), 1 μL of 0.1M dithiothreitol (Invitrogen, 18080044), 0.25 μL of RNasin Plus Ribonuclease Inhibitor (Promega, N2611), and 1 μL of SuperScript® III RT (Invitrogen, 18080044) was added to the ice-cold RNA sample, and NFW was added to adjust the volume to 20 μL. Reverse transcription was performed by incubation at 55°C for 1 hour, followed by heat treatment at 70°C for 15 minutes. The cDNA obtained from the reverse transcription was purified using the MinElute® PCR Purification Kit (QIAGEN, 28004). The concentration of the purified cDNA was measured using NanoDrop® 2000 (Thermo Scientific).

[0085] (4) qPCR Primers A to D were prepared as amplification primers for qPCR using the cDNA sample obtained in (3) above as a template. Primer A (SEQ ID NO: 12) and Primer B (SEQ ID NO: 13) were designed to amplify the 213-nucleotide-long first target region, which includes the ligation site linked by the circularization of linear RNA when circular RNA is produced. Primer C (SEQ ID NO: 14) and Primer D (SEQ ID NO: 15) were designed to amplify the 211-nucleotide-long second target region in circular RNA, which is 3' on the RNase H cleavage site and 5' on the ligation site.

[0086] The RNA sample before cleavage by RNase H described in (1) above includes not only circular RNA, which is linear RNA that has been circulated, but also linear RNA that has not been circulated.

[0087] Primers A and B described above can amplify the first target region contained in the reverse transcript obtained by reverse transcription of the cleavage product of circular RNA cleaved by RNase H. On the other hand, the reverse transcript obtained by reverse transcription of the cleavage product of non-circularized linear RNA cleaved by RNase H does not contain the first target region. Therefore, in amplification using primers A and B, only cDNA reverse transcribed from circular RNA is detected, while cDNA reverse transcribed from linear RNA is not detected (Figure 3).

[0088] In contrast, the second target region amplified by primers C and D includes not only the reverse transcript obtained by reverse transcription of the cleavage product of circular RNA cleaved by RNase H, but also the reverse transcript obtained by reverse transcription of the cleavage product of non-circularized linear RNA cleaved by RNase H. Therefore, amplification using primers C and D detects not only cDNA reverse transcribed from circular RNA, but also cDNA reverse transcribed from linear RNA (Figure 3). By quantifying using these two primer pairs, the relative abundance of circular RNA to all RNA, including circular RNA and linear RNA, can be determined.

[0089] The cDNA sample obtained in (3) above (0.1 ng to 1 ng), a pair of primers for amplifying the first or second target region, and KAPA SYBR FAST qPCR Master Mix (NIPPON Genetics, KK4600) were mixed to prepare the measurement sample. The prepared measurement sample was subjected to qPCR measurement using a real-time PCR system (QuantStudio® 12K Flex, Applied Biosystems).

[0090] Based on the results of qPCR measurements, the number of amplification cycles (Ct value) required for the target region to be amplified and reach the threshold was determined.

[0091] (5) Determination of the proportion of circular RNA The Ct value (hereinafter referred to as "Ct1") when using a primer pair (primer A and primer B) to amplify the first target region was 7.91. The Ct value (hereinafter referred to as "Ct2") when using a primer pair (primer C and primer D) to amplify the second target region was 7.01. Based on the values ​​of Ct1 and Ct2, the relative abundance of circular RNA in the RNA sample before cleavage by RNase H as described in (1) above is given by the following formula: Ratio of circular RNA (%) = 2 -(Ct2-Ct1) ×100(%) This can be determined by the following formula. Substituting the above Ct1 and Ct2 values ​​into this formula, the relative abundance of circular RNA was found to be 53.73%.

