Methods for RNA analysis
12492425 · 2025-12-09
Assignee
Inventors
- Alexander SCHWENGER (Tübingen, DE)
- Stefan Heinz (Tübingen, DE)
- Tilmann Roos (Tübingen, DE)
- Benyamin YAZDAN PANAH (Tübingen, DE)
- Veronika Wagner (Tübingen, DE)
Cpc classification
C12Q2521/30
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention is concerned with methods for analyzing RNA molecules. The provided methods involve conjugates for RNA cleavage comprising a chemical moiety with RNA cleaving activity and an oligonucleotide. The oligonucleotide is designed based on a target sequence present in an RNA molecule, and the cleavage of the RNA molecule is inter alia carried out at conditions allowing the hybridization of the oligonucleotide to the target 5 sequence. Thereby, the method is easily applicable to RNA molecules of any sequence. The method further involves the analysis of the RNA fragments obtained after cleavage to obtain information on the physical properties of the RNA molecule.
Claims
1. A method for analyzing an RNA molecule comprising the following steps: (i) providing an RNA molecule; (ii) providing at least one conjugate comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the sequence of said oligonucleotide is complementary to a target sequence of the RNA molecule; (iii) cleaving the RNA molecule provided in step (i) to obtain RNA fragments by contacting the RNA molecule with the at least one conjugate provided in step (ii) under conditions allowing the hybridization of said oligonucleotide to said target sequence and the cleavage of the RNA molecule; and (iv) determining a physical property of the RNA molecule by analyzing one or more of the RNA fragments obtained in step (iii), wherein the RNA molecule is an mRNA molecule and comprises a 5 cap structure and a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecule comprise one or more temperature shifts, wherein step (iii) of the method is as follows: (iii) cleaving the RNA molecule provided in step (i) to obtain RNA fragments by contacting the RNA molecule with the conjugate provided in step (ii) at a first temperature between about 5 C. and about 50 C., and at a second temperature between about 70 C. and about 90 C.; followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).
2. The method of claim 1, wherein cleaving the RNA molecule results in a 5 fragment, a 3 fragment and optionally one or more central fragments.
3. The method of claim 2, wherein the fragments are separated from each other before analyzing the one or more of the RNA fragments in step (iv).
4. The method of claim 3, wherein the fragments are separated by chromatography.
5. The method of claim 3, wherein the fragments are separated by electrophoresis.
6. The method of claim 3, wherein the 5 fragment is analyzed and/or the 3 fragment is analyzed.
7. The method of claim 6, wherein the 5 fragment is analyzed for one or more of (i) presence and/or integrity of the cap structure, (ii) methylation pattern; and (iii) orientation, by analytical HPLC and/or mass-spectrometry.
8. The method of claim 6, wherein the 5 fragment has a length of 1 to 100 nucleotides.
9. The method of claim 3, wherein the 3 fragment is analyzed.
10. The method of claim 9, wherein the 3 fragment comprises the sequence.
11. The method of claim 10, wherein the 3 fragment is analyzed for its nucleotide composition and/or length by complete hydrolysis of the 3 fragment followed by analysis of the individual nucleotides gained thereby by analytical HPLC and/or mass spectrometry.
12. The method of claim 10, wherein the 3 fragment has a length of 10 to 500 nucleotides.
13. The method of claim 6, wherein the RNA molecule is 300 to 9,000 nucleotides in length.
14. The method of claim 1, wherein the target sequence is present only once in the RNA molecule.
15. A method for analyzing a population of RNA molecules comprising the following steps: (i) providing a population of RNA molecules, wherein the population of RNA molecules comprises at least two different types of RNA molecules, wherein the different types of RNA molecules comprise an identical target sequence; (ii) providing a conjugate comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the sequence of said oligonucleotide is complementary to the target sequence; (iii) cleaving the population of RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugate provided in step (ii) under conditions allowing the hybridization of said oligonucleotide to said target sequence and the cleavage of the RNA molecules; and (iv) determining a physical property of the RNA molecules in the population by analyzing one or more of the RNA fragments obtained in step (iii), wherein the RNA molecules are mRNA molecules and each comprise a 5 cap structure and/or a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecules comprises one or more temperature shifts, wherein step (iii) of the method is as follows: (iii) cleaving the RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugates provided in step (ii) at a first temperature between about 5 C. and about 50 C., and at a second temperature between about 70 C. and about 90 C.; followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).
16. A method for analyzing a population of RNA molecules comprising the following steps: (i) providing a population of RNA molecules, wherein the population of RNA molecules comprises at least two different types of RNA molecules, wherein the different types of RNA molecules comprise different target sequences; (ii) providing at least two conjugates comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the oligonucleotide sequence of each conjugate is complementary to one of the different target sequences; (iii) cleaving the population of RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the at least two conjugates provided in step (ii) under conditions allowing the hybridization of said oligonucleotides to said target sequences and the cleavage of the RNA molecules; and (iv) determining a physical property of the RNA molecules in the population by analyzing one or more of the RNA fragments obtained in step (iii), wherein the RNA molecules are mRNA molecules and each comprise a 5 cap structure and/or a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecules comprises one or more temperature shifts, wherein step (iii) of the method is as follows: (iii) cleaving the RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugates provided in step (ii) at a first temperature between about 5 C. and about 50 C., and at a second temperature between about 70 C. and about 90 C.; followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).
17. The method of claim 4, wherein the chromatography is HPLC.
18. The method of claim 4, wherein the chromatography is affinity chromatography.
19. The method of claim 6, wherein the sequence of said oligonucleotide is complementary to the target sequence of the RNA molecule over a length of 5-25 nucleotides.
20. The method of claim 12, wherein the wherein the 3 fragment has a length 50 to 250 nucleotides.
21. The method of claim 20, wherein the RNA molecule is 300 to 9,000 nucleotides in length.
22. The method of claim 21, wherein the sequence of said oligonucleotide is complementary to the target sequence of the RNA molecule over a length of 5-25 nucleotides.
23. The method of claim 22, wherein the 3 fragment is analyzed to determine PolyA length.
24. The method of claim 23, wherein the chemical moiety with RNA cleaving activity is tris (2-aminobenzimidazol), 1H-Imidazo[1,2-a]imidazole, 5H-Benzimidazo[1,2-a]benzimidazol, Hexahydro-2H-pyrimido[1,2a]pyrimidin-2,8-dion, 2-Aminobenzimidazol, Imidazo[1,2-a]benzimidazol, or 2-Aminochinolin.
