METHODS FOR RNA ANALYSIS

Abstract

The present invention relates to the field of RNA analysis. In particular, the invention concerns the use of a catalytic nucleic acid molecule for the analysis of an RNA molecule. The invention concerns methods for analyzing the 5 terminal structures of an RNA molecule having a cleavage site for a catalytic nucleic acid molecule. In particular, the invention concerns a method for determining the presence of a cap structure in an RNA molecule having a cleavage site for a catalytic nucleic acid molecule, a method for determining the capping degree of a population of RNA molecules having a cleavage site for a catalytic nucleic acid molecule, a method for determining the orientation of the cap structure in a capped RNA molecule having a cleavage site for a catalytic nucleic acid molecule and a method for determining relative amounts of correctly capped RNA molecules and reverse-capped RNA molecules in a population of RNA molecules, wherein the population comprises correctly capped and/or reverse-capped RNA molecules that have a cleavage site for a catalytic nucleic acid molecule. Moreover, the present invention provides uses of a catalytic nucleic acid molecule.

Claims

1. A method for analyzing an RNA molecule having a cleavage site for a catalytic nucleic acid molecule, the method comprising the steps of: a) providing an RNA molecule having a cleavage site for a catalytic nucleic acid molecule, b) cleaving the RNA molecule with the catalytic nucleic acid molecule into a 5 terminal RNA fragment and at least one 3 RNA fragment by contacting the RNA molecule with the catalytic nucleic acid molecule under conditions allowing the cleavage of the RNA molecule, c) determining a physical property of the RNA molecule by analyzing the 5 terminal RNA fragment.

2. A method for analyzing a population of RNA molecules, wherein the population comprises at least one RNA molecule that has a cleavage site for a catalytic nucleic acid molecule, the method comprising the steps of: a) providing a sample containing the population of RNA molecules, b) cleaving the at least one RNA molecule having a cleavage site for the catalytic nucleic acid molecule with the catalytic nucleic acid molecule into a 5 terminal RNA fragment and at least one 3 RNA fragment by contacting the sample with the catalytic nucleic acid molecule under conditions allowing the cleavage of the RNA molecule, c) determining a physical property of the at least one RNA molecule having a cleavage site by analyzing the at least one 5 terminal RNA fragment obtained in step b), and d) measuring the relative amount of the at least one 5 terminal RNA fragment obtained in step b), thereby determining the relative amount of RNA molecules having said physical properties in the RNA population.

3. The method according to claim 1 or 2, wherein the catalytic nucleic acid molecule has been designed to be able to cleave the RNA molecule at a specific cleavage site.

4. The method according to any one of claims 1 to 3, wherein the RNA molecule having a cleavage site for the catalytic nucleic acid molecule has been designed to have a cleavage site for the catalytic nucleic acid molecule.

5. The method according to any one of claims 1 to 4, wherein the cleavage site of the catalytic nucleic acid molecule is located within 50 nucleotides from the 5 terminus of the RNA molecule.

6. The method according to any one of claims 1 to 5, wherein the catalytic nucleic acid molecule is a ribozyme, preferably selected from the group consisting of hammerhead ribozymes, hairpin ribozymes, and HDV ribozymes.

7. The method according to any one of claims 1 to 6, wherein the catalytic nucleic acid molecule is provided in step b) in trans.

8. The method according to any one of claims 1 to 7, wherein step b) comprises denaturation of the RNA molecule having a cleavage site for the catalytic nucleic acid molecule and annealing of the ribozyme to said RNA molecule.

9. The method according to any one of claims 1 to 8, wherein the sample containing the population of RNA molecules is generated by in vitro transcription, wherein the in vitro transcription is carried out in the presence of a cap analog, or by in vitro transcription and subsequent enzymatic capping.

10. The method according to any one of claims 1 to 9, wherein the cap analog is selected from the group consisting of G[5]ppp[5]G, m.sup.7G[5]ppp[5]G, m.sub.3.sup.2,2,7G[5]ppp[5]G, m.sub.2.sup.7,3-OG[5]ppp[5]G (3-ARCA), m.sub.2.sup.7,2-OGpppG (2-ARCA), m.sub.2.sup.7,2-OGppspG D1 (-S-ARCA D1) and m.sub.2.sup.7,2-OGppspG D2 (-S-ARCA D2).

