Analysis of RNA molecules using catalytic nucleic acids

Abstract

The present invention relates to methods for analyzing the structure of the 5' ends of RNA molecules in a population of RNA molecules using catalytic nucleic acids, for example to determine the presence or absence of a 5' cap structure.

Description

[Technical field]

[0001] The present invention relates to a method for analyzing a population of RNA molecules, which determines the amount of RNA molecules bearing a 5' cap structure. Other aspects of the invention relate to a method for determining the capping efficiency in a population of RNA molecules and a method for the quality control of capped RNA synthesis. [Background technology]

[0002] In vitro transcribed (IVT) mRNA-based therapeutics are emerging as novel biologics with a variety of applications, including recent vaccines against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as other infectious disease vaccine candidates, cancer immunotherapy, gene editing therapies and protein replacement therapies [1–6]. Ongoing preclinical and clinical trials using IVT mRNA require innovative solutions for quality control of IVT mRNA.

[0003] The presence of a cap structure at its 5' end is one of the key features of IVT mRNA that enhances its stability and translatability. Capped mRNAs are generally translated more efficiently compared to uncapped mRNAs [7-9]. After synthesis, enzymatic and co-transcriptional capping of IVT mRNA can be incomplete, resulting in the presence of a variable number of uncapped molecules in the final IVT mRNA.

[0004] Current approaches to determine the capping efficiency of IVT mRNA are of limited applicability. In one method, IVT mRNA is transcribed from a short DNA template in the presence of [α-32P]GTP and a cap analog. This generated IVT mRNA is digested with RNase T2, and the [ 32It has been shown that enzymatic capping of long mRNAs transcribed in the presence of [γ-32P]GTP releases a radioactive cap structure containing a [γ-32P]-labeled 3'-phosphate [10, 11]. However, this approach is only applicable to RNAs <50 nt in length. Others have determined the efficiency of enzymatic capping of long mRNAs transcribed in the presence of [γ-32P]GTP by measuring the decrease in mRNA radioactivity as the labeled gamma phosphate is eliminated upon successful capping

[12] . The main drawback of these methods is the use of radioactive materials for mRNA production.

[0005] In another approach, the capping efficiency of IVT mRNA of different lengths (>1,000 nt) was measured without using radioactivity. In this procedure, a complementary biotin-labeled oligonucleotide was annealed to the 5' end of the IVT mRNA, which was then cleaved by RNase H

[13] . The cleaved 5' end was purified using streptavidin-coated magnetic beads and then analyzed by liquid chromatography and mass spectrometry (LC-MS). Although this method avoids radioactive labeling of the mRNA, the RNase H cleavage site is not unique and the method is not completely reliable, since additional cleavage products of different lengths were also generated. Furthermore, the analysis of the cleaved fragments requires LC-MS, which affects the feasibility of the assay.

[0006] Recently, a biosensor has been developed to detect both capping levels and mRNA integrity.

[14] However, the biosensor method can only detect variations in capping levels in increments of at least 20%, making it difficult to use this method as an accurate analytical tool. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sahin U, Kariko K, Tureci O. mRNA-based therapeutics--developing a new class of drugs.Nat Rev Drug Discov.2014;13:759-80.doi:10.1038 / nrd4278.

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[0008] One aspect of the invention is a method for analyzing a population of RNA molecules, comprising the steps of: (a) contacting a catalytic nucleic acid molecule with a population of RNA molecules comprising one or more RNA molecules comprising a cleavage site for the catalytic nucleic acid molecule and a 5' cap structure under conditions that allow cleavage of the RNA molecules to generate a 5' terminal fragment and at least one 3' fragment; (b) at least partially separating the 5' end fragments obtained in step (a) from at least one 3' fragment to obtain a population of 5' end fragments; and (c) determining the amount of RNA molecules having a 5'-cap structure in the population of 5'-end fragments obtained in step (b); The present invention relates to a method comprising the steps of:

[0009] One aspect of the invention is a method for determining capping efficiency in a population of RNA molecules, comprising the steps of: (a) contacting a catalytic nucleic acid molecule with a population of RNA molecules comprising one or more RNA molecules comprising a cleavage site for the catalytic nucleic acid molecule and a 5' cap structure under conditions that allow cleavage of the RNA molecules to generate a 5' terminal fragment and at least one 3' fragment; (b) at least partially separating the 5' end fragments obtained in step (a) from at least one 3' fragment to obtain a population of 5' end fragments; and (c) determining the amount of RNA molecules having a 5'-cap structure in the population of 5'-end fragments obtained in step (b); The present invention relates to a method comprising the steps of:

[0010] One aspect of the invention is a method for analyzing an RNA molecule, comprising the steps of: (i) synthesizing an RNA molecule; (ii) capping the RNA synthesized in (i); and (iii) analyzing the RNA molecules by a method for analyzing a population of RNA molecules of the invention The present invention relates to a method comprising the steps of:

[0011] One aspect of the invention is a method for quality control of capped RNA synthesis, comprising: (i) synthesizing an RNA molecule; (ii) capping the RNA synthesized in (i); and (iii) analyzing the RNA molecules by a method for analyzing a population of RNA molecules of the invention The present invention relates to a method comprising the steps of:

[0012] One aspect of the present invention relates to a catalytic nucleic acid molecule, (i) a sequence selected from SEQ ID NOs: 1 to 25; (ii) a sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 25, and / or (iii) a fragment of (i) and / or (ii); Here, the catalytic nucleic acid molecule comprises a catalytic core.

[0013] One aspect of the present invention relates to the use of catalytic nucleic acid molecules as described herein in methods for analyzing a population of RNA molecules, methods for determining capping efficiency in a population of RNA molecules, methods for analyzing RNA molecules, and / or methods for quality control of capped RNA synthesis.

[0014] The step of at least partially separating the 5'-end fragments from the 3' fragment(s) before the step of determining the amount of RNA molecules having a 5'-cap structure is to provide a population enriched (at least partially purified) in 5'-end fragments, i.e., the amount of 5'-end fragments as a percentage of all cleaved and uncleaved RNA molecules present in the separated (or purified) enriched population obtained after the separation step is greater than the amount of 5'-end fragments as a percentage of all cleaved and uncleaved RNA molecules present in the population before the separation step. This enriched or partially purified population is then subjected to a step of determining the amount of RNA molecules having a 5'-cap structure. By providing this enriched or partially purified population first, the sensitivity and accuracy of the assay are increased. [Brief description of the drawings]

[0015] [Figure 1] Ribozyme-mediated cleavage to quantify the capping efficiency of in vitro transcribed mRNA. Ribozyme (Rz) anneals to IVT mRNA and cleaves the 5' end of IVT mRNA at 37°C in the presence of Mg++. Substrate cleavage results in a mixture of RNA: short capped and uncapped 5' cleavage products (5'CP), long 3' cleavage products (3'CP), long uncleaved RNA and Rz. The mixture is purified using a process with two silica-based columns, whereby long RNA and 3'CP are depleted on the first column membrane by using specific salt and ethanol conditions. The collected flow-through containing short capped 5'CP and uncapped 5'CP is applied to the second column, bound to the membrane and eluted in water. The purified 5'CP and Rz are visualized using 21% PAGE, 8M urea or analyzed by liquid chromatography and mass spectrometry (LC-MS), allowing quantification of the capping efficiency of IVT mRNA. [Diagram 2]Optimization of the molar ratio of ribozyme to IVT mRNA substrate. A fixed amount of U-containing or m1Ψ-containing uncapped mRNA was cleaved with increasing amounts of Rz1 and the resulting mixture was visualized using 21% PAGE, 8 M urea. The cleavage efficiency of Rz1 was assessed for increasing molar ratios of Rz to IVT mRNA substrate based on the ratio of uncleaved RNA (112 nt long) to 3' cleavage product (3'CP = 90 nt long). Molar ratios of Rz to RNA substrate from 1 to 10 were tested, resulting in approximately 50-70% cleavage. Rz was used as a control (ctrl). 5'CP, 5' cleavage product; nt, nucleotides. [Diagram 3] Ribozyme-mediated cleavage effectively evaluates the capping efficiency of IVT mRNA by visualization and quantification using denaturing polyacrylamide gel electrophoresis. Ribozyme (Rz)-mediated cleavage of U-containing or m1Ψ-containing RNA (using Rz1, Rz2 and Rz5): uncapped (-), enzymatically capped 0 (E0), enzymatically capped 1 (E1) and ARCA (A0) RNAs were purified on a silica-based column and then visualized using 21% PAGE, 8 M urea (purified, upper panel) or visualized using 21% PAGE, 8 M urea without silica-based column purification (unpurified, lower panel). Rz was used as a control in both the upper and lower panels, and in the lower panel uncleaved mRNA was used as an additional control (ctrl). 5'CP, 5' cleavage product (upper: capped, lower: uncapped); nt, nucleotides. The capping efficiency (%) of IVT mRNA visualized here is shown in Table 5. [Figure 4] Ribozyme-mediated cleavage effectively evaluates the capping efficiency of IVT mRNA of different lengths. Images show 21% PAGE, 8 M urea visualization of Rz5-cleaved and silica column-purified TEV m1Ψ-containing β-S-ARCA (D1) or CleanCap® Reagent AG (3'OMe) Cap 1 (CC1) capped IVT mRNA. IVT mRNA ranged in length from 1.1 kb to 9.4 kb. Rz, ribozyme; 5'CP, 5' cleavage product. [Diagram 5]Ribozyme-mediated cleavage assay detects increased capping efficiency following enzymatic capping of cotranscriptionally capped IVT mRNA. Cotranscriptionally D1-capped or D1+enzymatically capped (D1+E1), GCG transcription start site (TSS), hAg, U-containing IVT mRNA was cleaved with Rz1, silica column purified, and visualized using 21% PAGE, 8 M urea. Rz, ribozyme; 5'CP, 5' cleavage product. [Figure 6] The ribozyme-mediated cleavage assay is superior to the RNase H cleavage assay. The RNase H probe (P1) was hybridized and RNase H cleaved a set of U- or m1Ψ-containing RNAs: no cap (-), enzymatic cap 0 (E0), enzymatic cap 1 (E1), and ARCA (A0). The cleaved RNA fragment mixture was applied to a silica-based column for purification and visualized using 21% PAGE, 8 M urea (purified) or visualized using 21% PAGE, 8 M urea without silica-based column purification (unpurified). White arrow: additional +1 nt band; dashed box: RNA degradation caused by RNase H. The RNase H probe P1 or uncleaved RNA (ctrl) was used as controls. 5'CP, 5' cleavage product; nt, nucleotides. [Figure 7] LC-MS analysis of ribozyme-mediated cleavage products for quantification and characterization of capped products from Rz1-cleaved and silica column-purified, enzymatically capped and 2'-O-methylated (E1), human α-globin (hAg), m1Ψ-containing erythropoietin (EPO) mRNA. The enzymatic capping procedure yields 7MeGpppA(OMe)GGCGAACU*AGU*AU*U*CU*U*CU*GGU*Cp (MW=8,334) and 7MeGpppAGGCGAACU*AGU*AU*U*CU*U*CU*GGU*Cp (MW=8,319) in a 7:2 ratio, resulting from incomplete 2'O-methyl transfer. (A) UPLC profile and (B) MS profile. Rz, ribozyme; Cap, capped product; Capm, capped methylated product; Capu, capped unmethylated product; Cap+G, minor product; AU, arbitrary units. [Figure 8] LC-MS analysis of ribozyme-mediated cleavage products for quantification and characterization of capped products from Rz1-cleaved and silica column-purified, CleanCap® Reagent AG(3'OMe)=Cap1 (CC1), human alpha globin (hAg), m1Ψ-containing erythropoietin (EPO) mRNA. The expected capped product (MW=8,347) is detected at >99%. (A) UPLC profile and (B) MS profile. Rz, ribozyme; Cap, capped product; AU, arbitrary units. [Figure 9] Ribozyme-mediated cleavage assays using ribozymes targeting NCH-type sites (Rz6, Rz7, Rz8, Rz9 and Rz10; SEQ ID NOs: 6-10) that showed efficient cleavage and released short 5' capped and uncapped cleavage products. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0017] Preferably, the terms used herein are defined as set forth in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0018] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0019] In the following, elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the embodiments explicitly described. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by this description unless the context indicates otherwise.

[0020] The term "about" means approximately or near, and in the context of numerical values ​​or ranges described herein, preferably means + / - 10% of the recited or claimed numerical value or range.

[0021] The terms "a" and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not impose limitations on the scope of the invention as claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0022] Unless otherwise indicated, the term "comprises" is used in the context of this document to indicate that in addition to the members of the list introduced by "comprises", further members may optionally be present. However, it is contemplated as a particular embodiment of the invention that the term "comprises" encompasses the possibility that further members are not present, i.e., for the purposes of this embodiment, "comprises" should be understood to have the meaning of "consisting of."

[0023] Reference to a relative amount of a component characterized by a generic term is meant to refer to the total amount of all specific variants or members encompassed by said generic term. When a specific component defined by a generic term is identified as being present in a specific relative amount, and this component is further characterized as being a particular variant or member encompassed by the generic term, it is meant that other variants or members encompassed by the generic term are not additionally present such that the total relative amount of the components encompassed by the generic term exceeds the specified relative amount, and more preferably, no other variants or members encompassed by the generic term are present at all.

[0024] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention was not entitled to antedate such disclosure.

[0025] The presence of a cap structure on the 5' end of in vitro transcribed (IVT) mRNA determines its translation and stability, supporting its use in therapy. Both enzymatic and co-transcriptional capping can result in incomplete placement of the cap on the newly synthesized RNA molecule. IVT mRNA is rapidly emerging as a novel biologic, including recent vaccines against COVID-19 and other infectious diseases, as well as vaccine candidates for cancer immunotherapy and protein replacement therapy. Quality control methods required for the preclinical and clinical stages of development of these therapeutics are under development.

[0026] Capped mRNAs are generally translated more efficiently compared to uncapped mRNAs [7-9]. After synthesis, enzymatic and co-transcriptional capping of IVT mRNA can be incomplete, leading to the presence of a variable number of uncapped molecules in the final IVT mRNA. We have developed methods that allow for a rapid and simple quantitative measurement of capping efficiency. These methods can be used as quality control for mRNA-based therapeutics.