25. The method of claim 24, wherein the chemical moiety with RNA cleaving activity is tris(2-aminobenzimidazole).
26. The method of claim 6, wherein the chemical moiety with RNA cleaving activity is tris(2-aminobenzimidazol), 1H-Imidazo[1,2-a]imidazole, 5H-Benzimidazo[1,2-a]benzimidazol, Hexahydro-2H-pyrimido[1,2a]pyrimidin-2,8-dion, 2-Aminobenzimidazol, Imidazo[1,2-a]benzimidazol, or 2-Aminochinolin.
27. The method of claim 26, wherein the chemical moiety with RNA cleaving activity is tris(2-aminobenzimidazole).
28. The method of claim 15, wherein the chemical moiety with RNA cleaving activity is tris(2-aminobenzimidazol), 1H-Imidazo[1,2-a]imidazole, 5H-Benzimidazo[1,2-a]benzimidazol, Hexahydro-2H-pyrimido[1,2a] pyrimidin-2,8-dion, 2-Aminobenzimidazol, Imidazo[1,2-a]benzimidazol, or 2-Aminochinolin.
29. The method of claim 28, wherein the chemical moiety with RNA cleaving activity is tris(2-aminobenzimidazole).
30. The method of claim 16, wherein the chemical moiety with RNA cleaving activity is tris(2-aminobenzimidazol), 1H-Imidazo[1,2-a]imidazole, 5H-Benzimidazo[1,2-a]benzimidazol, Hexahydro-2H-pyrimido[1,2a]pyrimidin-2,8-dion, 2-Aminobenzimidazol, Imidazo[1,2-a]benzimidazol, or 2-Aminochinolin.
31. The method of claim 30, wherein the chemical moiety with RNA cleaving activity is tris(2-aminobenzimidazole).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.
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DEFINITIONS
(19) For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.
(20) As used in the specification and the claims, the singular forms of a and an also include the corresponding plurals unless the context clearly dictates otherwise.
(21) The term about in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of +10% and preferably +5%.
(22) It needs to be understood that the term comprising is not limiting. For the purposes of the present invention, the term consisting of is considered to be a preferred embodiment of the term comprising. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.
(23) The term nucleic acid means any DNA- or RNA-molecule and is used synonymous with polynucleotide. An oligonucleotide is a polynucleotide of a defined length, usually of a length of about 5 to about 100 nucleotides, but not limited thereto.
(24) The term DNA is the usual abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate and deoxy-cytidine-monophosphate monomers or analogs thereof which areby themselves-composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure. The backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the DNA-sequence. DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.
(25) The term RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers or analogs thereof, which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA-sequence. The term RNA generally refers to a molecule or to a molecule species selected from the group consisting of long-chain RNA, coding RNA, non-coding RNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), RNA oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, riboswitches, immunostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), circular RNA (circRNA), and a Piwi-interacting RNA (piRNA). Preferred in the context of the invention is any type of therapeutic RNA. mRNAs as defined in the following are particularly preferred for the present invention. Therapeutic RNA is to be understood as relating to RNA that is suitable for use in the human or animal body for a medical purpose, i.e. it has a clinical grade, particularly when it comes to parameters such as purity, integrity, as well as concerning the underlying production methods that must comply with (c) GMP conditions.
(26) The term messenger RNA (mRNA) refer to one type of RNA molecule. In vivo, transcription of DNA usually results in the so-called premature RNA which has to be processed into so-called messenger RNA, usually abbreviated as mRNA. Processing of the premature RNA, e.g. in eukaryotic organisms, comprises a variety of different posttranscriptional modifications such as splicing, 5-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of mRNA. The mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5 cap, a 5UTR, an open reading frame, a 3UTR and a poly(A) or a poly(C) sequence. In the context of the present invention, an mRNA may also be an artificial molecule, i.e. a molecule not occurring in nature. This means that the mRNA in the context of the present invention may, e.g., comprise a combination of a 5UTR, open reading frame, 3UTR and poly(A) sequence, which does not occur in this combination in nature.
(27) The term population of RNA molecules or RNA population as used herein refers to a plurality of RNA molecules comprised in one mixture or composition. Preferred in the context of the invention is a population of RNA molecules or RNA population involving any type of therapeutic RNAs.
(28) The term RNA in vitro transcription relates to a process wherein RNA is synthesized from a DNA template in a cell-free system (in vitro). DNA, preferably a linear DNA (e.g. linearized plasmid DNA, linearized dbDNA), is used as a template for the generation of RNA transcripts. A DNA template for RNA in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for RNA in vitro transcription, e.g. into plasmid DNA. Modified nucleotides may be incorporated during RNA in vitro transcription of the RNA.
(29) The term 3-untranslated region (3-UTR) as used herein refers to the part of an mRNA which is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the 3 terminus of the mRNA. In the context of the invention, the term 3-UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g. a poly(A) sequence (or poly(A) tail). A 3-UTR of the mRNA is not translated into an amino acid sequence. The 3-UTR sequence is generally encoded by the gene, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which is then further processed into mature mRNA in a maturation process. A 3-UTR corresponds to the sequence of a mature mRNA, which is located between the stop codon of the protein coding region, preferably immediately 3 to the stop codon of the protein coding region, and the poly(A) sequence of the mRNA.
(30) The term 5-untranslated region (5-UTR) as used herein refers to a particular section of messenger RNA (mRNA). It is located 5 of the open reading frame of the mRNA. Typically, the 5-UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites. The 5-UTR may be post-transcriptionally modified, for example by addition of a 5 cap structure. In the context of the present invention, the term 5-UTR typically refers to the sequence of an mRNA, which is located between the 5 cap structure and the start codon. Preferably, the 5-UTR is the sequence which extends from a nucleotide located 3 to the 5 cap structure to a nucleotide located 5 to the start codon of the protein coding region.
(31) The term 5-cap structure as used herein refers to a modified nucleotide, particularly a guanine nucleotide, added to the 5 end of an RNA molecule. The 5 cap may be added using a 5-5-triphosphate linkage. A 5 cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5 nucleotide of the nucleic acid carrying the 5 cap, typically the 5-end of an RNA. The naturally occurring 5 cap is m7GpppN. Further examples of 5cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4, 5 methylene nucleotide, I-(beta-D-erythrofuranosyl) nucleotide, 4-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3,4-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3-3-inverted nucleotide moiety, 3-3-inverted abasic moiety, 3-2-inverted nucleotide moiety, 3-2-inverted abasic moiety, 1,4-butanediol phosphate, 3-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3-phosphate, 3phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. Examples of 5 cap structures are cap1 (additional methylation of the ribose of the adjacent nucleotide of m7G), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7G), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7G), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7G).