11. The method according to any one of claims 1 to 10, wherein step c) comprises separating the RNA fragments and wherein the RNA fragments are separated by denaturing gel electrophoresis or liquid chromatography, preferably HPLC, FPLC or RPLC.

12. The method of any of claims 1 to 11, wherein the RNA molecule having a cleavage site for the catalytic nucleic acid molecule is an mRNA molecule.

13. The method of any of claims 1 to 12, wherein the RNA molecule having a cleavage site for the catalytic nucleic acid molecule comprises at least one modification.

14. The method according to any one of claims 1 to 13, wherein step c) comprises analysis of a structural feature or of a physical parameter of the 5 terminal RNA fragment.

15. The method according to any one of claims 1 to 14, wherein step c) comprises comparison of a structural feature or of a physical parameter of the 5 terminal RNA fragment, and the respective feature or parameter of a reference RNA fragment.

16. The method according to any one of claims 1 to 15, wherein step c) involves spectroscopic analysis, quantitative mass spectrometry, or sequencing.

17. The method according to any one of claims 1 to 16, wherein step c) comprises determining the presence or absence of a cap structure at the 5 terminus of the RNA molecule having a cleavage site for the catalytic nucleic acid molecule.

18. The method according to any one of claims 1 to 17, wherein the RNA molecule having a cleavage site for the catalytic nucleic acid molecule comprises a cap structure at the 5 terminus and step c) comprises determining the orientation of the cap.

19. The method according to any one of claims 2 to 18, wherein the population comprises at least one capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and wherein step d) comprises determining the relative amount of the at least one capped RNA molecule in the population.

20. The method according to claim 19, wherein the population comprises at least one capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and at least one non-capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and wherein step c) comprises separating capped 5 terminal RNA fragments and non-capped 5 terminal RNA fragments.

21. The method according to claim 19 or 20, wherein the amount of the capped and/or the amount of the non-capped 5 terminal RNA fragments are measured in step c) by spectroscopic methods, quantitative mass spectrometry, or sequencing.

22. The method according to any one of claims 19 to 21, wherein step d) comprises calculating the ratio of the amount of capped RNA molecules having a cleavage site for the catalytic nucleic acid molecule and the amount of non-capped RNA molecules having a cleavage site for the catalytic nucleic acid molecule in the population.

23. The method according to any one of claims 2 to 22, wherein the population comprises at least one correctly capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and wherein step d) comprises determining the relative amount of correctly capped RNA molecules having a cleavage site for the catalytic nucleic acid molecule in the population.

24. The method according to claim 23, wherein the population comprises at least one correctly capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and at least one reverse-capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule, and wherein step c) comprises separating correctly capped 5 terminal RNA fragments and reverse-capped 5 terminal RNA fragments.

25. The method according to claim 23 or 24, wherein the amount of correctly capped and/or the amount of the reverse-capped 5 terminal RNA fragments are measured in step d) by spectroscopic methods, quantitative mass spectrometry, or sequencing.

26. The method according to any one of claims 23 to 25, wherein step d) comprises calculating the ratio of the amount of correctly capped RNA molecules having a cleavage site for the catalytic nucleic acid molecule and the amount of reverse-capped RNA molecules having a cleavage site for the catalytic nucleic acid molecule in the population.

27. A method of determining the capping degree of a population of RNA molecules having a cleavage site for a catalytic nucleic acid molecule, the method comprising the steps of: a) providing a sample containing the population of RNA molecules, b) cleaving the RNA molecules with the catalytic nucleic acid molecule into a 5 terminal RNA fragment and at least one 3 RNA fragment by contacting the sample with the catalytic nucleic acid molecule under conditions allowing the cleavage of the RNA molecules, c) separating the RNA fragments obtained in step b), d) determining a measure for or measuring the amount of the capped and non-capped 5 terminal RNA fragments separated in step c) of said population of RNA molecules, and e) comparing said measures of capped and non-capped 5 terminal RNA fragments determined in step d), thereby determining the capping degree of said population of RNA molecules.