[0027] In the present embodiment, a catalytic nucleic acid molecule (particularly a ribozyme, Rz) was designed to specifically cleave the IVT mRNA at a unique position in close proximity to the 5'-end and release short 5' cleavage products, capped or uncapped, in the range of 10-30 nt. The well-defined capped or uncapped 5' cleavage products cleaved by the ribozyme differ from each other by the length of one nucleoside, in particular the length of the cap structure itself. The difference in length can be greater than one nucleotide when the length of the cap structure is longer than one nucleotide.

[0028] Compared to other methods for analyzing the 5' end of RNA molecules, the method of the present invention is improved in that it is more sensitive, more accurate and allows for greater reproducibility of results, making it particularly applicable to the quality control of mRNA-based therapeutics.

[0029] One aspect of the invention is a method for analyzing a population of RNA molecules, comprising the steps of: (a) contacting a catalytic nucleic acid molecule with a population of RNA molecules comprising one or more RNA molecules comprising a cleavage site for the catalytic nucleic acid molecule and a 5' cap structure under conditions that allow cleavage of the RNA molecules to generate a 5' terminal fragment and at least one 3' fragment; (b) at least partially separating the 5' end fragments obtained in step (a) from at least one 3' fragment to obtain a population of 5' end fragments; and (c) determining the amount of RNA molecules having a 5'-cap structure in the population of 5'-end fragments obtained in step (b); The present invention relates to a method comprising the steps of:

[0030] In some embodiments, the method is performed in a specified order of steps.

[0031] The term "nucleic acid" according to the present invention also includes nucleic acids on the nucleotide base, sugar or phosphate, as well as chemical derivatization of nucleic acids containing non-natural nucleotides and nucleotide analogues. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In general, a nucleic acid molecule or nucleic acid sequence refers to a nucleic acid, which is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared molecules and chemically synthesized molecules. According to the present invention, the nucleic acid may be in the form of a single-stranded or double-stranded linear molecule or a covalently closed circular molecule.

[0032] According to the present invention, a "nucleic acid sequence" refers to a sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can refer to an entire nucleic acid molecule (such as a single strand of an entire nucleic acid molecule) or a portion thereof (e.g., a fragment).

[0033] According to the present invention, the term "RNA" or "RNA molecule" refers to a molecule that comprises ribonucleotide residues, preferably consisting entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term "RNA" includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material, for example at one or more nucleotides of the RNA, for example at the termini (either or both) or internally of the RNA. Nucleotides in an RNA molecule may also include non-natural nucleotides or non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be called analogs, in particular analogs of naturally occurring RNA.

[0034] According to the present invention, RNA can be single-stranded or double-stranded. In some embodiments of the present invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule that is not associated with a complementary nucleic acid molecule (typically a non-complementary RNA molecule). Single-stranded RNA can contain self-complementary sequences that allow a portion of the RNA to fold back and form secondary structural motifs, including but not limited to base pairs, stems, stem loops, and bulges. Single-stranded RNA can exist as a negative strand [(-) strand] or a positive strand [(+) strand]. The (+) strand is the strand that contains or codes for genetic information. The genetic information can be, for example, a polynucleotide sequence that codes for a protein. When the (+) strand RNA codes for a protein, the (+) strand can directly serve as a template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of double-stranded RNA, the (+) strand and the (-) strand are two separate RNA molecules, and both of these RNA molecules associate with each other to form a double-stranded RNA ("duplex RNA").

[0035] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a source in nature, and has not been intentionally modified by humans in a laboratory, is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0036] The term "population of RNA molecules" refers to a plurality of RNA molecules. The population may be a mixture of two or more RNA molecule species, or may be a single RNA molecule species. The RNA molecules of one species of RNA molecules may have essentially the same nucleotide sequence. The population of RNA molecules may be obtained from any natural source or may be obtained synthetically. Any synthesis method known to those skilled in the art may be used. RNA synthesis may include in vitro transcription (IVT) or solid-phase synthesis. RNA synthesis may also include transcription and replication in a cell system. Synthesis includes capping of the RNA molecules, if not indicated herein as a separate step in the production of the RNA molecules. A "species of RNA molecules" may include capped and uncapped RNA molecules.

[0037] According to the present invention, "RNA replication" generally refers to the RNA molecule synthesized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The RNA molecule synthesized can be, for example, identical or complementary to the template RNA molecule. Generally, RNA replication can occur through the synthesis of a DNA intermediate, or directly through RNA-dependent RNA replication mediated by RNA-dependent RNA polymerase (RdRP).

[0038] An RNA molecule may contain a translatable nucleic acid sequence, i.e. a nucleic acid sequence that can be translated into a peptide or protein. For an RNA sequence to be "translatable", it requires the presence of a 5' cap structure.

[0039] "3' end of a nucleic acid" refers to its end with a free hydroxyl group according to the present invention. In a schematic diagram of a double-stranded nucleic acid, particularly DNA, the 3' end is always on the right side. "5' end of a nucleic acid" refers to its end with a free phosphate group according to the present invention. In a schematic diagram of a double-stranded nucleic acid, particularly DNA, the 5' end is always on the left side. 5' end 5'--P-NNNNNNN-OH-3' 3' end 3'-HO-NNNNNNN-P--5'

[0040] A "5' terminal fragment" of a nucleic acid molecule refers to a fragment of the nucleic acid molecule comprising the 5' terminal sequence of the nucleic acid molecule.

[0041] A "3' fragment" of a nucleic acid molecule relates to any fragment of a nucleic acid molecule that comprises the sequence of the nucleic acid molecule downstream of the sequence of the 5' fragment.

[0042] A "3' terminal fragment" of a nucleic acid molecule is a 3' fragment that comprises the 3' terminal sequence of a nucleic acid molecule.

[0043] In some embodiments, the 5'-end and 3'-fragments of nucleic acid molecules, particularly RNA molecules, can be obtained by cleavage with a catalytic nucleic acid molecule, resulting in a 5'-end fragment and at least one 3'-fragment. Cleavage at a single cleavage site results in one 5'-end fragment and one 3'-fragment (i.e., a 3'-end fragment). Cleavage at two or more cleavage sites results in one 5'-end fragment and two or more 3'-fragments.

[0044] In some embodiments, the sequences of the 5'-end fragment and at least one of the 3'-end fragments do not overlap with each other.

[0045] Any RNA population that includes one or more RNA molecules with a 5' cap structure can be subjected to the methods described herein. In some embodiments, the population of RNA molecules can be a population of mRNA molecules, self-replicating RNA, ncRNA and / or snRNA.

[0046] According to the present invention, the term "mRNA" means "messenger RNA" and relates to a transcript that is typically produced by using a DNA template and codes for a peptide or protein. Typically, an mRNA comprises a 5'-UTR, a protein coding region, a 3'-UTR, and a poly(A) sequence. An mRNA can be produced by in vitro transcription from a DNA template. Methods of in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. According to the present invention, an mRNA can be modified by stabilizing modifications and capping.

[0047] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" refers to a continuous or discontinuous sequence of adenylic acid residues typically located at the 3' end of an RNA molecule. A continuous sequence is characterized by consecutive adenylic acid residues. In nature, continuous poly(A) sequences are typical. Poly(A) sequences are not usually encoded by eukaryotic DNA, but are attached to the free 3' end of RNA by post-transcriptional template-independent RNA polymerase during eukaryotic transcription in the cell nucleus, although poly(A) sequences can also be encoded by DNA. Thus, an RNA molecule can encompass both DNA-encoded poly(A) sequences and / or enzymatically polyadenylated poly(A) sequences.

[0048] According to the present invention, the term "self-replicating RNA" includes any RNA that can replicate autonomously in a host cell. "Self-replicating RNA" includes RNA viruses that can have single-stranded RNA (ssRNA) genomes, including alphaviruses, flaviviruses, measles viruses (MV) and rhabdoviruses. Alphaviruses and flaviviruses have genomes of positive polarity, while the genomes of measles viruses (MV) and rhabdoviruses are negative-stranded ssRNA. Typically, self-replicating viruses are viruses that have a (+) strand RNA genome that can be directly translated after infection of cells, and this translation provides an RNA-dependent RNA polymerase that produces both antisense and sense transcripts from the infected RNA. In some embodiments, self-replicating RNA can be a type of mRNA.

[0049] Non-coding RNA (ncRNA) refers to RNA molecules that are not translated into a polypeptide or protein.

[0050] Small nuclear RNA (snRNA) refers to a class of small RNA molecules found in the nucleus of eukaryotic cells.

[0051] In some embodiments, in step (a), the catalytic nucleic acid molecule may be contacted with a population of RNA molecules obtained by in vitro transcription or solid phase synthesis.

[0052] The terms "transcription" and "transcribe" refer to the process in which a nucleic acid molecule with a specific nucleic acid sequence ("nucleic acid template") is read by an RNA polymerase, with the result that the RNA polymerase produces a single-stranded RNA molecule. During transcription, the genetic information in the nucleic acid template is transcribed. The nucleic acid template may be DNA; however, in the case of transcription, for example, from an RNA virus nucleic acid template, the template is typically RNA. The transcribed RNA can then be translated into a protein. According to the present invention, the term "transcription" includes "in vitro transcription (IVT)", which refers to a process in which RNA, in particular mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is applied to the production of the transcript. These cloning vectors are generally called transcription vectors and are encompassed by the term "vector" according to the present invention. The cloning vector is preferably a plasmid. According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0053] Methods of in vitro transcription are known to those skilled in the art, and various in vitro transcription kits are commercially available, for example as described in WO 2011 / 015347 A1.

[0054] The single-stranded nucleic acid molecule produced during transcription typically has a nucleic acid sequence that is the complementary sequence of the template.

[0055] According to the present invention, the term "template" or "nucleic acid template" or "template nucleic acid" generally refers to a nucleic acid sequence that can be replicated or transcribed.

[0056] "Nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer, where appropriate, to a nucleic acid sequence as part of an entire RNA molecule that is the product of transcription of a template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0057] The term "vector" is used herein in its most general sense and includes any intermediate vehicle for a nucleic acid, for example allowing said nucleic acid to be introduced into a prokaryotic and / or eukaryotic host cell and, where appropriate, integrated into the genome. Such vectors are preferably replicated and / or expressed within the cell. Vectors include plasmids, phagemids, viral genomes, and fractions thereof.

[0058] "Solid-phase synthesis" of nucleic acid molecules, particularly RNA molecules, refers to the chemical synthesis of nucleic acid molecules using nucleotide or oligonucleotide building blocks.Solid-phase synthesis of nucleic acid molecules is known to those skilled in the art.For example, phosphoramidite chemistry can be used.

[0059] In some embodiments, in an RNA molecule, the cleavage site may be located at least 5 nt, at least 10 nt, or at least 15 nt downstream of the 5' end of the RNA molecule.

[0060] In some embodiments, in an RNA molecule, the cleavage site may be located up to 50 nt or up to 35 nt downstream of the 5' end of the RNA molecule.

[0061] In some embodiments, the cleavage site may be located within 5-50 nucleotides downstream of the 5' end of the RNA molecule.

[0062] In some embodiments, the cleavage site may also be located within 10-50 nucleotides downstream of the 5' end of the RNA molecule.

[0063] In some embodiments, the cleavage site may also be located within 15-30 nucleotides downstream of the 5' end of the RNA molecule.

[0064] In some embodiments, the RNA molecule may comprise a 5'UTR sequence.Suitable 5'UTR sequences are known to those skilled in the art.For example, the 5'UTR may be selected from human alpha globin (hAg) 5'UTR and tobacco etch virus (TEV) 5'UTR.

[0065] The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule, such as an mRNA molecule. Untranslated regions (UTRs) can be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR).

[0066] A 3'-UTR is located at the 3' end of a gene, downstream of the stop codon of the protein coding region, if present, although the term "3'-UTR" preferably does not include the poly(A) tail. Thus, a 3'-UTR is upstream of the poly(A) tail (if present), e.g., immediately adjacent to the poly(A) tail.

[0067] A 5'-UTR, when present, is located at the 5' end of a gene, upstream of the start codon of the protein coding region. A 5'-UTR is downstream of the 5' cap (if present), e.g., immediately adjacent to the 5' cap.

[0068] In accordance with the present invention, 5' and / or 3' untranslated regions may be operably linked to an open reading frame such that these regions are associated with the open reading frame in a manner that enhances the stability and / or translation efficiency of an RNA that contains the open reading frame.

[0069] UTRs are involved in the stability and translation efficiency of RNA, such as mRNA. In addition to the structural modifications of 5' cap and / or 3' poly(A) tail described herein, both can be improved by selecting specific 5' and / or 3' untranslated regions (UTRs). Sequence elements within UTRs are generally understood to affect translation efficiency (mainly 5'-UTR) and RNA stability (mainly 3'-UTR). In order to increase the translation efficiency and / or stability of RNA replicon, it is preferred that an active 5'-UTR is present. Independently or additionally, it is preferred that an active 3'-UTR is present to increase the translation efficiency and / or stability of RNA replicon.

[0070] A 5'-UTR according to the present invention may comprise any combination of multiple nucleic acid sequences, optionally separated by a linker. A 3'-UTR according to the present invention may comprise any combination of multiple nucleic acid sequences, optionally separated by a linker.

[0071] The term "linker" according to the present invention relates to a nucleic acid sequence that is added between two nucleic acid sequences in order to link said two nucleic acid sequences. There is no particular limitation regarding the linker sequence.

[0072] The 3'-UTR typically has a length of 200-2000 nucleotides, e.g., 500-1500 nucleotides. The 3' untranslated regions of immunoglobulin mRNAs are relatively short (less than about 300 nucleotides), whereas the 3' untranslated regions of other genes are relatively long. For example, the 3' untranslated region of tPA is about 800 nucleotides long, the 3' untranslated region of factor VIII is about 1800 nucleotides long, and the 3' untranslated region of erythropoietin is about 560 nucleotides long. The 3' untranslated regions of mammalian mRNAs typically have a region of homology known as the AAUAAA hexanucleotide sequence. This sequence is likely a poly(A) attachment signal, and is often located 10-30 bases upstream of the poly(A) attachment site. The 3'-untranslated region may contain one or more inverted repeat sequences that can fold to provide a stem-loop structure that acts as a barrier against exoribonucleases or interacts with proteins known to enhance RNA stability (e.g., RNA-binding proteins).