[0092] (6) Verification of effects based on cleavage reaction by RNase H In (1) above, circular RNA was cleaved with RNase H to prevent the generation of reverse transcripts containing two or more copies of the target sequence. To verify that the quantitative performance of circular RNA is improved based on this RNase H cleavage reaction, a cDNA sample (hereinafter referred to as the "control cDNA sample") was prepared from circular RNA that was not cleaved with RNase H and subjected to qPCR measurement. Specifically, this control cDNA sample was prepared in the same manner as described in (1) to (3) above, except that RNase H was not added in the cleavage reaction in (1) above. This control cDNA sample was used as the target for qPCR measurement in the same manner as described in (4) above.

[0093] Figure 4 shows the results of qPCR measurements using primer pairs (primer A and primer B) to amplify the first target region, with and without RNase H cleavage of circular RNA (n=3). The solid and dashed lines represent the results, respectively. The dotted line in Figure 4 indicates the threshold used to calculate the Ct value.

[0094] The average Ct value when circular RNA was not cleaved with RNase H was 7.05. In contrast, the Ct value when circular RNA was cleaved with RNase H was 7.91, which was a higher Ct value. This difference in Ct value is 2 (7.9-7.05) This corresponds to 1.81 times the amount of template cDNA. This result indicates that when circular RNA is not cleaved with RNase H, the amount of circular RNA is overestimated by 81% compared to the actual amount, and that cleaving circular RNA with RNase H significantly improves the accuracy of quantification.

[0095] This disclosure also provides the following: (1) A method for producing a reverse transcript from circular RNA, A cutting step, comprising cutting the circular RNA using a cutting enzyme that cuts the circular RNA at a target cleavage site, and Reverse transcription step: Using the RNA cut in the above-mentioned cutting step as a template, reverse transcription is performed. Includes, The reverse transcription step is performed using a reverse transcription primer containing a base sequence complementary to the primer binding region, wherein the primer binding region does not include the target cleavage site. (2) The method according to (1), wherein the circular RNA is formed by the cyclization of an acyclic RNA. (3) The method according to (2), wherein the primer binding region is located upstream of the linkage site linked by the cyclic formation in the direction of reverse transcription. (4) A method for quantifying circular RNA in a sample, A cutting step, in which the circular RNA in the sample is cut using a cutting enzyme that cuts at a target cleavage site of the circular RNA, A reverse transcription step, in which the RNA cut in the aforementioned cutting step is used as a template for reverse transcription, and A quantitative step for quantifying the reverse transcript product obtained by the reverse transcription step. Includes, The reverse transcription step is performed using a reverse transcription primer containing a base sequence complementary to the primer binding region, wherein the primer binding region does not include the target cleavage site. (5) The method according to (4), wherein the quantitative step is to quantify the reverse transcript by quantitative nucleic acid amplification. (6) The method according to (4), wherein the circular RNA is formed by the cyclization of an acyclic RNA. (7) The method according to (6), wherein the primer binding region is located upstream of the linkage site linked by the cyclization in the direction of reverse transcription. (8) The quantitative nucleic acid amplification method includes amplifying and quantifying the first target region contained in the reverse transcript, The method according to (7), wherein the first target region consists of a nucleotide sequence complementary to the region containing the ligation site in the circular RNA. (9) The quantitative nucleic acid amplification method further includes amplifying and quantifying a second target region contained in the reverse transcript, The method according to (8), wherein the second target region is a region in the reverse transcript that does not contain the first target region. (10) The method according to (9), wherein the relative abundance of the circular RNA in the sample to all RNA, including the circular RNA and the acyclic RNA, is calculated based on the quantitative results of the first target region and the second target region quantified in the quantitative step. (11) The method according to (5), wherein the quantitative nucleic acid amplification method is a real-time detection method. (12) The method according to (1) or (4), wherein the cleaving enzyme is ribonuclease H, Cas13 protein, or Dicer protein. (13) The method according to (1) or (4), wherein the cleavage step cleaves two or more nucleoside bonds in the circular RNA. (14) The method according to (8), wherein the distance between the target cleavage site and the ligation site in the circular RNA is at least 12 nucleotides long. (15) The nucleic acid primer used to amplify the first target region is The circular RNA does not include the aforementioned linking site, and The method according to (14), wherein the base sequence complementary to the circular RNA does not include the site corresponding to the ligation site. (16) The method according to (4), wherein the sample is a pharmaceutical product. (17) The method according to (16), wherein the pharmaceutical product is an mRNA vaccine. [Prior art documents] [Patent Documents]