(32) The term cap analogue as used herein will be recognized and understood by the skilled person, and is e.g. intended to refer to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of a nucleic acid molecule, particularly of an RNA molecule, when incorporated at the 5-end of the nucleic acid molecule. Non-polymerizable means that the cap analogue will be incorporated only at the 5-terminus because it does not have a 5 triphosphate and therefore cannot be extended in the 3-direction by a template-dependent polymerase, particularly, by template-dependent RNA polymerase. Examples of cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g. GpppG); dimethylated cap analogue (e.g. m2,7GpppG), trimethylated cap analogue (e.g. m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g. m7Gpppm7G), or anti reverse cap analogues (e.g. ARCA; m7,2OmeGpppG, m7,2dGpppG, m7,3OmeGpppG, m7,3dGpppG and their tetraphosphate derivatives). Further cap analogues have been described previously (WO2008/016473, WO2008/157688, WO2009/149253, WO2011/015347, and WO2013/059475). Further suitable cap analogues in that context are described in WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789, WO2017/053297, WO2017/066782, WO2018075827 and WO2017/066797 wherein the disclosures referring to cap analogues are incorporated herewith by reference.
(33) Modified cap1 structures may be generated using tri-nucleotide cap analogue as disclosed in WO2017/053297, WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789, WO2017/066782, WO2018075827 and
(34) WO2017/066797. In particular, any cap structures derivable from the structure disclosed in claim 1-5 of WO2017/053297 may be suitably used to co-transcriptionally generate a modified cap1 structure. Further, any cap structures derivable from the structure defined in claim 1 or claim 21 of WO2018075827 may be suitably used to co-transcriptionally generate a modified cap1 structure.
(35) Preferred cap-analogues are the di-nucleotide cap analogues m7G (5) ppp (5) G (m7G) or 3-O-Me-m7G (5) ppp (5) G to co-transcriptionally generate cap0 structures. Further preferred cap-analogues are the tri-nucleotide cap analogues m7G (5) ppp (5) (2OMeA) pG or m7G (5) ppp (5) (2OMeG) pG to co-transcriptionally generate cap1 structures.
(36) 5-cap structures may also be formed via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes and/or cap-dependent 2-O methyltransferases) to generate cap0 or cap1 or cap2 structures. The 5-cap structure (cap0 or cap1) may be added using immobilized capping enzymes and/or cap-dependent 2-O methyltransferases using methods and means disclosed in WO2016/193226.
(37) The terms poly(A) sequence, poly(A) tail or 3-poly(A) tail as used herein will be recognized and understood by the skilled person, and are e.g. intended to be a sequence of adenosine nucleotides, typically located at the 3-end of an RNA, of up to about 1000 adenosine nucleotides. A poly(A) sequence is essentially homopolymeric, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides has essentially the length of 100 nucleotides. A poly(A) sequence may also be interrupted by at least one nucleotide different from an adenosine nucleotide, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide different from an adenosine nucleotide). A poly(A) sequence may also be segmented, e.g. may comprise more than one homopolymeric stretches of A nucleotides (e.g. at least 30A) and at least one spacer element (also comprising nucleotides different from an adenosine nucleotide). A poly(A) sequence, suitable located downstream of the 3 UTR as defined herein, may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides. A poly(A) sequence comprises typically about 50 to about 250 adenosines. A poly(A) sequence may be obtained from a DNA template during RNA in vitro transcription. A poly(A) sequence may also be obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA template. Alternatively, poly(A) sequences may be generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols known in the art, or alternatively, by using immobilized poly(A) polymerases e.g. using a methods and means as described in WO2016/174271.
(38) The term poly(C) sequence as used herein will be recognized and understood by the skilled person, and are for example intended to be a sequence of cytosine nucleotides, typically located at the 3-end of an RNA, of up to about 200 cytosine nucleotides. A poly(C) sequence, suitable located at the 3 terminus downstream of the 3 UTR as defined herein, comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. A poly(C) sequence in the RNA sequence of the present invention may be derived from a DNA template by RNA in vitro transcription. Alternatively, poly(C) sequences may be obtained in vitro by common methods of chemical synthesis, or enzymatically, without being necessarily transcribed from a DNA template.
(39) The term modified nucleotides as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to comprise nucleotides that comprise a modification. For example, any nucleotide different from G, C, U, T, A may be regarded as modified nucleotide. Such modified nucleotides may be incorporated during RNA in vitro transcription of the RNA (e.g. by using pseudouridine (w), N1-methylpseudouridine (m1w), or 5-methylcytosine, and 5-methoxyuridine instead of uracil in the nucleotide mixture of the transcription reaction). Modified nucleotides known in the art comprise 2-amino-6-chloropurineriboside-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-Methyl-inosine-5-triphosphate 4-thiouridine-5-triphosphate, 5-aminoallylcytidine-5-triphosphate, 5-aminoallyluridine-5-triphosphate, 5-bromocytidine-5-triphosphate, 5-bromouridine-5-triphosphate, 5-Bromo-2-deoxycytidine-5-triphosphate, 5-Bromo-2-deoxyuridine-5-triphosphate, 5-iodocytidine-5-triphosphate, 5-lodo-2-deoxycytidine-5-triphosphate, 5-iodouridine-5-triphosphate, 5-lodo-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-chloropurineriboside-5-triphosphate, 7-deazaadenosine-5-triphosphate, 7-deazaguanosine-5-triphosphate, 8-azaadenosine-5-triphosphate, 8-azidoadenosine-5-triphosphate, benzimidazole-riboside-5-triphosphate, N1-methyladenosine-5-triphosphate, N1-methylguanosine-5-triphosphate, N6-methyladenosine-5-triphosphate, O6-methylguanosine-5-triphosphate, pseudouridine-5-triphosphate, or puromycin-5-triphosphate, xanthosine-5-triphosphate. Particular preference is given to nucleotides for base modifications 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, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 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-thio-uridine, 1-taurinomethyl-4-thio-uridine, 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, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 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-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, 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-methylthio-N6-threonyl carbamoyladenosine, N6, N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, 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, 5-O-(1-thiophosphate)-adenosine, 5-O-(1-thiophosphate)-cytidine, 5-O-(1-thiophosphate)-guanosine, 5-O-(1-thiophosphate)-uridine, 5-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 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, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 2-O-methyl uridine, pseudouridine (w), N1-methylpseudouridine (m1w), 5-methylcytosine, and 5-methoxyuridine.