28. The method according to claim 27, wherein the catalytic nucleic acid molecule has been designed to be able to cleave the RNA molecules at a specific cleavage site.

29. The method according to claim 27 or 28, wherein the RNA molecules have been designed to have a cleavage site for the catalytic nucleic acid molecule.

30. The method according to any one of claims 27 to 29, wherein the cleavage site of the catalytic nucleic acid molecule is within the first 50 nucleotides of the 5-end of the RNA molecules.

31. The method according to any one of claims 27 to 30, wherein the catalytic nucleic acid molecule is a ribozyme, preferably selected from the group consisting of hammerhead ribozymes, hairpin ribozymes, and HDV ribozymes.

32. The method according to any one of claims 27 to 31, wherein the catalytic nucleic acid molecule is provided in step b) in trans.

33. The method according to any one of claims 27 to 32, wherein the sample containing the population of RNA molecules is generated by in vitro transcription in the presence of a cap analog or by in vitro transcription and subsequent enzymatic capping.

34. The method according to any one of claims 27 to 33, wherein the cap analog is selected from the group consisting of G[5]ppp[5]G, m.sup.7G[5]ppp[5]G, m.sub.3.sup.2,2,7G[5]ppp[5]G, m.sub.2.sup.7,3-OG[5]ppp[5]G (3-ARCA), m.sub.2.sup.7,2-OGpppG (2-ARCA), m.sub.2.sup.7,2-OGppspG D1 (-S-ARCA D1) and m.sub.2.sup.7,2-OGppspG D2 (-S-ARCA D2).

35. The method according to any one of claims 27 to 34, wherein the RNA fragments are separated in step c) by denaturing gel electrophoresis or liquid chromatography, preferably HPLC, FPLC or RPLC.

36. The method according to any one of claims 27 to 35, wherein the measure determined in step d) is the signal intensity of the capped and non-capped 5 terminal RNA fragments or the amount of the RNA fragments.

37. The method according to any one of claims 27 to 36, wherein the measure determined in step d) for the amount of the the capped and non-capped 5 terminal RNA fragments is determined by spectroscopic methods, quantitative mass spectrometry, or sequencing.

38. The method according to any one of claims 27 to 37, wherein in step e) the ratio of capped and non-capped 5 terminal RNA fragments is calculated.

39. The method of any of claims 1 to 38, wherein the RNA molecules are mRNA molecules.

40. The method according to any one of claims 27 to 39, wherein in step d) the relative amounts of the fragments separated in step c) are determined.

41. The method according to any one of claims 27 to 40, wherein the population comprises at least one capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and at least one non-capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule.

42. The method according to any one of claims 27 to 41, wherein step b) comprises denaturation of the RNA molecule having a cleavage site for the catalytic nucleic acid molecule and annealing of the ribozyme to the RNA molecule having a cleavage site for the catalytic nucleic acid molecule.

43. The method according to any one of claims 27 to 42, wherein step c) comprises separating the capped 5 terminal RNA fragments and the non-capped 5 terminal RNA fragments.

44. The method according to any one of claims 27 to 43, wherein the amount of the capped and the amount of the non-capped 5 terminal RNA fragments are measured in step d) by spectroscopic methods, quantitative mass spectrometry, or sequencing.

45. The method of any one of claims 27 to 44, wherein the orientation of the cap in the 5 terminal RNA fragment of a capped RNA molecule is determined.

46. The method according to any one of claims 27 to 45, wherein the at least one RNA molecule comprises at least one modification.

47. Use of a catalytic nucleic acid molecule for determining the capping degree of a population of RNA molecules, wherein the catalytic acid molecule is used to cleave the RNA molecules of the population into a 5 terminal RNA fragment and at least one 3 RNA fragment with a length useful for the determination of the capping degree.

48. The use according to claim 47, wherein the method for determining the capping degree further comprises at least one of the steps as defined in any of claims 27 to 46.