[0073] In some embodiments, the RNA molecule may contain at least one cleavage site for the catalytic nucleic acid molecule. In some embodiments, the RNA molecule has only one site that can be cleaved by the catalytic nucleic acid used in step (a).

[0074] In some embodiments, the catalytic nucleic acid can specifically cleave an RNA molecule at at least one cleavage site. Specific cleavage at a single cleavage site results in one 5'-end fragment and one 3'-fragment. Specific cleavage at two or more cleavage sites results in one 5'-end fragment and two or more 3'-fragments.

[0075] In some embodiments, the RNA molecule may include one cleavage site for the catalytic nucleic acid molecule. The cleavage site may be within a range of 5-50 nucleotides downstream of the 5' end of the RNA molecule, 10-50 nucleotides downstream of the 5' end of the RNA molecule, or 15-30 nucleotides downstream of the 5' end of the RNA molecule. Other cleavage sites may be located downstream of this range. To separate the 3' fragment from the 5' fragment, the at least one 3' fragment may be longer than the 5' fragment. For example, the at least one 3' fragment may have a length of 100, 200, 500 or even more nucleotides.

[0076] "Upstream" refers to the relative location of a first element of a nucleic acid molecule with respect to a second element of the nucleic acid molecule, where both elements are contained in the same nucleic acid molecule and the first element is located closer to the 5' end of the nucleic acid molecule than the second element of the nucleic acid molecule. The second element is then said to be "downstream" of the first element of the nucleic acid molecule. An element that is located "upstream" of a second element can be synonymously referred to as being located "5'" of the second element. For double-stranded nucleic acid molecules, designations such as "upstream" and "downstream" are given with respect to the "+" strand.

[0077] In some embodiments, the catalytic nucleic acid molecule of the present invention can cleave at a cleavage site in the 5'UTR sequence. For example, a catalytic nucleic acid molecule comprising a sequence selected from SEQ ID NOs: 1-4 and 6-25 recognizes a sequence in the 5'UTR of hAg, and a catalytic nucleic acid molecule comprising SEQ ID NO: 5 recognizes a sequence in the 5'UTR of TEV.

[0078] In some embodiments, the catalytic nucleic acid molecule may cleave at a 5'-NUH-3' cleavage site within the RNA molecule to generate a 5' fragment comprising a NUH>p 3' end, wherein: N is selected from G, A, C and U; and H is selected from A, C and U.

[0079] In some embodiments, the catalytic nucleic acid molecule may cleave at a 5'-NCH-3' cleavage site within the RNA molecule to generate a 5' fragment comprising a NCH>p 3' end, wherein: N is selected from G, A, C and U; and H is selected from A, C and U.

[0080] The NUH and NCH cleavage sites of catalytic nucleic acids are known to those of skill in the art.

[0081] As used herein, NUH>p 3' and NCH>p 3' indicate cleavage in an RNA molecule between the NUH or NCH sequence and the nucleotide immediately adjacent to the 3' side of this sequence. For example, a catalytic nucleic acid molecule comprising SEQ ID NO: 1, 3 or 4 cleaves the 3' side of the sequence GUC (Table 4 in Example 1). In another example, a catalytic nucleic acid molecule comprising SEQ ID NO: 2 cleaves the 3' side of the sequence GUA. In a further example, a catalytic nucleic acid molecule comprising SEQ ID NO: 5 cleaves the 3' side of the sequence ACA. Further examples of cleavage sites are disclosed in Table 1.

[0082] "Catalytic nucleic acid molecule" or "catalytic nucleic acid" refers to a nucleic acid molecule having nucleic acid cleavage activity. A catalytic nucleic acid may comprise a "catalytic core sequence" or "catalytic core" as well as 3' and 5' flanking sequences.

[0083] In some embodiments, the catalytic nucleic acid molecule may have a length of 30-60 nt or 35-50 nt, hi some embodiments, the catalytic core may be 20-25 nt, preferably 22 or 23 nt, in length.

[0084] In some embodiments, the flanking sequences may include "recognition sequences," i.e., sequences that specifically recognize a target sequence within an RNA molecule. In some embodiments, the flanking sequences may be 5-20 nt in length.

[0085] In some embodiments, catalytic nucleic acid molecule can be ribozyme or DNAzyme.Ribozyme and DNAzyme are known to those skilled in the art.The design and construction of catalytic nucleic acid molecule that specifically binds to target sequence in nucleic acid molecule and specifically cuts nucleic acid molecule are known to those skilled in the art.

[0086] "Ribozyme" refers to an antisense RNA molecule that combines the ability to recognize a specific target on an RNA substrate and cleave that target by acid-base catalysis. There are various types of ribozymes, the well-studied and often applied is the hammerhead type. The hammerhead RNA cleavage reaction is a phosphodiester isomerization of a 5'→3' diester to a 2',3'-cyclic phosphodiester, resulting in cleavage of the phosphate backbone. The first ribozymes were found in nature as RNAs capable of catalyzing self-cleavage, but in 1988 Haseloff&Gerlach (https: / / www.nature.com / articles / 334585a0.pdf) isolated their substrate and nuclease activity. They proposed a model for the design and synthesis of ribozymes, proposing minimal structural requirements for ribozyme-catalyzed RNA cleavage: (A) containing conserved sequences in the RNA substrate immediately adjacent to the cleavage site (the cleavage site, e.g., GUC and other triplets), (B) containing a naturally highly conserved sequence (core sequence) maintained in the ribozyme, and a region (C) consisting of adjacent helices with base pairing between the substrate and ribozyme RNA, allowing for correct positioning of the ribozyme on the substrate RNA and formation of a stable structure.

[0087] Examples of trans-cleaving ribozymes are described in US Pat. No. 6,656,731.

[0088] In some embodiments, the catalytic nucleic acid molecule can be a hammerhead ribozyme, a hairpin ribozyme, or a HDV ribozyme.

[0089] In some embodiments, the catalytic nucleic acid molecule can be a modified catalytic nucleic acid molecule. In some embodiments, the RNA molecule analyzed by the methods described herein can be a modified RNA molecule. In some embodiments, the catalytic nucleic acid molecule and / or the RNA molecule can comprise at least one modified nucleotide.

[0090] The modified RNA molecules and / or catalytic nucleic acid molecules as defined herein may contain nucleotide analogues / modifications, such as backbone, sugar or base modifications. A backbone modification in the context of the present invention is a modification in which the backbone phosphate of the nucleotides contained in the RNA molecules and / or catalytic nucleic acid molecules as defined herein is chemically modified. A sugar modification in the context of the present invention is a chemical modification of the sugar of the nucleotides of the RNA molecules and / or catalytic nucleic acid molecules as defined herein. Furthermore, a base modification in the context of the present invention is a chemical modification of the base portion of the nucleotides of the RNA molecules and / or catalytic nucleic acid molecules. In this context, the nucleotide analogues or modifications are preferably selected from nucleotide analogues that are applicable for transcription and / or translation.

[0091] Sugar Modification: Modified nucleosides and nucleotides that may be incorporated into the modified RNA molecules and / or catalytic nucleic acid molecules described herein may be modified at the sugar moiety. For example, the 2' hydroxyl group (OH) may be modified or replaced with a number of different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA), in which the 2' hydroxyl is linked, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, where the amino group, e.g., NRR, may be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), or the amino group can be attached to the sugar via a linker, the linker comprising one or more of the atoms C, N, and O. The sugar group can also comprise one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified RNA molecules can include nucleotides that contain, for example, arabinose as the sugar.

[0092] Backbone Modification: The phosphate backbone can be further modified with modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules and / or catalytic nucleic acid molecules described herein. The phosphate group of the backbone can be modified by replacing one or more of the oxygen atoms with different substituents. In addition, the modified nucleosides and nucleotides can include a complete replacement of the unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced with sulfur. Phosphate linkers can also be modified by replacing linking oxygens with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates).

[0093] Base modification: The modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules and / or catalytic nucleic acid molecules described herein can be further modified at the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove surface. In some embodiments, the major groove chemical modification can include an amino group, a thiol group, an alkyl group, or a halo group.

[0094] In certain embodiments of the invention, the nucleotide analogues / modifications are preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, 4-thiouridine ... 5-aminoallyl cytidine-5'-triphosphate, 5-aminoallyl uridine-5'-triphosphate, 5-bromo cytidine-5'-triphosphate, 5-brom uridine-5'-triphosphate, 5-bromo-2'-deoxy cytidine-5'-triphosphate, 5-bromo-2'-deoxy uridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxy cytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo -2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8- The base modification is selected from the group of base-modified nucleotides consisting of azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Particularly preferred is a nucleotide for base modification selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.In some embodiments, modified nucleosides include pyridin-4-one ribonucleosides, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine, 1 ... -4-thiouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydro-pseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thiopseudouridine.

[0095] In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl- These include pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0096] In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methyl-thio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0097] In some embodiments, the nucleotide can be modified on the major groove face and can include replacing the hydrogen on C-5 of uracil with a methyl or halo group. In certain embodiments, the modified nucleoside is 5'-0-(l-thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine, 5'-0-(l-thiophosphate)-guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(l-thiophosphate)-pseudouridine.

[0098] In further embodiments, the modified RNA molecule and / or catalytic nucleic acid molecule is 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, 5-methyl-uridine, pyrrolo-cytidine. , inosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0099] In certain preferred embodiments, the RNA molecule and / or catalytic nucleic acid molecule includes a modified nucleoside in place of at least one (eg, all) uridines, except as provided herein.

[0100] The term "uracil" as used herein refers to one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is: [ka] It is.

[0101] The term "uridine" as used herein refers to one of the nucleosides that can occur in RNA. The structure of uridine is: [ka] It is.

[0102] UTP (uridine 5'-triphosphate) has the following structure: [ka]

[0103] Modified uridines are also nucleosides that can occur in RNA. One such modified uridine is pseudo-UTP (pseudouridine 5'-triphosphate), which has the following structure: [ka]

[0104] "Pseudouridine" is an exemplary modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring through a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0105] Another exemplary modified nucleoside is N1-methyl-pseudouridine (1mψ), which has the structure: [ka] has.

[0106] N1-methylpseudoUTP has the following structure: [ka]

[0107] Another exemplary modified uridine is 5-methyl-uridine (m5U), which has the structure: [ka] has.

[0108] In certain preferred embodiments, one or more uridines in the RNA and / or catalytic nucleic acid molecules described herein are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0109] In certain preferred embodiments, the RNA molecule and / or catalytic nucleic acid molecule comprises a modified uridine in place of at least one uridine. In some embodiments, the RNA molecule and / or catalytic nucleic acid molecule comprises a modified uridine in place of each uridine.

[0110] In certain preferred embodiments, the modified uridines are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (1mψ), and 5-methyl-uridine (m5U). In some embodiments, the modified uridine comprises pseudouridine (ψ). In some embodiments, the modified uridine comprises N1-methyl-pseudouridine (1mψ). In some embodiments, the modified uridine comprises 5-methyl-uridine (m5U). In some embodiments, the RNA molecule and / or catalytic nucleic acid molecule may comprise two or more types of modified uridines, the modified uridines being independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (1mψ), and 5-methyl-uridine (m5U). In some embodiments, the modified uridine comprises pseudouridine (ψ) and N1-methyl-pseudouridine (1mψ). In some embodiments, the modified uridine comprises pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, modified uridines include N1-methyl-pseudouridine (1mψ) and 5-methyl-uridine (m5U). In some embodiments, modified uridines include pseudouridine (ψ), N1-methyl-pseudouridine (1mψ), and 5-methyl-uridine (m5U).

[0111] In certain preferred embodiments, the modified nucleoside that replaces one or more uridines, e.g., all uridines, in the RNA molecule and / or catalytic nucleic acid molecule is the following modified uridine: 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s4 Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5Um), 1-thio-uridine, deoxythymidine, 2'-F-arauridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0112] In one embodiment, the RNA molecule and / or catalytic nucleic acid molecule comprises other modified nucleosides or further modified nucleosides, such as modified cytidines, such as those described above. For example, in one embodiment, cytidine is partially or completely replaced with 5-methylcytidine, preferably completely, in the RNA. In one embodiment, the RNA molecule and / or catalytic nucleic acid molecule comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (1mψ), and 5-methyl-uridine (m5U). In one embodiment, the RNA molecule and / or catalytic nucleic acid molecule comprises 5-methylcytidine and N1-methyl-pseudouridine (1mψ). In some embodiments, the RNA molecule and / or catalytic nucleic acid molecule comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (1mψ) in place of each uridine.

[0113] In some embodiments, the catalytic nucleic acid molecule and / or RNA molecule may contain at least one N1-methylpseudouridine (m1Ψ) nucleotide.

[0114] In some embodiments, the catalytic nucleic acid molecule and / or RNA molecule may comprise at least one nucleoside selected from inosine, 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O-methyl uridine, and 2'-O-methyl cytidine.

[0115] In some embodiments, the catalytic nucleic acid and / or RNA molecule may comprise at least one deoxyribonucleotide.

[0116] In some embodiments, the catalytic nucleic acid molecule comprises: (i) a sequence selected from SEQ ID NOs: 1 to 25; (ii) a sequence having at least 80% identity, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% identity, to any one of SEQ ID NOs: 1 to 25, and / or (iii) A fragment of (i) and / or (ii) wherein the catalytic nucleic acid molecule comprises a catalytic core.

[0117] [Table 1] TIFF2025503044000009.tif98153Hammerhead Rz catalytic core sequence is underlined; hAg, human alpha globin; I, inosine; m, 2'-O-Met; TEV, tobacco etch virus; Rz, ribozyme; 5'UTR, 5' untranslated region.

[0118] [Table 2]

[0119] A "fragment" in reference to a nucleic acid sequence relates to a sequence that represents a part of the nucleic acid sequence, i.e. a nucleic acid sequence truncated at the 5'-end and / or the 3'-end. Preferably, a fragment of a nucleic acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% of the nucleotide residues from said nucleic acid sequence. In the present invention, fragments of RNA molecules that retain the stability of the RNA are preferred.