(40) The term fragment as used herein refers to a part of an underlying complete RNA molecule. Fragments in the context of the present invention are typically (i) a fragment comprising the 5 part of the underlying RNA molecule, (ii) a fragment comprising the 3 part of the underlying RNA molecule, and (iii) one or more central parts of the underlying RNA molecule.
(41) The term a physical property (or physical properties) as used herein refers to a physical property or to a structural feature of an RNA molecule. Where the plural (physical properties) is used, it may likewise refer to a single property or single feature. Preferably, the expression as used herein refers to a physical property or a structural feature of the RNA molecule, which distinguishes the RNA molecule from other, preferably structurally related, RNA molecule. A physical property or a structural feature may be capable of distinguishing the RNA molecule from a similar, preferably structurally related, RNA molecule lacking the physical property or a structural feature, more preferably from an RNA molecule, which is identical apart from the lacking physical property or the lacking structural feature. Typically, the distinct physical property reflects a structural feature, such as e.g. a distinct molecular weight, charge, specific nucleotide composition or nucleotide modification. As used herein, a physical property or a structural feature may be determined by standard analytical methods known in the art. A physical property or a structural feature may be determined after cleavage of the RNA molecule for one of the obtained fragments. The physical property or structural feature of the fragment obtained by cleavage of the RNA molecule reflects a physical property or a structural feature of the RNA molecule.
(42) The term LNA nucleotide as used herein refers to a modified RNA nucleotide. A LNA nucleotide is a locked nucleic acid. The ribose moiety of an LNA nucleotide may be modified with an extra bridge connecting the 2 oxygen and 4 carbon. This bridge locks the ribose in the 3-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in an oligonucleotide. LNA nucleotides hybridize with DNA or RNA. Oligomers comprising LNA nucleotides are synthesized chemically and are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. The presence of LNA nucleotides significantly increases the hybridization properties (melting temperature) of oligonucleotides.
(43) The term PNA nucleotide as used herein refers to a modified nucleic acid. DNA and RNA have a deoxyribose and ribose sugar backbone. The backbone of PNA is composed of repeating N-(2-aminoethyl)-glycine units and it is linked by peptide bonds. Therefore, PNAs are depicted like peptides, i.e. from N-terminus to C-terminus. PNAs exhibit a higher binding strength. Thus, long PNA oligomers are usually not required. The main concern of the length of the PNA-oligomers is to guarantee the specificity. PNA oligomers also show greater specificity in binding to complementary DNAs, with a PNA/DNA base mismatch being more destabilizing than a similar mismatch in a DNA/DNA duplex. This binding strength and specificity also applies to PNA/RNA duplexes. PNAs are not easily recognized by either nucleases or proteases and PNAs are also stable over a wide pH range.
(44) The term complementary means that a specific sequence is either completely (which may be preferred) or in most parts the complement sequence of an underlying sequence, in the present case of the target sequence. Thus, put in other words, a complement sequence is either 100% identical (which may be preferred) or is identical to a high degree to the complement sequence of an underlying sequence, in the present case of the target sequence. It has been set out above that the sequence of the oligonucleotide is complementary to the target sequence of the RNA molecule to such a degree that the hybridization will take place specifically between the target sequence of the RNA molecule and the oligonucleotide. Accordingly, the sequence of the oligonucleotide is complementary to the target sequence of the RNA molecule to such a degree that no hybridization between a non-target sequence of the RNA molecule and the oligonucleotide takes place. If the target sequence is e.g. 5-GGGAGAAAGCUUACC-3 (SEQ ID NO: 9), then the complement sequence is in the case of a 100% identity 5-GGTAAGCTTTCTCCC-3 (SEQ ID NO: 3). If the sequence has a lower identity and differs e.g. in a single nucleotide, it could e.g. be the sequence of 5-GGTAAGCTTACTCCC-3 (SEQ ID NO:10), which would nevertheless still hybridize specifically to the target sequence and thus be a complement sequence according to the present invention. It is generally preferred that the complement sequence of the oligonucleotide is 100% identical to the complement sequence of the underlying target sequence.
(45) The term hybridization as used herein refers to a single stranded DNA or RNA molecule with a specific sequences annealing to a complement sequence of a DNA or RNA molecule. Single stranded DNA can also hybridize with single stranded RNA to result in a DNA/RNA hybrid. Usually, a double-stranded DNA or RNA or a hybrid is stable under physiological conditions. An increase in temperature will usually cause the two hybridized or annealed strands to separate into single strands. A decrease in temperature causes the single stranded DNA and/or RNA molecules to anneal or hybridize to each other. Hybridization involves the formation of base pairs between A and T (or U) nucleotides and G and C nucleotides of the specific sequence and the complement sequence. Hybridization is usually carried out under stringent conditions, preferably under high stringency conditions. The term high stringency conditions is to be understood such that a specific sequence specifically hybridizes to a complement sequence in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions which distinguish an oligonucleotide with an exact complement sequence, or an oligonucleotide containing only a few mismatched nucleotides (e.g. 1, 2, 3, 4 or 5 mismatched nucleotides), from a random sequence that happens to have a few small complement regions (comprised of e.g. 3 to 4 nucleotides) to the specific sequence. Such small regions of complementarity melt more easily than a longer complement sequence of preferably about 10 to about 25 nucleotides, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50 C. to about 70 C. Such high stringency conditions tolerate little, if any, mismatch between a specific sequence and a complement sequence. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
(46) The term target sequence as used herein corresponds to a specific sequence of the RNA molecule. It may be a specific sequence of the RNA molecule that is present only once in the RNA molecule, such as e.g. the specific sequence of 5-GGGAGAAAGCUUACC-3 (SEQ ID NO: 9). However, the target sequence may also provide for some flexibility, e.g. in that one or more positions in the sequence are not flexible, which is in the above exemplary sequence e.g. 5-GGGAGAWAGCUUACC-3 (SEQ ID NO: 11), where W is A or U. Accordingly, a complement sequence can also be flexible at the one or more positions, see also above. It is generally preferred that the target sequence is a specific sequence without flexibility.