49. Use of a catalytic nucleic acid molecule in a method for analyzing an RNA molecule having a cleavage site for the catalytic nucleic acid molecule.

50. Use of a catalytic nucleic acid molecule in a method for analyzing a population of RNA molecules, wherein the population comprises at least one RNA molecule having a cleavage site for a catalytic nucleic acid molecule.

51. Use of a catalytic nucleic acid molecule according to claim 49 or 50, wherein the method comprises determining the presence or absence of a cap structure in the RNA molecule having a cleavage site for the catalytic nucleic acid molecule.

52. Use of a catalytic nucleic acid molecule according to any one of claims 49 to 51, wherein the RNA molecule having a cleavage site for the catalytic nucleic acid molecule has a cap structure at the 5 terminus and wherein the method comprises determining the orientation of said cap structure.

53. The use of a catalytic nucleic acid molecule according any one of claims 50 to 52, wherein the population comprises at least one capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and wherein the method comprises determining the relative amount of capped RNA molecules having a cleavage site for the catalytic nucleic acid molecule in the population of RNA molecules.

54. The use of a catalytic nucleic acid according any one of claims 50 to 53, wherein the population comprises at least one correctly capped RNA molecule having a cleavage site for the catalytic nucleic acid molecule and wherein the method comprises determining the relative amount of correctly capped RNA molecules having a cleavage site for the catalytic nucleic acid molecule in the population of RNA molecules.

55. The use of a catalytic nucleic acid according to any one of claims 49 to 54, wherein the method comprises at least one of the features as defined in any one of claims 1 to 26 or claims 27 to 46.

56. 5 terminal RNA fragment obtainable by the method according to any one of claims 1 to 26 or claims 27 to 46.

57. RNA molecule consisting of 10 to 20 nucleotides, wherein the RNA molecule comprises a cap structure at its 5 terminus and the sequence NUH at its 3-terminus, wherein N is selected from G, A, C and U; and H is selected from A, C and U.

58. The 5 terminal RNA fragment according to claim 56 or the RNA molecule according to claim 57 having the general structure
5-cap-N.sub.10-NUH-3.

59. The 5 terminal RNA fragment according to claim 56 or 58, or the RNA molecule according to claim 57 or 58, wherein the 5 terminal RNA fragment or the RNA molecule comprises or consists of SEQ ID NO: 6, which optionally comprises a cap structure at the 5 terminus.

60. Use of the 5 terminal RNA fragment according to any one of claim 56, 58 or 59, or the RNA molecule according to any one of claim 57, 58 or 59, in a method for analyzing an RNA molecule.

61. The use according to claim 60, wherein the method further comprises at least one of the features as defined in any one of claims 1 to 26 or claims 27 to 46.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0303] 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.

[0304] FIG. 1: G/C optimized mRNA sequence coding for Homo sapiens New York Esophageal Squamous Cell Carcinoma 1 antigen (HsNY-ESO-1; SEQ ID NO: 3).

[0305] FIG. 2: G/C optimized mRNA sequence coding for Photinus pyralis Luciferase (PpLuc; SEQ ID NO: 4).

[0306] FIG. 3: G/C optimized mRNA sequence coding for Homo sapiens prostate stem cell antigen (HsPSCA; SEQ ID NO: 5).

[0307] FIGS. 4A-B: Schematic diagram of non-capped (A) and capped mRNA (B).

[0308] FIG. 5: Diagram of hammerhead ribozyme annealed to target RNA sequence (highlighted in bold).

[0309] FIG. 6: Hammerhead ribozyme HHNUH2d annealed to 5 UTR of target mRNA sequence (highlighted in bold). Recognition site marked by circle and AUG start codon underlined. Cleavage at the indicated site yields a 13mer 5fragment of non-capped RNA (or a 14mer fragment if the RNA is capped).

[0310] FIGS. 7A-B: Separation of capped and non-capped RNA fragments by denaturing polyacrylamide gel electrophoresis (dPAGE). RNAs were synthesized in the absence () or presence (+) of a cap analog as described in Example 2 and subsequently incubated without () or with (+) hammerhead (HH) ribozyme HHNU2d as described in Example 3. (A) Full gel and (B) enlarged part of gel with capped and non-capped RNA fragments.