[0120] In some embodiments, a fragment of a catalytic nucleic acid molecule described herein can comprise the catalytic core of a catalytic nucleic acid molecule of any one of SEQ ID NOs: 1-25.

[0121] In some embodiments, the catalytic core may comprise SEQ ID NO:26 or 27 (Table 2), or a sequence having at least 80% identity, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:26 and / or 27.

[0122] In some embodiments, in any one of SEQ ID NOs: 1-25, Am can be independently selected from A and 2'-O-methyl adenosine, Gm can be independently selected from G and 2'-O-methyl guanosine, Um can be independently selected from U and 2'-O-methyl uridine, and / or Cm can be independently selected from C and 2'-O-methyl cytidine.

[0123] In some embodiments, the catalytic nucleic acid molecules of the present invention can be RNA molecules, in particular ribozymes.

[0124] The term "nucleic acid variant" includes deletion, addition, mutation, substitution and / or insertion of single or multiple nucleotides compared to the reference nucleic acid. Deletion includes removal of one or more nucleotides from the reference nucleic acid. Addition variants include 5'- and / or 3'-terminal fusion of one or more nucleotides, for example 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides. In the case of substitution, at least one nucleotide in the sequence is removed and at least one other nucleotide is inserted in its place (such as transversion and transition). Mutations include abasic sites, crosslinked sites, and chemically altered or modified bases. Insertion includes addition of at least one nucleotide to the reference nucleic acid.

[0125] According to the present invention, a "nucleotide change" may refer to a deletion, addition, mutation, substitution and / or insertion of a single or multiple nucleotides compared to a reference nucleic acid. In some embodiments, a "nucleotide change" is selected from the group consisting of a deletion of a single nucleotide, an addition of a single nucleotide, a mutation of a single nucleotide, a substitution of a single nucleotide and / or an insertion of a single nucleotide compared to a reference nucleic acid. According to the present invention, a nucleic acid variant may contain one or more nucleotide changes compared to a reference nucleic acid.

[0126] A variant of a specific nucleic acid sequence can preferably have at least one functional property of the specific sequence, and is preferably functionally equivalent to the specific sequence, e.g., is a nucleic acid sequence that exhibits properties identical or similar to those of the specific nucleic acid sequence.

[0127] Preferably, the "degree of identity" or "identity" or "% identity" between a given nucleic acid sequence and a nucleic acid sequence that is a variant of said given nucleic acid sequence is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98% or 99%. The degree of identity is preferably given over a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In a preferred embodiment, the degree of identity is given over the entire length of the reference nucleic acid sequence.

[0128] "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0129] The term "% identical" is intended to refer in particular to the percentage of nucleotides that are identical in optimal alignment between the two sequences being compared, said percentage being purely statistical, and the differences between the two sequences may be randomly distributed over the entire length of the sequences, and the sequences being compared may contain additions or deletions compared to the reference sequence in order to obtain optimal alignment between the two sequences. Comparison of two sequences is usually performed by comparing said sequences over a segment or "comparison window" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2:482, using the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, and using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or with the aid of computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0130] The percent identity is obtained by determining the number of identical positions where the compared sequences match, dividing this number by the number of positions compared and multiplying this result by 100.

[0131] For example, one may use the BLAST program "BLAST 2 sequences" available at the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi.

[0132] "nt" is an abbreviation for a single nucleotide or for multiple nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0133] In some embodiments, in step (a), the catalytic nucleic acid molecule may be contacted with a population of RNA molecules that have been capped by enzymatic capping or / and co-transcriptional capping in the presence of a capping analog.

[0134] The terms "5' cap," "cap," "5' cap structure," and "cap structure" are used synonymously and can refer to the dinucleotide found at the 5' end of some eukaryotic primary transcripts, such as precursor messenger RNAs. A 5' cap is a structure in which an (optionally modified) guanosine is attached to the first nucleotide of an mRNA molecule via a 5'-5' triphosphate linkage (or a modified triphosphate linkage in the case of certain cap analogs). These terms can refer to a conventional cap or a cap analog.

[0135] "RNA containing a 5' cap" or "RNA with a 5' cap" or "RNA modified with a 5' cap" or "capped RNA" refers to RNA that includes a 5' cap. For example, providing an RNA with a 5' cap can be achieved by in vitro transcription of a DNA template in the presence of said 5' cap, and said 5' cap is co-transcriptionally incorporated into the generated RNA strand, or RNA can be generated, for example, by in vitro transcription, and a 5' cap can be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked to the 5' position of the subsequent base ("second base") of the RNA molecule via a phosphodiester bond.

[0136] The term "conventional 5' cap" refers to a naturally occurring 5' cap, preferably a 7-methylguanosine cap, in which the guanosine of the cap is a modified guanosine, the modification consisting of methylation at the 7 position.

[0137] In the context of the present invention, the term "5' cap analog" refers to a molecular structure that is similar to a conventional 5' cap, but that has been modified such that it has the ability to stabilize RNA when bound to RNA, preferably in vivo and / or within a cell. A cap analog is not a conventional 5' cap.

[0138] For eukaryotic mRNA, the 5' cap has been generally described to be involved in the efficient translation of mRNA: In general, in eukaryotes, translation is initiated only at the 5' end of a messenger RNA (mRNA) molecule, unless an internal ribosome entry site (IRES) is present. Eukaryotic cells can provide a 5' cap to RNA during transcription in the nucleus: newly synthesized mRNA is usually modified with a 5' cap structure, for example, once the transcript reaches a length of 20-30 nucleotides. First, the 5' terminal nucleotide pppN (ppp stands for triphosphate; N stands for any nucleoside) is converted intracellularly to 5'GpppN by a capping enzyme with RNA 5'-triphosphatase and guanylyltransferase activity. GpppN is then methylated intracellularly by a second enzyme with (guanine-7)-methyltransferase activity to give monomethylated m 7 A GpppN cap may be formed. In one embodiment, the 5' cap used in the present invention is a natural 5' cap.

[0139] In the present invention, naturally occurring 5' capped dinucleotides typically include unmethylated capped dinucleotides (G(5')ppp(5')N; also referred to as GpppN) and methylated capped dinucleotides ((m 7 G(5')ppp(5')N;m 7 m 7GpppN (N is G) is represented by the following formula: [ka]

[0140] The capped RNA of the present invention can be prepared in vitro and therefore does not depend on the capping mechanism in the host cell. The most frequently used method for making capped RNA in vitro is the synthesis of all four ribonucleoside triphosphates and m 7 G(5')ppp(5')G(m 7 The first step is to transcribe a DNA template with either bacterial or bacteriophage RNA polymerase in the presence of a cap dinucleotide such as GpppG. The RNA polymerase then catalyzes the transcription of the m-phosphate of the α-phosphate of the next template nucleoside triphosphate (pppN). 7 Transcription is initiated by nucleophilic attack of the 3'-OH of the guanosine moiety of GpppG, forming intermediate m 7 This results in GpppGpN (where N is the second base of the RNA molecule). Formation of the competing GTP-initiated product pppGpN is suppressed by setting the cap-to-GTP molar ratio at 5-10 during in vitro transcription.

[0141] In preferred embodiments of the present invention, the 5' cap (if present) is a 5' cap analog. These embodiments are particularly suitable when the RNA is obtained by in vitro transcription, e.g., in vitro transcribed RNA (IVT-RNA). Cap analogs were first described to facilitate large-scale synthesis of RNA transcripts by in vitro transcription.

[0142] For messenger RNA, several cap analogs (synthetic caps) have been commonly described so far, all of which can be used in the context of the present invention. Ideally, a cap analog associated with higher translation efficiency and / or increased resistance to in vivo degradation and / or increased resistance to in vitro degradation will be selected.

[0143] Preferably, a cap analog is used that can be incorporated into an RNA strand in only one orientation. Pasquinelli et al. (1995, RNA J. 1:957-967) showed that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit for the initiation of transcription, such that approximately 40-50% of capped transcripts have the cap dinucleotide in the reverse orientation (i.e., the initial reaction product is Gpppm). 7 In comparison to RNA with a correct cap, RNA with a reverse cap is not functional for translation of a nucleic acid sequence into a protein. Therefore, it is important to incorporate the cap in the correct orientation, i.e., m 7 It would be desirable to obtain RNA with a structure essentially corresponding to GpppGpN, etc. Reverse incorporation of cap dinucleotides has been shown to be inhibited by replacement of either the 2'-OH or 3'-OH groups of the methylated guanosine units (Stepinski et al., 2001, RNA J. 7:1486-1495; Peng et al., 2002, Org. Lett. 24:161-164). RNA synthesized in the presence of such "anti-reverse cap analogs" will not retain the traditional 5'-capped methylated guanosine units. 7 It is translated more efficiently than RNA in vitro transcribed in the presence of GpppG. For this purpose, one cap analog in which the 3'OH group of the methylated guanosine unit is replaced with OCH3 has been described, for example, by Holtkamp et al., 2006, Blood 108:4009-4017 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analog (ARCA)). ARCA is a suitable cap dinucleotide according to the present invention. [ka]

[0144] In one embodiment, the RNA of the present invention is inherently resistant to decapping. This is important because, in general, the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is limited by natural degradation of the mRNA. One in vivo pathway of mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase that includes a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the alpha and beta phosphate groups of the triphosphate bridge. In the present invention, cap analogs that are less susceptible or less susceptible to this type of cleavage may be selected or may exist. A suitable cap analog for this purpose is represented by the formula (I): [ka] A cap dinucleotide according to the formula: In the formula, R 1 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; R 2 and R 3 is independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R 2 and R 3 together form OXO, where X is selected from the group consisting of optionally substituted CH, CHCH, CHCHCH, CHCH(CH), and C(CH), or R 2 is R 2 is bonded to the hydrogen atom at the 4' position of the ring to form -O-CH2- or -CH2-O-, R 5 is selected from the group consisting of S, Se, and BH3; R 4 and R 6 is independently selected from the group consisting of O, S, Se, and BH3.

[0145] n is 1, 2, or 3.

[0146] R 1 , R 2 , R3, R 4 , R 5 , R 6 Preferred embodiments of the above are disclosed in WO 2011 / 015347 A1 and may be selected according to circumstances in the present invention.

[0147] For example, in one embodiment, the RNA of the present invention comprises a phosphorothioate cap analog, which has one of the three non-bridging O atoms of the triphosphate chain replaced with an S atom, i.e., R 4 , R 5 or R 6 is a specific cap analogue in which one of R is S. Phosphorothioate cap analogues have been described by J. Kowalska et al., 2008, RNA, 14:1119-1131, as a solution to the undesired decapping process and thus to increase the stability of RNA in vivo. In particular, the replacement of the sulfur atom in the β-phosphate group of the 5' cap with an oxygen atom results in stabilization against Dcp2. In its embodiment which is preferred in the present invention, R of formula (I) 5 is S and R 4 and R 6 is O.

[0148] In a further embodiment, the RNA of the invention comprises a phosphorothioate cap analog in which a phosphorothioate modification of the RNA 5' cap is combined with an "anti-reverse cap analog" (ARCA) modification. Respective ARCA-phosphorothioate cap analogs are described in WO 2008 / 157688 A2, all of which can be used in the RNA of the invention. In that embodiment, R 2 or R 3 At least one of is not OH, preferably R 2 and R 3One of the groups is methoxy (OCH3), and the other is R 2 and R 3 The other of is preferably OH. In a preferred embodiment, an oxygen atom is replaced by a sulfur atom in the β phosphate group (hence, R 5 is S and R 4 and R 6 is O). The phosphorothioate modification of ARCA is thought to ensure that the α, β, and γ phosphorothioate groups are correctly positioned within the active sites of cap-binding proteins in both the translational and decapping machinery. At least some of these analogs are essentially resistant to pyrophosphatases Dcp1 / Dcp2. Phosphorothioate-modified ARCA was described to have a much higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate groups.

[0149] Particularly preferred cap analogs of the present invention are 2’ 7,2’-O Gpp s pG is referred to as β-S-ARCA (WO 2008 / 157688 A2; Kuhn et al., 2010, Gene Ther. 17:961-971). Thus, in one embodiment of the present invention, the RNA of the present invention is modified with β-S-ARCA. β-S-ARCA is represented by the following structure: [ka]

[0150] Generally, replacement of oxygen atoms with sulfur atoms in the bridging phosphates results in phosphorothioate diastereomers designated D1 and D2 based on their elution patterns in HPLC. Briefly, the "D1 diastereomer of β-S-ARCA" or "β-S-ARCA(D1)" is the diastereomer of β-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA(D2)). Determination of stereochemical configuration by HPLC is described in WO 2011 / 015347 A1.

[0151] In a first particularly preferred embodiment of the invention, the RNA of the invention is modified with the β-S-ARCA (D2) diastereomer. The two diastereomers of β-S-ARCA differ in their susceptibility to nucleases. It has been shown that RNA carrying the D2 diastereomer of β-S-ARCA is almost completely resistant to Dcp2 cleavage (only 6% cleavage compared to RNA synthesized in the presence of an unmodified ARCA 5' cap), whereas RNA with a β-S-ARCA (D1) 5' cap shows moderate susceptibility to Dcp2 cleavage (71% cleavage). It has further been shown that increased stability against Dcp2 cleavage correlates with increased protein expression in mammalian cells. In particular, it has been shown that RNA with a β-S-ARCA (D2) cap is translated more efficiently in mammalian cells than RNA with a β-S-ARCA (D1) cap. Thus, in one embodiment of the invention, the RNA of the invention is modified with the P2 diastereomer of β-S-ARCA. β The substituents R of formula (I) correspond to the stereochemical configuration at the atoms 5 In this embodiment, the R of formula (I) is modified with a cap analogue characterized by a stereochemical configuration at the P atom that includes 5 is S and R 4 and R 6 is O. Furthermore, R in formula (I) 2 or R 3 At least one of is preferably not OH, and preferably is R 2and R 3 One of the groups is methoxy (OCH3), and the other is R 2 and R 3 The other is preferably OH.