(47) The term chemical moiety with RNA cleaving activity as used herein is defined as a moiety allowing for the hydrolysis of an RNA-phosphodiester bond of an RNA backbone. In principle, the hydrolysis of the RNA backbone may be catalyzed in three different ways: (I) by deprotonation of the 2-OH-group attacking the phosphorus atom as a nucleophile, (II) by protonation of the 5-OH-group acting as a leaving group, or (III) by stabilization of the transitionally formed dianionic phosphorane. Thus, a chemical moiety with RNA cleaving activity should be able to serve as both acid and base catalyst. In the context of the present invention, the term chemical moiety with RNA cleaving activity does not comprise naturally occurring ribonuclease activities of ribozymes, DNAzymes, RNAse, other RNA nucleases etc. Accordingly, the term chemical moiety with RNA cleaving activity has to be understood as an artificial moiety with the capability of cleaving RNA.
(48) The term sequence identity as used herein means that two sequences are identical if they exhibit the same length and order of nucleotides. The percentage of identity typically describes the extent, to which two sequences are identical, i.e. it typically describes the percentage of nucleotides that correspond in their sequence position to identical nucleotides of a reference sequence. For the determination of the degree of identity, the sequences to be compared are considered to exhibit the same length, i.e. the length of the longest sequence of the sequences to be compared. This means that a first sequence consisting of 8 nucleotides is 80% identical to a second sequence consisting of 10 nucleotides comprising the complete first sequence. In other words, in the context of the present invention, identity of sequences preferably relates to the percentage of nucleotides of a sequence, which have the same position in two sequences having the same length.
(49) The term reactor as used herein refers to a vessel wherein a cleavage of an RNA molecule or a population of RNA molecules, optionally combined with a separation, is carried out under specified conditions.
(50) It is noted that the provided methods generally achieve a high cleavage efficiency. Thus, in one embodiment, the method results in cleavage of at least 50% of the RNA molecules. In one embodiment, the method results in cleavage of at least 60% of the RNA molecules. In one embodiment, the method results in cleavage of at least 70% of the RNA molecules. In one embodiment, the method results in cleavage of at least 80% of the RNA molecules. In one embodiment, the method results in cleavage of at least 90% of the RNA molecules. In one embodiment, the method results in cleavage of 95% of the RNA molecules. In one embodiment, the method results in cleavage of at least 99% of the RNA molecules.
Detailed Description of the Findings Underlying the Present Invention
(51) The inventors found that a conjugate comprising a chemical moiety with RNA cleavage activity and an oligonucleotide complementary to a target sequence of an RNA molecule to be analyzed efficiently cleaves an RNA molecule comprising the target sequence. The inventors further found that an RNA molecule can be efficiently cleaved with multiple conjugates at the same time. Further, the derived RNA fragments can subsequently be analyzed for their physical properties. Surprisingly, the conjugates are stable even at high temperatures. This stability allows using the conjugates in methods involving multiple temperature cycles facilitating multiple rounds of hybridization, cleavage and denaturation, thus resulting in a high conversion efficiency. Furthermore, the conjugates may be re-used after separating them from the fragment(s).
(52) Thus, the present inventors found a method for analyzing an RNA molecule, wherein the RNA molecule can easily be cut at a single or at multiple sites without the need to adopt the RNA molecule itself since the cleavage reaction is sequence-specific with respect to the sequence of the RNA molecule. In other words, it is possible to carry out the cleavage at a desired site of the RNA molecule simply by designing the oligonucleotide accordingly, which is comprised in the conjugate together with the chemical moiety with RNA cleavage activity.
(53) The present invention provides an advantageous method for analyzing an RNA molecule. The method can be applied to an RNA molecule (which may also be referred to as a population of identical RNA molecules) as well as to a population of different RNA molecules (such as in particular mixtures comprising different RNA molecules) without requiring separation of the different RNA molecules prior to analysis. The provided method further allows determining physical properties of the RNA molecule or the different RNA molecules in a population. The analysis may be directed to one specific physical property, e.g. the analysis of the 5 cap structure or the 3 region. The analysis may also be directed to several physical properties, e.g., the analysis of the 5 cap structure and the composition of the 3 region.
(54) Importantly, the provided method allows determining different physical properties of an RNA molecule at the same time. Depending on the desired analysis, the method may be adapted by including additional separation and/or purification steps to ensure an accurate analysis. For example, for analyzing the 3 fragment, the cleavage of the RNA molecule may be followed by suitable purification steps directed at separating the 3 fragment from the 5 fragment and/or any central fragments and the conjugate. A suitable method for purifying a 3 fragment is e.g. oligo dT-based capturing. Other approaches for separating/purifying fragments are encompassed as well. For example, a 5 fragment and a 3 fragment of different sizes can be purified by HPLC due to their size difference. HPLC also allows removing the conjugate depending on its size.
(55) The method can be further adapted by immobilizing the conjugate on a support and incubating the RNA molecule with the support. Advantageously, in this setup, the conjugate will not be comprised in the resulting fragment fraction(s). Other embodiments are generally also conceivable where the RNA molecule is immobilized on a support (e.g. by oligo-dT based capturing which will bind the 3 end of the RNA molecule to the support). The conjugate (designed to cleave upstream of the 3 end coupled to the support) may be incubated with the immobilized RNA molecule resulting in cleavage. While the 3 fragment will stay on the support, the 5 fragment and any central fragments will be in the elution fraction. The 3 fragment can subsequently be eluted for the solid support. Also by using this approach, the 3 fragment and the 5 fragment are separated from each other.
(56) The provided method is further advantageous as it can in principle be applied to RNA molecules of any sequence and length. The oligonucleotide of the conjugate can easily be designed based on the desired cleavage site within the RNA molecule. Hence, cleavage at virtually any site of an RNA molecule is possible. Therefore, the present method can easily be adapted depending on the RNA molecule to be analyzed and the physical property to be determined.
(57) Taken together, these features make the provided method, means and uses highly advantageous for RNA analysis, in particular in the field of therapeutic RNAs, where the RNAs are administered to the human and/or animal body. As the provided method gives precise answers regarding the physical properties of an RNA molecule, the method is highly suitable for determining compliance of an RNA molecule (or a population thereof, in particular a mixture of RNA molecules) with regulatory requirements.
EXAMPLES
(58) The following Examples are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.
Example 1: Preparation of RNA
(59) A DNA Sequence was Introduced into a Modified pUC19 Derived Vector Backbone to Comprise a 3-UTR, a Histone-stem-loop structure, a stretch of adenine nucleotides (A64), and a stretch of cytosine nucleotides (C30) at the 3-terminal end. The DNA plasmid was linearized and transcribed in vitro using DNA dependent RNA polymerase in the presence of a nucleotide mixture and cap analog. Obtained RNA was purified using RP-HPLC. The RNA sequence is provided in the sequence listing and in Table 1.