[0311] FIGS. 8A-B: Separation of capped 5 mRNA fragments, non-capped 5 mRNA fragments, 3 mRNA fragments and hammerhead ribozyme by HPLC. The mRNA sample was prepared by mixing 60% enzymatically capped mRNA coding for Photinus pyralis Luciferase (PpLuc) and 40% non-capped mRNA coding for Photinus pyralis Luciferase (PpLuc). Subsequently, this sample was incubated with the hammerhead (HH) ribozyme HHNU2d as described in Example 3 and analysed by HPLC. [0312] (A) Full chromatogram showing the separation of the hammerhead (HH) ribozyme from the 3 mRNA fragment. [0313] (B) Enlarged area of the chromatogram showing the separation of capped and non-capped 5 mRNA fragments.

[0314] FIGS. 9A-D: Resolution of co-transcriptionally capped, non-capped, and enzymatically capped 5 mRNA fragments separated by dPAGE and HPLC. Photinus pyralis luciferase (PpLuc) RNAs were synthesized in the absence (no cap) or presence (cotx) of a cap analog, non-capped RNAs were subsequently enzymatically capped (Ecap) as described in Example 2. The RNAs were incubated with hammerhead (HH) ribozyme HHNU2d as described in Example 3 and analysed by dPAGE and HPLC. [0315] (A) Enlarged part of a dPAGE gel with capped (cotx, Ecap) and non-capped (no cap) RNA fragments. Two bands were detected for co-transcriptionally capped RNA. [0316] (B-D) Enlarged area of the HPLC chromatogram showing the separation of capped and non-capped 5 mRNA fragments. (B) Non-capped PpLuc RNA, (C) enzymatically capped PpLuc RNA originating from (B), (D) co-transcriptionally capped PpLuc RNA. Five peaks were detected for co-transcriptionally capped mRNA.

[0317] FIG. 10: Fractionation of co-transcriptionally capped 5 mRNA fragments via HPLC. Photinus pyralis luciferase (PpLuc) RNAs were synthesized in the presence of a cap analog as described in Example 2, incubated with hammerhead (HH) ribozyme HHNU2d as described in Example 3, and analyzed via HPLC. Fractions were collected over the course of time as indicated on the x-axis of the diagram. Double peaks at 13.30-13.80 minutes and 14.30-14.80 minutes could not be separated via HPLC and were thus pooled prior to MALDI analysis (sample S1=fractions 12-14, S2=21-24, S3=28-30).

[0318] FIGS. 11A-B: MALDI-TOF spectrum for samples S1 and S2 obtained after HPLC separation of ribozyme-cleaved 5 terminal RNA fragments of co-transcriptionally capped Photinus pyralis luciferase (PpLuc) RNAs. Analyses were performed as described in Example 3.

[0319] FIGS. 12A-D: Overlay of HPLC analyses of co-transcriptionally capped, non-capped, and enzymatically capped 5 terminal RNA fragments. Photinus pyralis luciferase (PpLuc) RNAs were synthesized in the absence (no cap) or presence of a cap analog, non-capped RNAs were subsequently enzymatically capped (Ecap) as described in Example 2. In addition, enzymatically capped Photinus pyralis Luciferase (PpLuc) RNAs lacking the initial 5 guanosine (Ecap-G1) were synthesised analogously. The RNAs were incubated with hammerhead (HH) ribozyme HHNU2d as described in Example 3 and analysed by HPLC.

[0320] FIGS. 13A-D: Quantitation of different RNA populations. Peak areas (mAU*min) for non-capped, correctly capped and reverse-capped RNA populations (FIG. 12 D, full-length and minus1G (n1) RNA) were determined using Chromeleon software. Relative proportions were plotted: (A) distribution of single populations; (B) combined capped versus non-capped populations; (C) combined minusG1 RNA (correctly capped and reverse-capped) and combined full-length RNA (correctly capped and reverse-capped) versus non-capped; (D) combined correctly capped RNA (full-length and minus1G), combined reverse-capped RNA (full-length and minusG1) versus non-capped RNA.