[0096] [Patent Document 1] International Publication No. 2024 / 032321 [Non-patent literature]

[0097] [Non-Patent Document 1] Jeck WR and Sharpless NE, Nat Biotechnol, 2014, 32(5):453-61. [Non-Patent Document 2] Wesselhoeft, RA, et al., Nature communications, 2018, 9:2629. [Non-licensed document 3] Wesselhoeft RA, et al., Mol. Cell., 2019, 74(3):508-520.e4.

Non-licensed Document 4

Claims

1. A method for producing a reverse transcript from circular RNA, A cutting step, comprising cutting the circular RNA using a cutting enzyme that cuts the circular RNA at a target cleavage site, and Reverse transcription step: Using the RNA cut in the above-mentioned cutting step as a template, reverse transcription is performed. Includes, The reverse transcription step is performed using a reverse transcription primer containing a base sequence complementary to the primer binding region, wherein the primer binding region does not include the target cleavage site.

2. The method according to claim 1, wherein the circular RNA is formed by the cyclization of an acyclic RNA.

3. The method according to claim 2, wherein the primer binding region is located upstream of the linkage portion linked by the cyclic formation in the direction of reverse transcription.

4. A method for quantifying circular RNA in a sample, A cutting step, in which the circular RNA in the sample is cut using a cutting enzyme that cuts at a target cleavage site of the circular RNA, A reverse transcription step, in which the RNA cut in the aforementioned cutting step is used as a template for reverse transcription, and A quantitative step for quantifying the reverse transcript product obtained by the reverse transcription step. Includes, The reverse transcription step is performed using a reverse transcription primer containing a base sequence complementary to the primer binding region, wherein the primer binding region does not include the target cleavage site.

5. The method according to claim 4, wherein the quantitative step involves quantifying the reverse transcript by quantitative nucleic acid amplification.

6. The method according to claim 4, wherein the circular RNA is formed by the cyclization of an acyclic RNA.

7. The method according to claim 6, wherein the primer binding region is located upstream of the linkage portion linked by the cyclic formation in the direction of reverse transcription.

8. The quantitative nucleic acid amplification method includes amplifying and quantifying a first target region contained in the reverse transcript, The method according to claim 7, wherein the first target region consists of a nucleotide sequence complementary to the region containing the ligation site in the circular RNA.

9. The quantitative nucleic acid amplification method further includes amplifying and quantifying the second target region contained in the reverse transcript, The method according to claim 8, wherein the second target region is a region in the reverse transcript that does not include the first target region.

10. The method according to claim 9, wherein the relative abundance of the circular RNA in the sample to all RNA, including the circular RNA and the acyclic RNA, is calculated based on the quantitative results of the first target region and the second target region quantified in the quantitative step.

11. The method according to claim 5, wherein the quantitative nucleic acid amplification method is a real-time detection method.

12. The method according to claim 1 or 4, wherein the cleaving enzyme is ribonuclease H, Cas13 protein, or Dicer protein.

13. The method according to claim 1 or 4, wherein the cleavage step cleaves two or more nucleoside bonds in the circular RNA.

14. The method according to claim 8, wherein the distance between the target cleavage site and the ligation site in the circular RNA is at least 12 nucleotides long.

15. The nucleic acid primer used to amplify the first target region is The circular RNA does not include the aforementioned linking site, and The method according to claim 14, wherein the base sequence complementary to the circular RNA does not include the site corresponding to the ligation site.

16. The method according to claim 4, wherein the sample is a pharmaceutical product.

17. The method according to claim 16, wherein the pharmaceutical product is an mRNA vaccine.

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