(60) TABLE-US-00001 TABLE1 Constructusedintheexperiment Constructsize RNAID:R4032 SEQIDNOs 646 GGGAGAAAGCUUACCAUGGGCGCCCCCACCCUGCCGCCGGCCUGGCAGCCG SEQIDNO:1 UUCCUCAAGGACCACCGCAUCUCGACCUUCAAGAACUGGCCGUUCCUGGAGG GCUGCGCGUGCACCCCGGAGCGGAUGGCCGAGGCCGGCUUCAUCCACUGCC CCACCGAGAACGAGCCGGACCUGGCCCAGUGCUUCUUCUGCUUCAAGGAGCU GGAGGGCUGGGAGCCGGACGACGACCCGAUCGAGGAGCACAAGAAGCACAGC AGCGGCUGCGCCUUCCUGAGCGUGAAGAAGCAGUUCGAGGAGCUGACGCUC GGGGAGUUCCUGAAGCUGGACCGGGAGCGGGCCAAGAACAAGAUCGCGAAG GAGACCAACAACAAGAAGAAGGAGUUCGAGGAGACCGCCAAGAAGGUGCGGC GGGCCAUCGAGCAGCUGGCCGCCAUGGACUGACCACUAGUUAUAAGACUGAC UAGCCCGAUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUA AUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAUGCAUCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAAGGCU CUUUUCAGAGCCACCAGAAUU
Example 2: Conditions for Analytical HPLC
(61) For analysis, RNA samples were diluted to 0.1 or 0.05 g/L using water for injection (WFI). 10 l to 20 L of diluted RNA samples were injected into the HPLC column (monolithic poly(styrene-divinylbenzen) matrix or AQUITY UPLC OST C18 matrix). The IP RP HPLC analysis was performed using the following conditions: Buffer A (0.1 M TEAA (pH 7.0)); Buffer B (0.1 M TEAA (pH 7.0) containing 25% acetonitrile). Gradients are indicated in respective Figures. Chromatograms were recorded at a wavelength of 260 nm. Evaluation of obtained chromatograms was done using Chromeleon software. Equipment used for analytical HPLC is provided in Table 2.
(62) TABLE-US-00002 TABLE 2 Materials used for analytical HPLC U3000 UHPLC-System Thermo Scientific HPLC column (monolithic Thermo Scientific poly(styrene-divinylbenzen) matrix AQUITY UPLC OST C18 column Waters Corporation 2.1 50 mm, 1.7 m particle size WFI Fresenius Kabi, Ampuwa Acetonitril (MS-grade) Fisher Scientific 0.1M TEAA in WFI (Eluent A) CureVac AG 25% ACN in 0.1M TEAA (Eluent B) CureVac AG
Example 3: Reaction Optimizations
(63) The inventors found that an oligonucleotide-conjugate harboring a 5 terminal Tris(2-aminobenzimidazole) moiety efficiently cuts a long RNA construct. Accordingly, an oligonucleotide-conjugate with a Tris(2-aminobenzimidazole) moiety may be used in a method for analyzing the 3 and/or 5 terminus of an RNA. As test RNA, the RNA construct with SEQ ID NO: 1 was used.
(64) The following conjugate with 5 terminal Tris(2-aminobenzimidazole) moiety (Cutter) was used:
(65) 5-Cutter-CGGCTCCCAGCCCTC-3 (SEQ ID NO: 2)
(66) The oligonucleotide was designed to be complementary to a target region located in the RNA sequence. After successful cleavage of the RNA (646 nucleotides), a fragment of approximately 222 nucleotide in size, and a fragment of approximately 424 nucleotide in size was expected to be obtained.
(67) TABLE-US-00003 Sequenceoftheexpected222fragment,with complementaryregionhighlightedinbold (SEQIDNO:5): GGGAGAAAGCUUACCAUGGGCGCCCCCACCCUGCCGCCGGCCUGGCAGCC GUUCCUCAAGGACCACCGCAUCUCGACCUUCAAGAACUGGCCGUUCCUGG AGGGCUGCGCGUGCACCCCGGAGCGGAUGGCCGAGGCCGGCUUCAUCCAC UGCCCCACCGAGAACGAGCCGGACCUGGCCCAGUGCUUCUUCUGCUUCAA GGAGCUGGAGGGCUGGGAGCCG Sequenceoftheexpected424fragment (SEQIDNO:6): GACGACGACCCGAUCGAGGAGCACAAGAAGCACAGCAGCGGCUGCGCCUU CCUGAGCGUGAAGAAGCAGUUCGAGGAGCUGACGCUCGGGGAGUUCCUGA AGCUGGACCGGGAGCGGGCCAAGAACAAGAUCGCGAAGGAGACCAACAAC AAGAAGAAGGAGUUCGAGGAGACCGCCAAGAAGGUGCGGCGGGCCAUCGA GCAGCUGGCCGCCAUGGACUGACCACUAGUUAUAAGACUGACUAGCCCGA UGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUAAUAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAUGCAUCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAAG GCUCUUUUCAGAGCCACCAGAAUU
(68) Oligonucleotide conjugate and RNA were incubated in a 50 mM Tris-Puffer (pH 8.0) for about 19 h. To avoid thermal degradation of the RNA, the incubation temperature was set to about 20 C. Different amounts of oligonucleotide conjugate were tested (1 eq (ng/ng), 2 eq (ng/ng), 4 eq (ng/ng), 8 eq (ng/ng)) (eq=mass equivalent).
(69) To improve the hybridization of oligonucleotide conjugate to the RNA, a temperature cycle was introduced (20 C.->80 C. for 20 sec->20 C.), followed by final incubation step at 20 C. for 19 h. The different reaction products were analyzed using analytical HPLC. The results are shown in
(70) As exemplarily shown in
(71)
(72) To further optimize the procedure, thermal cycles were introduced during the incubation period. Accordingly, after an initial cycle (20 C.->80 C. for 20 sec->20 C.) the reaction was incubated for 1 h at 20 C. That procedure was repeated 8 times, followed by a final incubation step of 11 h at room temperature. The result is shown in
(73) As
(74) An increase in incubation time to about 70 h led to a conversion efficiency of about 90% (using 5 eq oligonucleotide conjugate). Again, conversion of the RNA could not be further improved by further increasing the amount of oligonucleotide conjugate in the reaction (10 eq, 15 eq, 20 eq, 25 eq, 30 eq, 35 eq, 40 eq). Further optimizations may be required to reduce the incubation time. The results are shown in
(75) Conclusion/Discussion:
(76) The results show that a conjugate comprising the chemical moiety with RNA cleaving activity and the oligonucleotide can be used for sequence specific digestion of a long RNA construct. Further optimizations that may improve the conversion efficiency of the RNA into the cleavage products may be the temperature profile of the reaction, the buffer conditions of the reaction, the sequence of the oligonucleotide (e.g. implementation of LNA, PNA nucleotides), and/or the implementation of a oligonucleotide conjugate feeding step.