EXAMPLES

[0321] The examples shown in the following 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 Hammerhead Ribozymes

[0322] 1. A hammerhead ribozyme can be directed to cleave 3 of any NUH sequence as shown in FIG. 5 (N=G,A,C,U; H=A,C,U) (Haseloff and Gerlach, 1988. Nature 334: 585-591; McCall et al., 2000. Molecular Biotechnology 14: 5-17). The schematic diagram of FIG. 5 shows how helix I and helix III anneal to the target RNA sequence.

[0323] 2. The trans-acting hammerhead ribozyme HHNUH2d was designed to target the 5 region of the RNA sequences shown in FIGS. 1 to 3, forming helix III with mRNA positions 1-12, and helix I with mRNA positions 14-18 (FIG. 6). The 5 region of the target RNA sequence contains two possible recognition sites, NUH1 (positions 10-12) and NUH2 (positions 11-13), of which NUH2 is the preferred target site.

[0324] Sequence of the trans-acting hammerhead ribozyme HHNUH2d (SEQ ID NO: 2):

TABLE-US-00005 5-GCAUGGCUGAUGAGGCCUCGACCGAUAGGUCGAGGCCGAAAAGCU UUCUCCC-3

[0325] 3. The ribozyme HHNUH2d was synthesized and HPLC purified by Biomers.net GmbH (Ulm, Germany), and 200 g were resolved on a preparative denaturing 10 cm8 cm1.0 mm acrylamide gel for purification (8 M urea (Applichem), 20% acrylamid:bisacrylamid 19:1 (Applichem), 1TBE, 1% APS (AppliChem), 0.1% TEMED (AppliChem); 180 V, 2 hours, Mini-PROTEAN Tetra Cell (BioRad)). The ribozyme band was identified by UV shadowing (E-BOX VX2 gel documentation system with 312 nm-UV Transilluminator (Peqlab)) over a TLC plate (Kieselgel 60 F254, Merck), excised and eluted from the gel slice in 10 mM Tris/HCl, pH 7.5 (room temperature, 16 hours). The supernatant was filtered through Corning Costar Spin-X columns (Sigma) (1 minute, 16.000 g, room temperature), and RNAs were precipitated (300 mM NaOAc, pH 5, 75% ethanol, 16 hours, 20 C.). Following centrifugation (30 minutes, 16.000 g, 4 C.), pellets were washed in 75% ethanol (invert, centrifuge 5 minutes, 16.000 g, 4 C.), dried and re-dissolved in H.sub.2O.

Example 2: Preparation of the mRNA

[0326] 1. Preparation of DNA and mRNA Constructs

[0327] For the present example DNA sequences encoding HsNY-ESO-1 mRNA according to SEQ ID NO: 3 (FIG. 1), PpLuc mRNA according to SEQ ID NO: 4 (FIG. 2) and HsPSCA RNA according to SEQ ID NO: 5 (FIG. 3) were prepared and used for subsequent in vitro transcription reactions.

[0328] According to a first preparation, the DNA sequences coding for the above mentioned mRNAs were prepared. The constructs were prepared by modifying the wild type coding sequence by introducing a GC-optimized sequence for stabilization, followed by a stabilizing sequence derived from the alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)), a stretch of 64 adenosines (poly-A-sequence), a stretch of 30 cytosines (poly-C-sequence), and a histone stem loop. In FIGS. 1 to 3 the sequences of the corresponding mRNAs are shown.

[0329] The 5 region of the target RNA sequence contains two possible recognition sites, NUH1 (positions 10-12) and NUH2 (positions 11-13), of which NUH2 is the preferred target site. Cleavage occurs 3 the H of the NUH recognition site.

[0330] 2. In Vitro Transcription

[0331] The respective DNA plasmids prepared according to paragraph 1 were transcribed in vitro using T7 RNA polymerase.