[0152] In a second particularly preferred embodiment, the RNA of the present invention is modified with the β-S-ARCA(D1) diastereomer. This embodiment is particularly suitable for the transfer of capped RNA into immature antigen-presenting cells, such as for vaccination purposes. It has been demonstrated that the β-S-ARCA(D1) diastereomer is particularly suitable for increasing the stability of the RNA, increasing the translation efficiency of the RNA, extending the translation of the RNA, increasing the total protein expression of the RNA, and / or increasing the immune response against the antigen or antigen peptide encoded by said RNA, when the capped RNA is transferred into immature antigen-presenting cells (Kuhn et al., 2010, Gene Ther. 17:961-971). Thus, in an alternative embodiment of the present invention, the RNA of the present invention is modified with the P1 diastereomer of β-S-ARCA. β The substituents R of formula (I) correspond to the stereochemical configuration at the atoms 5 The cap analogs according to formula (I) are characterized by the stereochemical configuration at the P atom including: 5 The stereochemical configuration at the P atom is that of the D1 diastereomer of β-S-ARCA. β Any cap analogue described in WO 2011 / 015347 A1 that corresponds to the stereochemical configuration at the atoms may be used in the present invention. 5 is S and R 4 and R 6 is O. Furthermore, R in formula (I) 2 or R 3 At least one of is preferably not OH, and preferably is R 2 and R 3 One of the groups is methoxy (OCH3), and the other is R2 and R 3 The other is preferably OH.

[0153] In one embodiment, the RNA of the present invention is modified with a 5' cap structure according to formula (I), in which any one of the phosphate groups is replaced by a boranophosphate group or a phosphoselenoate group. Such caps have increased stability both in vitro and in vivo. Optionally, each compound has a 2'-O- or 3'-O-alkyl group (alkyl is preferably methyl); each cap analog is called BH3-ARCA or Se-ARCA. Compounds particularly suitable for capping mRNA include β-BH3-ARCA and β-Se-ARCA, which are described in WO 2009 / 149253 A2. For these compounds, the P of the D1 diastereomer of β-S-ARCA is β The substituents R of formula (I) correspond to the stereochemical configuration at the atoms 5 The stereochemical configuration at the P atom containing is preferred.

[0154] In one embodiment, the 5' cap can be a trinucleotide AU (Cap 1) having the following structure: [ka]

[0155] In some embodiments, the capping analog is G[5']ppp[5']G, m 7 G[5']ppp[5']G, m3 2,2,7 G[5']ppp[5']G, m2 7,3’-O G[5']ppp[5']G(3'-ARCA), m2 7,2’-O GpppG(2'-ARCA), m2 7,2’-O Gpp s pG D1 (β-S-ARCA D1), m2 7,2’-O Gpp spG D2 (β-S-ARCA D2), m7(3'OMeG)(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG (3'OMe), Catalog Number: N-7413), m7G(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG, Catalog Number: N-7113), CleanCap® Reagent AU (N-7114), m7G(5')ppp(5')(2'OMeA)pU.

[0156] In some embodiments, in step (a), the population of RNA molecules may be contacted with an excess of catalytic nucleic acid molecules. For example, in step (a), the population of RNA molecules may be contacted with catalytic nucleic acid molecules at a molar ratio of RNA molecules to catalytic nucleic acid molecules of about 1:1 to about 1:20, or about 1:1 to about 1:10. For example, the molar ratio of RNA molecules to catalytic nucleic acid molecules may be about 1:2.5, about 1:5, or about 1:10. A ratio of about 1:2.5 is preferred.

[0157] In some embodiments, the length of the 5' end fragment allows for discrimination between capped and uncapped 5' end fragments, hi some embodiments, the capped and uncapped 5' end fragments may differ in length by 1-3 nucleotides.

[0158] In some embodiments, the 5'-end fragment obtained in step (a) may have a length of at least 5 nt, at least 10 nt, or at least 15 nt. In some embodiments, the 5'-end fragment obtained in step (a) may have a length of up to 35 nt or up to 50 nt. In some embodiments, the length of the 5'-end fragment may be in the range of 5-50 nt, 10-50 nt, or 15-30 nt.

[0159] In some embodiments, the 5'-end fragment is preferably subjected to step (b) in a reaction mixture comprising the reactants of the cleavage reaction of step (a).

[0160] In some embodiments, in step (a), the 5' end fragment may be obtained in a mixture with at least one 3' fragment, catalytic nucleic acid molecule and / or non-cleaved RNA molecule.

[0161] The cleavage in step (a) of the method described herein may be essentially complete. In some embodiments, in step (a), the 5'-end fragment may be obtained in a mixture with at least one 3' fragment and a catalytic nucleic acid molecule.

[0162] The cleavage in step (a) of the method described herein may be incomplete. In some embodiments, in step (a), the 5'-end fragment may be obtained in a mixture with at least one 3' fragment, catalytic nucleic acid molecule, and non-cleaved RNA molecule.

[0163] In one embodiment, in step (b), the 5'-end fragments and at least one 3'-fragment present in the population of cleaved RNA molecules obtained in step (a) are at least partially separated or purified from each other. This separation (purification) step results in a population enriched in 5'-end fragments, i.e. the amount of 5'-end fragments as a percentage of all cleaved and uncleaved RNA molecules present in the separated (or purified) enriched population obtained in step (b) is greater than the amount of 5'-end fragments as a percentage of all cleaved and uncleaved RNA molecules present in the population obtained in step (a). In some embodiments, the enriched population of 5'-end fragments is in solution. In one embodiment, the enriched population of 5'-end fragments obtained in step (b), e.g. in solution, can be used in the method of the invention for analyzing the 5'-cap structure of RNA molecules.

[0164] In some embodiments, the percentage increase in the amount of 5' end fragments can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the percentage increase is in the range of 50%-95%, 60%-95%, 70%-95%, 80%-95%, 90%-95%, or 95%-99%. In some embodiments, the percentage increase is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold or more.

[0165] In some embodiments, step (b) may comprise subjecting the mixture obtained in step (a) to chromatography using a silica-based stationary phase under conditions allowing at least partial separation of the 5'-terminal fragment from at least one 3' fragment, uncleaved RNA molecule and / or catalytic nucleic acid molecule.

[0166] Any silica-based material suitable for chromatography may be used, for example for the column and / or membrane. Suitable silica-based materials are known to those skilled in the art.

[0167] In some embodiments, step (b) may comprise two separate chromatographic steps, each using a silica-based stationary phase. Examples 1 and 2 describe typical protocols for silica-based chromatography using two separate columns.

[0168] In some embodiments, in the first chromatographic step, the long-chain RNA, including the uncleaved full-length RNA and the long 3' fragment, may remain on the solid phase, while the short 5'-end fragments are collected in the flow-through fraction. The mobile phase may be a mixture of the sample obtained in step (a), alcohol and an aqueous buffer in a ratio that allows at least partial separation of the 5'-end fragment from the uncleaved RNA molecules and / or at least one 3' fragment.

[0169] In some embodiments, in the first chromatographic step, the ratio of alcohol to aqueous buffer is less than 1, i.e., an excess of aqueous buffer is used in view of the alcohol. The volume ratio of alcohol to aqueous buffer can range from about 2:3 to about 3:4, with an excess of aqueous buffer in view of the alcohol. In some embodiments, the mobile phase can contain the sample obtained in step (a) and the alcohol / buffer mixture in a volume ratio ranging from about 1:5 to about 1:7.

[0170] In some embodiments, the 5'-end fragment can be bound to a solid phase in the second chromatographic step. The mobile phase can be a mixture of the sample obtained in the first chromatographic step, alcohol and an aqueous buffer in a ratio that allows the 5'-end fragment to bind to the solid phase.

[0171] In some embodiments, in the second chromatographic step, the ratio of alcohol to aqueous buffer is greater than 1, i.e., an excess of alcohol is used in view of the aqueous buffer. The volume ratio of alcohol to aqueous buffer can be in the range of about 5:1 to about 20:1, e.g., about 10:1. In some embodiments, the mobile phase can contain the sample obtained in the first chromatographic step and the alcohol / buffer mixture in a volume ratio in the range of about 2:1 to about 1:1.

[0172] The 5' fragment bound to the second column can be eluted with water. Suitable protocols are known to those of skill in the art.

[0173] In some embodiments, in the two chromatographic steps, the alcohol is independently selected from the group consisting of C 1-6 Alcohol, preferably C 1-3 It may be an alcohol, such as methanol, ethanol, 1-propanol or 2-propanol, or a mixture thereof. Ethanol is preferred.

[0174] The aqueous buffer may be any suitable buffer known to those of skill in the art.

[0175] "At least partially separating" or "at least partial separation" in step (b) of the method of the present invention relates to complete or incomplete separation of the 5' end fragment from at least one 3' fragment and / or any other components of the reaction mixture obtained in step (a).

[0176] In some embodiments, step (b) may also comprise subjecting the mixture obtained in step (a) to PAGE under conditions allowing at least partial separation of the 5'-end fragments from at least one 3' fragment and / or uncleaved RNA molecule. The PAGE may further comprise (i) isolating at least one band of interest from the PAGE gel, said at least one band comprising the 5'-end fragment, and (ii) eluting the 5'-end fragments from the at least one isolated band obtained in step (i).

[0177] In some embodiments, step (b) may also include contacting the mixture obtained in step (a) with oligo-dT nucleotides under conditions that allow at least partial separation of the 5'-end fragments from at least one 3' fragment and / or non-cleaved RNA molecule. The oligo-dT nucleotides may bind RNA molecules that contain poly(A) sequences to a solid phase. mRNA molecules typically contain poly(A) sequences. In some embodiments, the oligo-dT nucleotides may be bound to plastic or magnetic beads, or are bound to biotin. In some embodiments, the beads form a column.

[0178] According to the invention, in one embodiment, the poly(A) sequence comprises or essentially consists of or consists of at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, and preferably up to 500, preferably up to 400, preferably up to 300, preferably up to 200, in particular up to 150 A nucleotides, in particular up to about 120 A nucleotides. In this context, "essentially consists of" means that most of the nucleotides in the poly(A) sequence, typically at least 50%, preferably at least 75% by number of nucleotides in the "poly(A) sequence", are A nucleotides (adenylic acid), while allowing the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e. 100% of the number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0179] In some embodiments, the catalytic nucleic acid molecule may be labeled, for example, with biotin.

[0180] In some embodiments, the catalytic nucleic acid molecule may be bound to a surface, which may be a magnetic or plastic bead or particle.

[0181] In some embodiments, the separating step (b) may further comprise separating the 5'-end fragment from the catalytic nucleic acid molecule under conditions that allow at least partial separation of the 5'-end fragment from the catalytic nucleic acid molecule. In some embodiments, the separating may comprise contacting the mixture of step (a) with a substance that binds to the labeled catalytic nucleic acid molecule under conditions that allow at least partial separation of the 5'-end fragment from the labeled catalytic nucleic acid molecule. The label may be biotin.

[0182] In some embodiments, steps (b) and (c) may be separate steps. As described herein, in step (b), the 5'-end fragments obtained in step (a) are at least partially separated from at least one 3'-fragment to obtain a population of 5'-end fragments. In some embodiments, in step (b), the capped 5'-end fragments are preferably not separated from the uncapped 5'-end fragments.

[0183] In some embodiments, in step (c), the amount of RNA molecules having a 5' cap structure may be determined in the population of 5' end fragments obtained in step (b). In some embodiments, in step (c), the capped 5' end fragments may be distinguished from the uncapped 5' end fragments, for example, by chromatographic methods. In some embodiments, the capped 5' end fragments may be 1, 2 or 3 nucleotides longer than the uncapped 5' end fragments.

[0184] In some embodiments, step (c) may include gel electrophoresis, spectroscopy, mass spectrometry, liquid chromatography and / or sequencing. In some embodiments, the gel electrophoresis may be PAGE. In some embodiments, the mass spectrometry may be LC-MS. In some embodiments, the liquid chromatography may be HPLC or UPLC.

[0185] In some embodiments, different methods may be used in steps (b) and (c), for example, step (b) may use chromatography using a silica-based stationary phase, oligo-dT-based separation, elution from PAGE, and / or a separation method using immobilized catalytic nucleic acid, while step (c) may use PAGE, mass spectrometry, HPLC, and / or UPLC.

[0186] In some embodiments, step (c) may include determining the amount of capped and uncapped 5' end fragments. The amount of capped and uncapped 5' end fragments may determine the capping efficiency.

[0187] In some embodiments, the percentage of capped 5'-end fragments may be calculated relative to the total amount of 5'-end fragments. The percentage may be calculated as mole % or weight %.

[0188] In some embodiments, the method of the present invention may further comprise a step (d) of analyzing the cap structure in the capped 5'-end fragment. The analysis of the cap structure may comprise the analysis of the bond between the capping analog and the RNA molecule. The capping analog may comprise two 3' positions (referred to herein as the "first" and "second" 3' positions) that may both have the ability to bind to the 5' end of the RNA molecule. In the first position, the ribose may be bound to a guanosine (G) and in the second position, the ribose may be bound to m7G. The term "orientation" or "correct orientation" refers to the binding of the RNA molecule to the first 3' position that results in efficient translation of the mRNA, and "reverse orientation" refers to the binding of the RNA molecule to the second 3' position that results in reduced translation efficiency. The analysis of the cap structure may comprise the determination of the amount of correctly oriented cap structure and / or the percentage of correctly oriented cap structure relative to the total amount of capped molecules. For example, treatment of a correctly oriented capped 5' end fragment with pyrophosphatase results in cleavage of m7G from the 5' end fragment, and treatment of a reverse capped 5' end fragment with pyrophosphatase results in cleavage of G from the 5' end fragment, allowing discrimination between the correctly and reverse orientations.

[0189] Yet another aspect of the invention is a method for determining capping efficiency in a population of RNA molecules, comprising the steps of: (a) contacting a catalytic nucleic acid molecule with a population of RNA molecules comprising one or more RNA molecules comprising a cleavage site for the catalytic nucleic acid molecule and a 5' cap structure under conditions that allow cleavage of the RNA molecules to generate a 5' terminal fragment and at least one 3' fragment; (b) at least partially separating the 5' end fragments obtained in step (a) from at least one 3' fragment to obtain a population of 5' end fragments; and (c) determining the amount of RNA molecules having a 5'-cap structure in the population of 5'-end fragments obtained in step (b); The method includes:

[0190] Embodiments of steps (a), (b) and (c) are described herein in relation to the methods of analyzing a population of RNA molecules of the invention.