Example 4: Specific Digestion of the 3 Terminus Comprising a Poly(A) and a Poly(C) Stretch
(77) The inventors found that a conjugate harboring a 5 terminal Tris(2-aminobenzimidazole) moiety can be used for sequence specific cleavage of the 3 terminus of an RNA. As test RNA, the RNA construct with SEQ ID NO: 1 was used.
(78) The following conjugate with 5 terminal Tris(2-aminobenzimidazole) modification (Cutter) was used: 5-Cutter-CTCGGTGCAAGGAGGGGAG-3 (SEQ ID NO: 4)
(79) The oligonucleotide was designed to be complementary to a region in the 3 terminus of the RNA. After successful cleavage of the RNA (646 nucleotides), a 3 terminal fragment of 134 nucleotides in size, and a fragment of 512 nucleotides in size were expected to be obtained.
(80) TABLE-US-00004 Sequenceoftheexpected512ntfragment,with complementaryregionhighlightedinbold (SEQIDNO:7): GGGAGAAAGCUUACCAUGGGCGCCCCCACCCUGCCGCCGGCCUGGCAGCC GUUCCUCAAGGACCACCGCAUCUCGACCUUCAAGAACUGGCCGUUCCUGG AGGGCUGCGCGUGCACCCCGGAGCGGAUGGCCGAGGCCGGCUUCAUCCAC UGCCCCACCGAGAACGAGCCGGACCUGGCCCAGUGCUUCUUCUGCUUCAA GGAGCUGGAGGGCUGGGAGCCGGACGACGACCCGAUCGAGGAGCACAAGA AGCACAGCAGCGGCUGCGCCUUCCUGAGCGUGAAGAAGCAGUUCGAGGAG CUGACGCUCGGGGAGUUCCUGAAGCUGGACCGGGAGCGGGCCAAGAACAA GAUCGCGAAGGAGACCAACAACAAGAAGAAGGAGUUCGAGGAGACCGCCA AGAAGGUGCGGCGGGCCAUCGAGCAGCUGGCCGCCAUGGACUGACCACUA GUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCUCCUCCCCU CCUUGCACCGAG Sequenceoftheexpected3terminal134nt fragment(SEQIDNO:8): AUUAAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAUGCAUCCCCCCCCCCCCCCCCCCCCCCCCC CCCCCCAAAGGCUCUUUUCAGAGCCACCAGAAUU
(81) RNA and oligonucleotide conjugate (1 eq, 2 eq, 10 eq, equimolare) were incubated in 50 mM Tris-Puffer (pH 8.0) for 48 h (including 2 temperature cycles carried out as in Example 3). The obtained products were analyzed on HPLC. The analytical HPLC showed 4 distinct peaks: a peak corresponding to the 19 nt oligonucleotide conjugate, a peak corresponding to the 134 nt 3 terminal fragment, a peak corresponding to the 512 nt fragment and a peak corresponding to the target RNA (646nt fragment). The chromatogram is shown in
(82) Conclusion/Discussion:
(83) As shown in
(84) As a next step, the 134 nt size fraction can be isolated (e.g. fractionation of HPLC) and subjected to total RNA hydrolysis. The obtained nucleoside hydrolysate can be analyzed on HPLC or MS (total hydrolysis approach is exemplified in WO 2017/149139).
Example 5: Digestion of an RNA Using Multiple Oligonucleotide Conjugates
(85) The inventors found that several oligonucleotide-conjugates can be used in a simultaneous reaction, showing that a simultaneous analysis of the 3 terminus and the 5 terminus is feasible. As test RNA, the RNA construct with SEQ ID NO: 1 was used.
(86) The following conjugates with 5 terminal Tris(2-aminobenzimidazole) moiety (Cutter) were used simultaneously in one reaction: 5-Cutter-CGGCTCCCAGCCCTC-3 (SEQ ID NO: 2); 5-Cutter-GGTAAGCTTTCTCCC-3 (SEQ ID NO: 3); 5-Cutter-CTCGGTGCAAGGAGGGGAG-3 (SEQ ID NO: 4).
(87) RNA and the above oligonucleotide conjugates were incubated in 50 mM Tris-Puffer (pH 8.0) for 92 h (including 2 temperature cycles carried out as in Example 3). One amount of oligonucleotide conjugate was tested (2 eq)). After successful cleavage of the RNA (646 nucleotides), a 15 nt fragment (5 terminus), a 207 nt fragment, a 290 nt fragment, and a 134 nt fragment (3 terminus) were expected to be obtained.
(88) The obtained products were analyzed on HPLC. The analytical HPLC showed that several peaks appeared, wherein the peaks should represent (i) the four expected cleavage products, (ii) the oligonucleotide conjugate (cutter), (iii) the undigested RNA, and (iv) cleavage intermediates, as indicated in
(89) Conclusion/Discussion:
(90) Besides the expected RNA cleavage products (15nt fragment (5 terminus), 207nt fragment, 290nt fragment and 134nt fragment (3 terminus)), full length RNA (646nt), oligonucleotide conjugate (cutter), and cleavage intermediates were detected. The results exemplarily show that a simultaneous digestion of an RNA using oligonucleotide conjugates is feasible, thus generally allowing fingerprinting approaches as well as a simultaneous analysis of the 5 cap and 3 tail.
Example 6 (Prophetic): Method for the Analysis of RNA Using a Solid Phase Approach
(91) An oligonucleotide conjugate harboring a 5 terminal tris(2-aminobenzimidazole) moiety is additionally functionalized at the 3 terminus and immobilized on a solid phase.