[0191] Yet another aspect of the invention is a method for analyzing an RNA molecule, comprising the steps of: (i) synthesizing an RNA molecule; (ii) capping the RNA synthesized in (i); and (iii) analyzing the RNA molecules by a method for analyzing a population of RNA molecules of the invention, as described herein. The method includes:

[0192] Any known RNA synthesis method may be used in step (i).In some embodiments, the RNA molecule may be synthesized by in vitro transcription and / or solid-phase synthesis, as described herein in connection with the method for analyzing a population of RNA molecules of the present invention.

[0193] In some embodiments, in step (ii), the RNA synthesized in step (i) may be capped by enzymatic capping or / and co-transcriptional capping, as described herein in connection with the methods for analyzing a population of RNA molecules of the invention.

[0194] Yet another aspect of the present invention is a method for quality control of capped RNA synthesis, comprising the steps of: (i) synthesizing an RNA molecule; (ii) capping the RNA synthesized in (i); and (iii) analyzing the RNA molecules by a method for analyzing a population of RNA molecules of the invention, as described herein. The method includes:

[0195] Any known RNA synthesis method may be used in step (i).In some embodiments, the RNA molecule may be synthesized by in vitro transcription and / or solid-phase synthesis, as described herein in connection with the method for analyzing a population of RNA molecules of the present invention.

[0196] In some embodiments, in step (ii), the RNA synthesized in step (i) may be capped by enzymatic capping or / and co-transcriptional capping, as described herein in connection with the methods for analyzing a population of RNA molecules of the invention.

[0197] In one embodiment, the separating and determining steps are not performed simultaneously using a PAGE gel or HPLC.

[0198] Yet another aspect of the present invention is a method for producing a medicament for use in a method for the preparation of a medicament (i) a sequence selected from SEQ ID NOs: 1 to 25; (ii) a sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 25, and / or (iii) A fragment of (i) and / or (ii) wherein the catalytic nucleic acid molecule comprises a catalytic core.

[0199] SEQ ID NOs: 1-25 are described herein in the context of methods for analyzing populations of RNA molecules of the invention.

[0200] In some embodiments, the catalytic nucleic acid molecule described in this aspect of the invention can be an isolated catalytic nucleic acid molecule. As used herein, "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. The term "isolated nucleic acid" means, according to the present invention, that the nucleic acid is (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid that is available for manipulation by recombinant techniques.

[0201] The term "recombinant" in the context of the present invention means "produced through genetic engineering." Preferably, a "recombinant" such as a recombinant cell in the context of the present invention is not naturally occurring.

[0202] In some embodiments, the catalytic nucleic acid molecules of the present invention can be RNA molecules, in particular ribozymes.

[0203] Yet another aspect of the present invention is a method for producing a medicament for use in a method for the preparation of a medicament (i) the sequence of SEQ ID NO: 26 or SEQ ID NO: 27, (ii) a sequence having at least 90% identity, preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26 and / or SEQ ID NO: 27. It is a nucleic acid molecule comprising:

[0204] In some embodiments, the nucleic acid molecules described in this aspect of the invention may be isolated nucleic acid molecules.

[0205] In some embodiments, the nucleic acid molecule of this aspect may be an RNA molecule.

[0206] In some embodiments, a nucleic acid molecule of this aspect may comprise the catalytic core of a ribozyme.

[0207] In some embodiments, the nucleic acid molecule of this aspect can be a ribozyme.

[0208] In some embodiments, a nucleic acid molecule of this aspect may include at least one modified nucleotide, as described herein.

[0209] Yet another aspect of the present invention is the use of a catalytic nucleic acid molecule as described herein in a method of the present invention for analyzing a population of RNA molecules, a method of the present invention for determining the capping efficiency in a population of RNA molecules, a method of the present invention for analyzing RNA molecules, and / or a method of the present invention for the quality control of capped RNA synthesis, as described herein.

[0210] The present invention will now be described in detail and illustrated by figures and examples, which are used for illustrative purposes only and are not intended to be limiting. The descriptions and examples make further embodiments, which are also encompassed by the present invention, accessible to those skilled in the art. EXAMPLES

[0211] Example 1 In this example, a ribozyme (Rz) was designed to specifically cleave IVT mRNA at a unique position in close proximity to the 5' end, releasing short 5' cleavage products ranging from 10 to 30 nt, either capped or uncapped. The well-defined 5' cleavage products cleaved by the ribozyme from capped mRNA differ from those cleaved from uncapped RNA by only one nucleoside in length, especially in the cap structure itself.

[0212] These products were purified using silica-based columns and visualized / quantified using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC-MS). Using this technique, we determined the capping efficiency of IVT mRNAs with different features, including different cap structures, diverse 5' untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assay we developed is rapid and reliable for the analysis of capping efficiency for R&D purposes and as a general quality control for mRNA-based therapeutics.

[0213] RNA from cleavage reactions can be directly analyzed by denaturing polyacrylamide gel electrophoresis (PAGE). This example demonstrates that purifying RNA from cleavage reactions using a silica-based column and loading the resulting cleaned short 5' cleavage product RNA onto a gel results in better reproducibility and improved visualization and quantification.

[0214] RNA is electrophoresed under conditions where differences in 5' cleavage products are detectable. Stained and visualized 5' cleavage products released from capped or uncapped mRNA are quantified by their gel band intensity to assess capping efficiency. Short purified 5' cleavage products were also analyzed using LC-MS to allow further characterization such as determination of methylation status or low abundance of capped products.

[0215] 1. Materials and Methods 1.1. Template for in vitro transcription Templates for in vitro transcription were generated by linearizing plasmids containing different coding sequences flanked by sequences corresponding to the 5' untranslated region (UTR) of human alpha globin (hAg) or the 5' leader of the tobacco etch virus (TEV), a constant 3' UTR, and a 100 nt-long poly(A) tail [15, 16]. Linearization was performed with the restriction enzymes EarI or BbsI (both from New England Biolabs).

[0216] 1.2. In vitro transcription and capping of RNA RNAs ranging in size from 100 nt to 9.4 kb were synthesized using the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific). Reactions included UTP to generate standard IVT mRNA, or N1-methylpseudouridine 5'-triphosphate (m1ΨTP) (TriLink) for nucleoside-modified mRNAs in which 100% of the uridines were replaced with m1Ψ. In a subset of RNAs, the sequences of the first three transcribed nucleotides were GCG, GGA, AGC, AGG, or AGA. Cap analogs, ARCA-G (TriLink, N-7003), β-S-ARCA (D1, BioNTech SE)

[17] , or CleanCap® Reagent AG (3'OMe) (TriLink, N-7413) were added to the transcription reaction to generate mRNAs with cap 0 (A0), cap 0 (D1), or cap 1 (CC1), respectively. The synthesized mRNA was enzymatically capped using Vaccinia Virus Capping Enzyme (New England Biolabs) according to the manufacturer's instructions to generate RNA with cap 0 (E0) or cap 1 (E1). RNA quality was tested using 1.4% agarose gel electrophoresis

[18] , and RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific).

[0217] 1.3. Design of ribozymes and RNase H probes Five hammerhead ribozymes were designed for this study (Table 3).

[0218] [Table 3] TIFF2025503044000017.tif52153Hammerhead Rz catalytic core sequence is underlined; hAg, human alpha globin; I, inosine; m, 2'-O-Met; TEV, tobacco etch virus; Rz, ribozyme; 5'UTR, 5' untranslated region.

[0219] Rz1, Rz2, Rz3 and Rz4 cleave the hAg 5'UTR after GUC, GUA, GUC and GUC triplets, respectively [19, 20]. Rz3 differs from Rz1 by exhibiting a 4 nt shorter 3' arm, while Rz4 was designed with a 4 nt longer 5' arm and a 1 nt shorter 3' arm. Rz5 contains an inosine (I) and was engineered to cleave the TEV 5'UTR after the ACA triplet

[21] . To increase the stability of the annealed Rz:target complex, 2'-O-methylated nucleotides (Nm) were incorporated into the last 7 or 19 nucleotide positions of Rz2 and Rz5, respectively. An RNase H probe with a deoxynucleotide (dN) incorporated (5'-dGdAdC dCdAdG AmAmGm AmAmUm AmCmUm Am-3') was designed according to Beverly et al.

[13] . Ribozymes and RNase H probes were synthesized by Metabion, and their quality was confirmed by electrospray ionization time-of-flight mass spectrometry (ESI-TOF).

[0220] 1.4. Ribozyme-mediated mRNA cleavage Ribozyme cleavage reactions contained 0.2-0.6 μM mRNA and a 2.5-fold molar excess of ribozyme over mRNA substrate in 10 mM Tris and 10 mM MgCl2. The ribozyme was first annealed to the mRNA in a reaction mixture without MgCl2 by first incubating at 95°C for 2 min and then at room temperature for 5 min. The cleavage reaction was initiated by adding MgCl2, carried out at 37°C for 1 h, and then either further processed or frozen at -20°C. After testing a range of 1.0-10.0-fold molar excess of ribozyme over mRNA substrate, a 2.5-fold molar excess of ribozyme over mRNA was found to be optimal.

[0221] 1.5. mRNA cleavage by RNase H RNase H cleavage assays were performed by annealing a 5-fold molar excess of RNase H probe to the mRNA substrate by incubating at 92°C for 2 min, followed by stepwise cooling (65°C for 2 min, 55°C for 2 min, and 40°C for 2 min) in a buffer containing 50 mM Tris and 100 mM NaCl. After annealing, 125 μM RNase H (New England Biolabs) and 10 mM MgCl2 were added to the reaction mixture, followed by incubation at 37°C for 1 h. Reactions were either further processed or frozen at -20°C.

[0222] 1.6. Purification of short truncated RNA fragments using silica-based columns Cleaved short RNA fragments from the mixture of cleaved or uncleaved long RNA fragments present in the ribozyme or RNase H-mediated cleavage reaction were purified by adapting the procedure for RNA isolation using the RNeasy Mini Kit (Qiagen). First, 100 μl of the cleavage reaction mixture was mixed with 350 μl of RLT buffer (lysis buffer from the RNeasy Mini Kit) and 250 μl of 100% ethanol and applied to the column. Long RNAs, including uncleaved full-length mRNA and long 3' cleavage fragments, remained on the column, whereas short 5' cleavage products were collected in the flow-through fraction. Next, 50 μl of RLT buffer and 500 μl of 100% ethanol were added to the collected flow-through fraction (700 μl) and the mixture was applied successively to a second silica column in two aliquots of 625 μl with intervening centrifugations at 9,600 × g for 15 s in a Heraeus Fresco 17 Centrifuge (Thermo Scientific) while maintaining the temperature at 22 °C-23 °C throughout all centrifugation steps. Under these conditions, the short 5' cleavage products and the ribozymes bound to the column were washed with 500 μl of RPE buffer (wash buffer from the RNeasy Mini Kit) and centrifuged at 9,600 × g for 15 s, followed by a washing step with 500 μl of 100% ethanol and centrifugation at 9,600 × g for 2 min. To remove the remaining ethanol, the column was transferred to a clean collection tube and centrifuged at 17,000 × g for 1 min. The column was then transferred to a clean 1.5 ml tube and the short RNA fragments were eluted by adding 30 μl of RNase-free water to the column, followed by centrifugation at 13,800×g for 1 min. Typically, 20 μg of RNA was purified per column for RNAs <5 kb long and 60 μg of RNA for RNAs >5 kb long.

[0223] 1.7. Purification of cleaved short fragments by elution from polyacrylamide gels As a second option, purification of the cleaved short fragments was performed according to a method modified from Nilsen 2013

[22] , whereby they were separated and then eluted from a 21% polyacrylamide gel prepared with 8 M urea. Samples were separated in two minigels run in parallel in a Bio-Rad Mini-Protean Tetra Cell in Tris-borate-EDTA (TBE) buffer. The first gel was diluted 1:10,000 in TBE buffer and stained with SYBR® Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific S11494) used as a reference, and the corresponding bands of interest were excised from a second unstained gel run in parallel. The excised gel fragments were transferred to 400 μl of elution buffer (20 mM Tris-HCl, 3 M sodium acetate, 1 mM EDTA, 0.25% SDS), frozen on dry ice for 15 min, and stored at room temperature overnight to release the RNA into solution. After centrifugation at 17000×g for 10 min, the supernatant contained the RNA, which was extracted with an equal volume of acid-phenol / chloroform followed by chloroform. Isopropanol precipitation was performed and the recovered RNA was dissolved in 10 μl of RNase-free water.

[0224] 1.8. Visualization and analysis of cleaved short fragments by PAGE RNA fragments cleaved by ribozymes or RNase H were diluted 1:1 in Gel Loading Buffer II (Ambion) 1) immediately after cleavage, 2) after cleavage and silica column purification, or 3) after purification from gel as described above. For optimal loading, reaction volumes were constant between samples, and the concentration of purified short RNA fragments was 10–70 ng / μl. RNA samples were denatured at 65°C for 10 min, then separated for 2–2.5 h using 21% PAGE, 8 M urea in TBE buffer, stained with SYBR® Gold, and visualized by ultraviolet light using a Gel Doc EZ system (Bio-Rad). Images were analyzed using the Volume tool in Image Lab 5.0 software (Bio-Rad). Images of RNA bands corresponding to 1) the slower moving capped 5' cleavage product RNA, 2) the one nucleotide shorter and faster moving uncapped 5' cleavage product RNA, and 3) the background in the same region were selected. To quantify the capping efficiency, the relative intensity of the bands corresponding to capped and uncapped RNA was measured for each RNA sample and background values ​​were subtracted. The total intensity of the two bands combined was considered as 100%. The calculated value of the band corresponding to capped RNA represents the capping efficiency.