(92) Click-chemistry approach: The 3 terminus of the oligonucleotide conjugate comprising a 5 terminal Tris(2-aminobenzimidazole) modification is functionalized with an alkyne (e.g., ethynyl). Next, the obtained oligonucleotide conjugate is subjected to an azide functionalized matrix. Cu(I)-catalyzed azide-alkyne cycloaddition of ethynyl of the oligonucleotide with the azide group of the matrix is performed using BaseClick-Kit biotin (baseclick GmbH) according to the manufacturer's instructions. To prevent damage of the oligonucleotide conjugate by copper ions, the matrix is washed with 70% EtOH and/or 70% EtOH+10 mM EDTA in order to complex the copper ions.
(93) The column comprising immobilized oligonucleotide conjugate is used for cleavage of an RNA construct. To achieve optimal cleavage efficiency, the column is used in an (HP) LC setup, using a flow rate that allows sufficient contact and cleavage of the RNA. Optionally, the digested RNA is collected and re-subjected to the column until sufficient cleavage is obtained (almost 100%). Eventually, the final cleavage product is subjected to HPLC analysis and/or MS and/or total hydrolysis.
(94) Biotin-Streptavidin approach: The 3 terminus of the oligonucleotide conjugate comprising a 5 terminal Tris(2-aminobenzimidazole) modification is functionalized with a biotin moiety. Next, the obtained oligonucleotide conjugate is subjected to a streptavidin matrix. The column comprising immobilized oligonucleotide conjugate is used for cleavage of an RNA construct. To achieve optimal cleavage efficiency, the column is used in an (HP) LC setup, using a flow rate that allows sufficient contact and cleavage of the RNA. Optionally, the digested RNA is collected and re-subjected to the column until sufficient cleavage is obtained (almost 100%). Eventually, the final cleavage product is subjected to HPLC analysis and/or MS and/or total hydrolysis.
Example 7: Fingerprinting of an RNA Molecule Using One Oligonucleotide Conjugate
(95) The present example shows that a fingerprinting approach using oligonucleotide conjugates is suitable to distinguish RNA molecules, in particular, similar RNA molecules.
(96)
(97) A finger-printing approach using an oligonucleotide conjugate with multiple cleavage sites within the RNA molecules to be analyzed was developed to distinguish these three different RNA species (RNA 1, RNA 2, RNA 3). The inventors used one oligonucleotide conjugate that has been adapted to hybridize multiple times in the three different RNA molecules. In particular, the oligonucleotide conjugate used for the present experiment comprises four nucleotides that hybridize with complementary RNA motifs within the target RNA sequence. The oligonucleotide includes three DNA nucleotides and one LNA nucleotide exhibiting stronger RNA binding. The oligonucleotide conjugate further comprises a (Tris(2-aminobenzimidazole) at the 5end for RNA cleavage.
(98) Generally, an oligonucleotide conjugate can be designed for any kind of RNA molecule and any mixture of RNA molecules, depending on the RNA motif at the desired cleavage site(s). For example, in fingerprinting approaches it may be beneficial to use short RNA motifs as binding sites on the target RNA, as such short RNA motifs occur with higher frequency. The introduction of LNA nucleotides exhibiting stronger binding may be used to ensure proper hybridization of the oligonucleotide conjugates on these short RNA motifs.
(99) Each target RNA was digested using said oligonucleotide conjugate under the following conditions. Reaction buffer: 50 mM Tris, 1 mM EDTA, 50 mM NaCl, pH 8.0. Molar ratio of each of the at least one conjugates to the RNA molecule: 8:1 Temperature profile: 25 C.->80 C. for 20 sec->25 C. for 2.5h Temperature cycles: 6
(100) The obtained cleavage products were analyzed on analytical HPLC to obtain unique RNA fingerprints based on the size distribution of the cleavage products (see
(101) An RNA fingerprint approach either using an oligonucleotide conjugate with multiple cleavage sites within the RNA molecules to be analyzed as described in Example 7, or using more than one oligonucleotide conjugate as described in Example 5, can be used for determining the identity of an RNA e.g. after RNA production. Accordingly, the method can be used as a quality control method to determine the identity of an RNA.
Example 8: Determination of Optimal Cleavage Temperatures
(102) The present example shows that the conversion efficiencies can be improved by increasing the cleavage temperature. Under the conditions of the present Example, the optimal cleavage temperature was in a range of about 35 C. to about 45 C. Furthermore, the example shows that the analysis can surprisingly be performed over various reaction cycles without degrading the RNA and/or without degrading the oligonucleotide conjugate.
(103) Experimental Procedure:
(104) 100 pmol RNA of interest (SEQ ID NO: 1) was incubated with 6 equimolar of DNA oligonucleotide conjugate (SEQ ID NO: 2 to perform an RNA analysis assay under the following conditions: Reaction buffer: 50 mM Tris, 1 mM EDTA, 50 mM NaCl, pH 8.0 Reaction cycles Hybridization temperature: 25 C. (annealing temperature) Temperature shift up to 85 C., hold for 15 sec (denaturing temperature) Down to cleavage temperature: CT=25 C., CT=30 C., CT=35 C., CT=40 C., CT=45 C., CT=50 C. . . . cleavage temperature CT for 2.5 hours Cycle was repeated 6 times Reaction time in total: 15 hours
(105) The assay was performed with 6 different cleavage temperatures CT (CT=25 C., CT=30 C., CT=35 C., CT=40 C., CT=45 C., CT=50 C.) to determine the optimal temperature for cleavage of an RNA target.
(106) Each cleavage product was subjected to analytical HPLC (exemplary chromatograms shown in
(107) TABLE-US-00005 TABLE 3 Analysis of the target RNA peak to determine cleavage efficiency Non-cleaved Thermal target degradation Thermal RNA (% Cleavage Conversion of target degradation educt peak) temperature efficiency RNA of cutter 34 25 C. 66% 22 30 C. 78% 12 35 C. 88% 8 40 C. 92% 5 45 C. 95% (+) 10 50 C. 90% +
(108) The results of the experiments, summarized in Table 3, show that the conversion efficiency can be increased to almost 100% by increasing the cleavage temperature. Conversion efficiency refers to the amount of RNA molecule cleaved by the oligonucleotide conjugate. Thermal degradation of target RNA refers to unspecific RNA degradation. Depending on the stability of the RNA molecule to be analyzed, the composition of the oligonucleotide conjugate and the desired conversion efficiency, suitable denaturation, hybridization and cleavage temperatures can be selected. For example, LNA nucleotides require higher denaturation temperatures than DNA nucleotides, and can therefore operate at higher cleavage temperatures. In the present experiment, the optimal cleavage temperature was in a range of about 35 C. and 45 C.