[0225] 1.9. LC-MS analysis of ribozyme cleavage products LC-MS was performed using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters) coupled to a Xevo TQ-S mass spectrometer (Waters) equipped with an electrospray source operated in negative ionization mode. MS spectra were acquired over the range m / z 400–2,000. All samples were chromatographed on an Acquity Beh C18 column (Waters, 2.1 × 50 mm; 1.7 μm particle size) at a column temperature of 60 °C. Analytes were separated with a gradient of 16.6 mM triethylamine (TEA; VWR), 100 mM hexafluoroisopropanol (HFIP; Sigma) and 10% methanol, Ultra LC-MS grade (Carl Roth) as buffer A and 16.6 mM TEA, 100 mM HFIP and 95% methanol as buffer B at a flow rate of 0.3 ml / min. The gradient applied for oligonucleotides >20 nt in length was 0% buffer B in 1.5 min, 0 to 7% in 3.5 min, 7 to 15% in 6.25 min, and 15 to 40% in 4.5 min.

[0226] 2.Results 2.1. Ribozyme assay to quantify IVT mRNA capping efficiency Human α-globin (hAg) and TEV 5' untranslated region (5'UTR) are among the most widely used 5'UTRs for therapeutic IVT mRNAs [16, 23, 24]. We designed ribozymes targeting the 5'UTR sequence of hAg or TEV (Table 4, see Methods section). All five ribozymes designed here form well-described hammerhead structures and were predicted to cleave target RNAs after defined nucleotide triplets [19, 25]. The ribozymes cleave most efficiently after AUC or GUC triplets and can also target other triplets, but with reduced cleavage efficiency: GUA, AUA, CUC>AUU, UUC, UUA>GUU, CUA>UUU, CUU

[21] . Rz1, Rz3 and Rz4 were designed to cleave after GUC, and Rz2 to cleave after GUA. Rz5 contained an inosine (I) that allows recognition and cleavage after the ACA triplet in the TEV 5′UTR [ 21 ].

[0227] [Table 4] Recognition sequences are underlined; cleavage sites are indicated as N>; CP, cleavage product; hAg, human alpha globin; TEV, tobacco etch virus; Rz, ribozyme; 5'UTR, 5' untranslated region.

[0228] Each of the ribozyme-mediated cleavage reactions produced short capped and uncapped RNA 5' cleavage products that differed from each other only in length due to the cap structure, with the capped RNA being exactly one nucleotide longer. The ribozymes were designed to cleave products 10-30 nt long from the target RNA, while the capped RNA was 35-47 nt long, allowing for separation and distinction of the 5' cleavage products from the ribozyme (Table 4). The Rz2 and Rz5 sequences complementary to the target RNA also contained 2'-O-methylated nucleotides to enhance cleavage by increasing the stability of the double-stranded structure formed.

[0229] To quantify the capping efficiency, Rz was annealed to the mRNA substrate (Figure 1). ++ In the presence of ions, the 5' end of the IVT mRNA is cleaved, resulting in a mixture of short capped and / or uncapped 5' cleavage products, long 3' cleavage products, long uncleaved RNA, and Rz. Using optimized conditions, the short Rz cleavage fragments are purified using a silica-based MinElute Qiagen column and then visualized using 21% PAGE, 8M urea or LC-MS analysis (Figure 1). In this case, the 5' cleavage products are purified along with the ribozyme for further visualization / quantification. Purification of the 5' cleavage products is necessary for LC-MS analysis and also improves visualization of the RNA using 21% PAGE, 8M urea.

[0230] An alternative purification approach was performed on the PAGE-separated samples by extracting and eluting the 5' cleavage products from the gel (see Methods section, data not shown). This process results in the removal of both the uncleaved and 3'-cleaved RNA products from the mixture as well as the ribozyme, which may be an advantage if LC-MS analysis is planned using overlapping Rz and 5' cleavage products. However, this purification step is experimentally time-consuming and cannot be easily scaled up. Using this method, it is possible to purify 2-4 samples in parallel in 2 days, whereas the use of silica-based columns allows the purification of 12 samples in parallel in less than 1 day with the option of scaling up.

[0231] Here, we describe an assay to assess capping efficiency, which consists of a ribozyme cleavage reaction, purification of the cleaved fragments, and visualization of capped and uncapped products using 21% PAGE, 8M urea, or LC-MS analysis.

[0232] 2.2. Optimization of Ribozyme Cleavage Assay To identify the optimal molar ratio of Rz to RNA substrate for the cleavage reaction, a 112-nt long U- or m1Ψ-containing mRNA substrate was chosen. The use of the aforementioned short substrates allowed the detection and differentiation of uncleaved RNA, 5' and 3' cleavage products, and Rz separated on the same gel. Figure 2 shows the 112-nt long uncleaved RNA, 22 and 90 nt long 5' and 3' cleavage products, and 39 nt long Rz1 detected using 21% PAGE, 8 M urea. Increasing the molar concentration of Rz over the RNA substrate was tested for both U- and m1Ψ-containing RNA. A 2.5-fold molar excess of Rz over the RNA substrate was chosen and used for all subsequent experiments.

[0233] For further optimization, various temperature settings during the cleavage reaction were tested. Reactions were carried out at 25°C, 37°C and 50°C with Rz1, Rz3 and Rz4 as detailed in section 1.4.

[0234] In this experiment, temperature did not have a significant effect on Rz cleavage, and the same capping efficiency results were obtained using different temperature settings. However, reactions performed at 50°C resulted in more significant degradation (data not shown). Takagi et al. and Sawata et al. found that below 25°C, the product dissociation step became rate-limiting, and at a temperature of 37°C, no burst kinetics were detected, and ribozyme chemical cleavage was the rate-limiting step [26, 27]. Considering their findings and ours, cleavage reactions at 37°C were used in subsequent experiments.

[0235] 2.3.2 Quantification of capping efficiency after visualization of 5' cleavage products using 1% PAGE, 8M urea To measure capping efficiency, short capped and uncapped 5' cleavage products were visualized using 21% PAGE, 8M urea. As a proof-of-principle experiment, cleavage reactions were performed using mRNA encoding erythropoietin (EPO). GCG transcription start site (TSS) and hAg 5'UTR-containing IVT mRNA was cleaved using Rz1 and Rz2. GGA TSS and TEV 5'UTR-containing IVT mRNA encoding EPO was cleaved using Rz5. The following U- and m1Ψ-containing RNAs were tested: uncapped RNA (-), enzymatically capped RNA without 2'-O-methylation (E0), enzymatically capped and 2'-O-methylated RNA (E1), or ARCA cotranscriptionally capped RNA (A0). Using 21% PAGE, 8M urea, bands representing the ribozyme (Rz) and short capped and uncapped 5' cleavage products were detected at the expected sizes (Figure 3).

[0236] 3'-end cleavage products or uncleaved long RNAs were observed in 21% PAGE, 8M urea gels after ribozyme-mediated cleavage reactions without a silica-based column purification step. Depletion of long RNA fragments and an increase in signals representing short capped and uncapped 5' cleavage products were observed after purification using a silica-based column. As expected, visualization with 21% PAGE, 8M urea showed the presence of bands at the bottom of the gel in all uncapped control (-) RNA samples. Furthermore, in all enzymatically capped (E0 and E1) samples, capped high intensity bands were observed in the majority of cases, whereas uncapped bands were not observed or had lower intensity, indicating high capping efficiency of enzymatically capped RNA. In contrast, both capped and uncapped bands were detected with comparable intensity in ARCA (A0) samples, regardless of the RNA or Rz used.

[0237] Approximately 84-100% capping efficiency was detected for all 12 enzymatically capped (E0 and E1) samples, independent of the 5' end and ribozyme used, whereas the ARCA (A0) samples showed capping efficiencies of 34-53% for the hAg 5'UTR and 67-77% for the TEV 5'UTR-containing RNA (Table 5). When comparing the % capping efficiency of purified E0 RNA and the corresponding 2'-O-methylated E1 RNA, a difference of + / - 1% was observed in 5 out of 6 cases, indicating high reproducibility of the assay.

[0238] [Table 5] enzymatically capped without 2'-O-methylation (E0), enzymatically capped and 2'-O-methylated (E1), ARCA cotranscriptionally capped (A0), unmodified / uridine-containing (U), m1Ψ-containing (m1Ψ), hAg, human α-globin; TEV, tobacco etch virus.

[0239] Capping efficiency of silica column purified RNA was consistently less variable when compared to ribozyme-cleaved RNA that was not silica column purified. In 10 out of 12 cases using different batches of purified RNA, both E0 and E1 showed capping efficiencies of 90-96%, whereas for the same RNA that was not silica column purified, the observed capping efficiency range was 88-100% (Table 5). Thus, purification using silica columns is recommended as a standard part of the procedure, not only for LC-MS analysis, but also for visualization and quantification of capping efficiency using 21% PAGE, 8 M urea.

[0240] 2.4. Capping assay by ribozyme-mediated cleavage effectively evaluates the capping efficiency of variably capped IVT mRNAs of different lengths To test whether ribozyme-mediated cleavage could detect capping of IVT mRNAs of different lengths, Rz5 cleavage reactions were performed (without modification of the method described above) on five IVT mRNAs ranging from 1.1 kb to 9.4 kb in length. The 1.1 kb-long β-S-ARCA (D1)-capped TEV 5'UTR, m1Ψ-containing RNA showed a capping efficiency of 61%, whereas the 2.3-9.4 kb-long CleanCap® Reagent AG (3'OMe), cap 1 (CC1) RNAs showed capping efficiencies of 81-92% (Figure 4). No correlation was observed between capping % and RNA length. The method described herein successfully evaluated the capping efficiency of IVT mRNAs of different lengths capped using diverse cap structures.

[0241] 2.5. Ribozyme-mediated cleavage assay detects increased capping efficiency following further enzymatic capping of cotranscriptionally capped RNA To further test the performance of the ribozyme-mediated cleavage assay, the co-transcriptionally capped D1 U-containing GCG TSS hAg 5'UTR IVT mRNA was subsequently subjected to enzymatic capping (E1). The co-transcriptionally D1 capped IVT mRNA initially exhibited a capping efficiency of 67%, but after subsequent E1 capping, the capping efficiency increased to 94% (Figure 5). This finding confirms that a significant portion of the 5' cleavage products at D1 are in fact uncapped and do not potentially represent T7 RNA polymerase skipping the first G of the transcription start, resulting in the same RNA fragment that is 1 nt shorter than the capped fragment and equal in size to the uncapped fragment.

[0242] 2.6. Superior performance of the ribozyme-mediated cleavage assay over the RNase H cleavage assay To compare ribozyme-mediated and RNase H-mediated cleavage assays, we developed six RNase H probes that can anneal to the hAg 5'UTR sequence. RNase H cleavage reactions were performed as described in the Methods section, and the probes were screened (data not shown). A probe (P1) containing a stretch of six DNA nucleotides (dNs) and ten 2'-O-methylated RNA nucleotides was selected for superior performance (Table 6).

[0243] [Table 6] DNA nucleotides in bold, dN; m, 2'-O-Met; hAg, human α-globin.

[0244] The 5'-ends of hAg GCG-initiated enzymatically capped (E0 or E1) or cotranscriptionally ARCA-capped (A0) U-containing and m1Ψ-containing RNAs were RNase H cleaved (Figure 6). RNase H cleavage confirmed the high capping efficiency for enzymatic RNAs previously detected by ribozyme-mediated cleavage (purified hAg GCG: 75-85%). For ARCA samples, a considerably lower capping efficiency of 37-47% was obtained (Figure 6, Table 7), which is consistent with the data presented in section 2.3.

[0245] In contrast to ribozyme-mediated cleavage, RNase H cleavage resulted in additional bands. The bands were 1 nt longer (21 or 22 nt long) and appeared adjacent to or overlapping with the expected short RNA fragments of 20 nt and 21 nt corresponding to the uncapped and capped enzymatic 5' cleavage products, respectively, in all U-containing samples, including uncapped RNA (Figure 6). This finding indicates that RNase H cleaves at two positions: the expected position after the fourth DNA nucleotide and with lower efficiency at the position after the fifth DNA nucleotide, thereby complicating the capping efficiency analysis of U-containing samples (Figure 6). Furthermore, in all RNase H-cleaved samples, a large spread of nonspecific long RNA fragments was observed that were not present in the no-enzyme control (Figure 6). These nonspecific long RNA fragments of various sizes could not be depleted by silica-based column purification. Their presence made the quantification of capping efficiency from 21% PAGE, 8M urea gels less reproducible, which may result in complex LC-MS analysis.

[0246] Thus, RNase H cleavage using a specific probe can be used to quantify capping efficiency using 21% PAGE, 8M urea. However, in contrast to RNase H cleavage, ribozyme-mediated cleavage results in a single-site cleavage and does not result in a smear of nonspecific long fragments, allowing reliable quantification using 21% PAGE, 8M urea or LC-MS analysis.

[0247] [Table 7] TIFF2025503044000022.tif21153Enzymatically capped without 2'-O-methylation (E0), enzymatically capped and 2'-O-methylated (E1), ARCA cotranscriptionally capped (A0), unmodified / uridine-containing (U), m1Ψ-containing (m1Ψ); hAg, human α-globin.

[0248] 2.7. LC-MS Analysis of Capping Efficiency Using Ribozyme-Mediated Cleavage Assay As proof of principle, LC-MS analysis was performed on ribozyme-cleaved and silica-based column purified short 5' cleavage products from enzymatically capped 2'-O-methylated (E1) or CleanCap® Reagent AG(3'OMe)(CC1)AGG TSS, hAg 5'UTR, m1Ψ-containing IVT mRNA. UPLC and MS profiles of E1-capped IVT mRNA (Figure 7) showed the following: 68% of predicted major products (7MeGpppA(Ome)GGCGAACU*AGU*AU*U*CU*U*CU*GGU*C>p), 20% unmethylated products (7MeGpppAGGCGAACU*AGU*AU*U*CU*U*CU*GGU*C>p), · and 1% further product (+G).

[0249] The detection of 89% E1-capped product (for AGG TSS IVT mRNA) is consistent with the 95-96% detected for E1-capped RNA using 21% PAGE, 8 M urea; furthermore, different batches and GCG TSS RNA were used for PAGE.

[0250] The UPLC and MS profile of CleanCap® Reagent AG(3'OMe), N-7413 TriLink(CC1)EPO mRNA (Figure 8) showed >99% capping, which is also consistent with the >90% capping efficiency obtained by screening >100 CC1-capped IVT mRNAs using 21% PAGE, 8M urea (Figure 4, data not shown). Thus, the ribozyme-mediated cleavage assay combined with silica-based column purification is highly compatible with LC-MS analysis. As shown here, application to LC-MS creates further possibilities for characterizing the 5' cleavage products, such as determining the methylation status or identifying low abundance capped products.

[0251] 3. Discussion Quality control of mRNA vaccines and therapeutics is necessary at every stage of development from preclinical trials to clinical application, as well as to support marketing approval. The cap structure on the mRNA molecule determines the mRNA translation and thus correlates with the therapeutic efficacy of the mRNA [7-9]. In the process of mRNA production, capping of mRNA can be performed enzymatically or co-transcriptionally. Both strategies result in a mixture of capped and uncapped mRNA molecules. In this study, we developed an assay to detect capping efficiency based on a ribozyme-mediated cleavage reaction. After this reaction, the next step is silica-column purification and visualization and quantification using 21% PAGE, 8 M urea or LC-MS. Visualization using 21% PAGE, 8 M urea allows for the analysis of a large number of mRNA samples in parallel without the need for specific equipment. Moreover, the method described here is compatible with LC-MS, allowing a detailed characterization in which not only the percentage of capping but also the methylation status and potential minor capped by-products can be detected from the same sample.

[0252] As discussed previously, current approaches to assess capping efficiency have limitations, such as the need for radioactive labeling [10–12] or rather insensitive detection of capping levels

[14] . Beverly et al. developed an assay to assess capping efficiency based on RNase H cleavage of a specific biotin-labeled probe followed by purification with streptavidin-coated magnetic beads and LC-MS analysis

[13] . Here, we directly compared our developed ribozyme-mediated cleavage assay targeting the hAg 5'UTR with an RNase H cleavage assay designed for the same 5'UTR. As expected, the ribozyme cleaved only at one position in the IVT mRNA, and after silica-based purification, only three short RNA fragments (the ribozyme and the capped and uncapped 5' cleavage products) were observed on the gel, allowing accurate quantification. In contrast, RNase H cleaved at two positions, resulting in the capped and uncapped 5' cleavage products as well as an additional unexpected band 1 nt longer that also appeared in the uncapped RNA control sample. This made quantification using the RNase H assay cumbersome. These results are consistent with the observations of Beverly et al., who also reported two cleavage sites after RNase H cleavage and biotin labeling analysis

[13] . Furthermore, we observed that RNase H cleavage also produced a large number of nonspecific long cleaved RNA fragments that would be expected to adversely affect quantification. We conclude that the ribozyme cleavage-based assay developed in this study shows significant advantages compared to other assays for capping detection.

[0253] The design of the ribozymes used in these assays to quantify capping efficiency depends on the 5'-terminal sequence of the target mRNA. Thus, the ribozyme cleavage site is required in a structurally accessible region located 10-30 nt from the 5'-end of the mRNA. In this study, hammerhead ribozymes were designed to cleave after GUC (Rz1, Rz3, Rz4), GUA (Rz2) or ACA (Rz5) triplets. In addition to such triplets, other NUH triplets (H stands for A, C or U) that can be targeted by hammerhead ribozymes can also be used (NUH cleavage efficiency, in descending order, is AUC>AUA, CUC>AUU, UUC, UUA>GUU, CUA>UUU, CUU

[21] ). Incorporation of an inosine in the ribozyme recognition sequence (as in Rz5) allows additional targeting of NCH triplets (e.g. ACA). The possibility of targeting both the canonical NUH triplet and the non-canonical NCH site significantly improves the versatility of this assay by allowing the selection of the most accessible cleavage site within the 5'UTR

[21] . Using the assay developed and optimized here, we were able to reproducibly quantify the capping efficiency of U- or m1Ψ-containing IVT mRNAs containing different 5'UTRs and with diverse caps of different lengths. The 89-100% capping efficiency observed after using the vaccinia virus capping enzyme system confirms previous findings of 88-98% capping reported by Beverly et al.

[13] . Furthermore, we show that diverse cap structures can result in relatively consistent and diverse capping efficiencies between different IVT mRNA batches generated using the same cap structure. For example, enzymatic capping and co-transcriptionally capping with CleanCap® Reagent AG(3'OMe) (TriLink) typically yielded capping efficiencies of >90%, whereas co-transcriptional capping with ARCA-G (TriLink) or β-S-ARCA (D1) resulted in lower capping efficiencies ranging from 34 to 77%.

[0254] Taken together, the ribozyme-mediated cleavage assay developed in this study is a useful assay for easy, rapid and reliable analysis of capping efficiency for research and development purposes or as a quality control for hAg and TEV 5'UTR-containing mRNA-based therapeutics. Using the same methods for ribozyme assay design, purification of short fragments, and visualization or quantification by gel electrophoresis or LC-MS, ribozyme assays targeting other 5'UTRs may be developed, allowing for broader applicability in the mRNA therapeutic field.

[0255] Example 2 Two-step silica-based column chromatography of catalytic nucleic acid (e.g. ribozyme) cleaved IVT mRNA Catalytic nucleic acid (e.g. ribozyme) cleaved IVT mRNA mixture is mixed with aqueous buffer, then ethanol (buffer volume >= ethanol volume) is added and applied to a first silica-based column. After the centrifugation step, the uncleaved full-length mRNA and the long RNAs, including the long 3' cleavage fragments, remained on the column, whereas the short 5' cleavage products were collected in the flow-through fraction. · Buffer and ethanol were then added to the collected flow-through fraction (aqueous buffer < ethanol volume) and the mixture was applied to a second silica column. Under these conditions, the short 5' cleavage products bound to the column were washed and centrifuged in multiple steps until eluted from the column. Purification of short 5' end products improves reproducibility of UPLC and / or LC-MS analysis and improves visualization and quantification from polyacrylamide gels.

[0256] Example 3 In an experimental protocol similar to that described in Example 1 above, this example compares the ribozyme used in Example 1 (Rz1) with five other ribozymes, each targeting an NCH cleavage site, e.g., ACA, CCA, CCC. In particular, ribozymes Rz6-Rz10 (SEQ ID NOs: 6-10) and Rz1 were used to cleave a capped RNA molecule (CC1) and an uncapped RNA molecule (ppp). The results shown in Figure 9 demonstrate the efficient cleavage and release of short 5' cleavage products, capped or uncapped, ranging from 10 to 31 nucleotides using ribozymes targeting NCH-type sites in RNA molecules.

Claims

1. 1. A method for analyzing a population of RNA molecules, comprising: (a) contacting a catalytic nucleic acid molecule with a population of RNA molecules comprising one or more RNA molecules comprising a cleavage site for said catalytic nucleic acid molecule and a 5' cap structure under conditions that allow cleavage of said RNA molecules to generate a 5' terminal fragment and at least one 3' fragment, wherein said generated 5' terminal fragment is obtained in a mixture of said at least one 3' fragment, said catalytic nucleic acid molecule and / or uncleaved RNA molecules; (b) at least partially separating the 5'-end fragments obtained in step (a) from the at least one 3'-fragment to obtain a population of 5'-end fragments, (i) step (b) comprises subjecting the mixture obtained in step (a) to PAGE under conditions that allow at least partial separation of the 5'-terminal fragment from the at least one 3'-fragment and / or the uncleaved RNA molecules, and further comprises (i) isolating at least one band of interest from the PAGE gel, wherein the at least one band comprises the 5'-terminal fragment, and (ii) eluting the 5'-terminal fragment from the isolated at least one band obtained in step (i); or (ii) step (b) comprises contacting the mixture obtained in step (a) with oligo-dT nucleotides under conditions that allow at least partial separation of the 5'-terminal fragment from the at least one 3'-fragment and / or uncleaved RNA molecule; process; and (c) determining the amount of RNA molecules having the 5' cap structure in the population of 5'-end fragments obtained in step (b). A method comprising:

2. The method of claim 1, wherein the population of RNA molecules is a population of mRNA molecules, self-replicating RNA, ncRNA and / or sRNA.

3. 3. The method of claim 1, wherein in step (a), the catalytic nucleic acid molecule is contacted with a population of RNA molecules obtained by in vitro transcription or solid-phase synthesis.

4. in the RNA molecule, the cleavage site is located at least 5 nt downstream of the 5' end of the RNA molecule, and / or 3. The method of claim 1 or 2, wherein the cleavage site in the RNA molecule is located up to 50 nt downstream of the 5' end of the RNA molecule.

5. The method of claim 1 or 2, wherein the RNA molecule comprises a 5'UTR.

6. 6. The method of claim 5, wherein the 5'UTR is selected from a human alpha globin (hAg) 5'UTR and a TEV 5'UTR.

7. 3. The method of claim 1 or 2, wherein the RNA molecule comprises at least one cleavage site for the catalytic nucleic acid molecule.

8. the catalytic nucleic acid molecule (i) cleaving at a cleavage site in the 5'UTR sequence, or (ii) cleaving the RNA molecule at a 5'-NUH-3' cleavage site to generate a 5' fragment containing a NUH>p 3' end, wherein: N is selected from G, A, C and U; and H is selected from A, C and U, or (iii) cleaving the RNA molecule at a 5'-NCH-3' cleavage site to generate a 5' fragment containing an NCH>p 3' end, wherein: N is selected from G, A, C and U; and H is selected from A, C and U, or (iv) is a ribozyme or a DNAzyme; or (v) (i) a sequence selected from SEQ ID NOs: 1 to 25; (ii) a sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 25, and / or (iii) a fragment of (i) and / or (ii), wherein the catalytic nucleic acid molecule comprises a catalytic core.

9. 3. The method of claim 1 or 2, wherein the amount of 5'-end fragments as a percentage of all cleaved and uncleaved RNA molecules present in the population obtained after step (b) is greater than the amount of 5'-end fragments as a percentage of all cleaved and uncleaved RNA molecules present in the population before step (b), providing an enriched or at least partially purified population of 5'-end fragments.

10. 3. The method of claim 1 or 2, wherein the catalytic nucleic acid molecule is a hammerhead ribozyme, a hairpin ribozyme, or an HDV ribozyme.

11. The method of claim 1 or 2, wherein the catalytic nucleic acid molecule and / or the RNA molecule comprises at least one modified nucleotide.

12. Am is independently selected from A and 2'-O-methyladenosine; Gm is independently selected from G and 2'-O-methylguanosine; Um is independently selected from U and 2'-O-methyluridine, and / or Cm is independently selected from C and 2'-O-methylcytidine; The method of claim 8.

13. The method of claim 1 or 2, wherein in step (a), the catalytic nucleic acid molecule is contacted with a population of RNA molecules capped by enzymatic capping or / and co-transcriptional capping in the presence of a capping analog.

14. The capping analog is G[5']ppp[5']G,m ７ G[5']ppp[5']G,m ３ ２，２，７ G[5']ppp[5']G, m ２ ７，３’－Ｏ G[5']ppp[5']G (3'-ARCA), m ２ ７，２’－Ｏ GpppG (2'-ARCA), m ２ ７，２’－Ｏ Gpp ｓ pG D1 (β-S-ARCA D1), m ２ ７，２’－Ｏ Gpp ｓ 14. The method of claim 13, wherein the nucleotide sequence is selected from pG D2 (β-S-ARCA D2), m7(3'OMeG)(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG(3'OMe)), and m7G(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG).

15. In step (a), contacting the population of RNA molecules with an excess of catalytic nucleic acid molecules; or 3. The method of claim 1, wherein in step (a), the population of RNA molecules is contacted with the catalytic nucleic acid molecules at a molar ratio of RNA molecules to catalytic nucleic acid molecules of about 1:1 to about 1:

20.

16. The method of claim 1 or 2, wherein the length of the 5'-end fragments allows for discrimination between capped and uncapped 5'-end fragments.

17. The method of claim 16, wherein the capped and uncapped 5'-end fragments differ in length by 1 to 3 nucleotides.

18. the 5'-end fragment obtained in step (a) has a length of at least 5 nt; or The method of claim 1 or 2, wherein the 5'-end fragment obtained in step (a) has a length of up to 50 nt.

19. 3. The method of claim 1 or 2, wherein the oligo-dT nucleotides are bound to plastic or magnetic beads or to biotin.

20. 20. The method of claim 19, wherein the beads form a column.

21. The method of claim 1 or 2, wherein the catalytic nucleic acid molecule is labeled.

22. 22. The method of claim 21, wherein the label is biotin.

23. The method of claim 1 or 2, wherein the catalytic nucleic acid molecule is attached to a surface.

24. 24. The method of claim 23, wherein the surface is a magnetic or plastic bead or particle.

25. 3. The method of claim 1 or 2, wherein the separating step (b) further comprises separating the 5'-terminal fragment from the catalytic nucleic acid molecule under conditions that allow for the at least partial separation of the 5'-terminal fragment from the catalytic nucleic acid molecule.

26. 26. The method of claim 25, wherein said separating comprises contacting said mixture of step (a) with a substance that binds to said labeled catalytic nucleic acid molecule under conditions that allow said at least partial separation of said 5' end fragment from said labeled catalytic nucleic acid molecule.

27. The method of claim 1 or 2, wherein in step (b), the capped 5'-end fragments are not separated from the uncapped 5'-end fragments.

28. 3. The method of claim 1 or 2, wherein steps (b) and (c) are separate steps.

29. 3. The method of claim 1 or 2, wherein the capped 5'-end fragment is 1, 2, or 3 nucleotides longer than the uncapped 5'-end fragment.

30. 3. The method of claim 1 or 2, wherein step (c) comprises gel electrophoresis, spectroscopy, mass spectrometry, liquid chromatography and / or sequencing.

31. 31. The method of claim 30, wherein the gel electrophoresis is PAGE.

32. The method of claim 1 or 2, wherein the amount of RNA molecules having the 5' cap structure is determined in the at least partially purified 5' end fragments obtained in step (b).

33. In step (c), the amount of the capped 5'-end fragments and the uncapped 5'-end fragments is determined; or The method of claim 1 or 2, wherein in step (c), the percentage of capped 5'-end fragments is calculated relative to the total amount of 5'-end fragments.

34. (d) analyzing the cap structure in the capped 5'-end fragment.

3. The method of claim 1 or 2, further comprising:

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