[0332] 3. In Vitro Transcription in the Presence of Cap Analog

[0333] For the production of 5-capped RNAs using cap analog, transcription was carried out in 5.8 mM m7G(5.sup.)ppp(5.sup.)G Cap analog, 4 mM ATP, 4 mM CTP, 4 mM UTP, and 1.45 mM GTP (all Thermo Fisher Scientific).

[0334] 4. In Vitro Transcription of Non-Capped RNAs

[0335] For the production of non-capped, 5 triphosphate RNAs, transcription was carried out in the presence of 4 mM of each ATP, GTP, CTP and UTP (all Thermo Fisher Scientific).

[0336] 5. Enzymatic Capping of mRNA

[0337] Enyzmatic capping was performed using the ScriptCap m.sup.7G Capping System (Cell Script) according to the manufacturer's instructions. In brief, per reaction, 60 g of non-capped RNAs were heat-denatured (10 minutes, 65 C.) in a volume of 68.5 l and immediately cooled on ice (5 minutes). Following addition of reaction components (1 ScriptCap Capping buffer, 1 mM GTP, 0.1 mM SAM, 1000 U/ml ScripGuard RNase Inhibitor, 400 U/ml ScriptCap Capping Enzyme) to a final volume of 100 l, reactions were incubated for 1 hour at 37 C. RNAs were precipitated in 2.86 M LiCl for 16 hours at 20 C., followed by centrifugation (30 minutes, 16.000 g, 4 C.). Pellets were washed in 0.5 reaction volumes 75% ethanol (invert, centrifuge 5 minutes, 16.000 g, 4 C.), dried and re-dissolved in H.sub.2O.

[0338] Subsequently the mRNA was purified using PureMessenger (CureVac, Tbingen, Germany; WO2008/077592A1).

TABLE-US-00006 TABLE 2 Target RNAs Length of Description Sequence SEQ (Name) (nucleotides) ID NO Experiment HsNY-ESO-1 mRNA 760 3 Capped mRNA (used for PAGE, FIG. 7) PpLuc mRNA 1870 4 co-transcriptionally capped mRNA (used for HPLC/PAGE) PpLuc mRNA 1870 4 enzymatically capped mRNA (used for HPLC/PAGE) PpLuc mRNA 1870 4 non-capped RNA (used for HPLC/PAGE as no cap control) HsPSCA mRNA 589 5 non-capped RNA (used for PAGE, FIG. 7 as no cap control)

Example 3: Cap Analysis Assay

[0339] 1. Principle of the Assay

[0340] The hammerhead ribozyme HHNUH2d of example 1 was incubated with the in vitro transcribed RNAs of example 2 (Table 2) and the cleavage products were separated by denaturing polyacrylamide gel electrophoresis (PAGE) or high performance liquid chromatography (HPLC).

[0341] 2. Ribozyme Cleavage Reaction

[0342] Reaction scales for gel analysis were usually 1 (10 pmol RNA). For HPLC analysis, 15 reaction (150 pmol RNA) were set up, allowing a more sensitive detection and thus a more precise determination of the respective mRNA populations. Per reaction, 10 pmol of HHNUH2d and 10 pmol of the respective substrate RNA were annealed in 0.625 mM EDTA in a total volume of 6 l (2 min at 95 C., 0.1 C./sec to 25 C., 10 min at 25 C.). After addition of 4 l of 100 mM MgCl.sub.2, 125 mM Tris/HCl, pH 7.5 (final concentration 40 mM MgCl.sub.2, 50 mM Tris/HCl), the reaction was incubated at 25 C. for 1 hour. For analysis via polyacrylamide gel electrophoresis (PAGE), the 1 reaction was stopped with 30 l 95% formamide, 20 mM EDTA. For HPLC analysis, the 15 reaction was stopped with 24 l 250 mM EDTA (final concentration 40 mM).

[0343] 3. Gel Separation, Quantification of Cleavage Products and Calculation of Capping Degree

[0344] Stopped reactions were heat-denatured (heated to 80 C. for 2 minutes, immediately put on ice for 5 minutes) and separated on a 10 cm8 cm1.5 mm 20% denaturing PAGE (8 M urea (AppliChem), 20% acrylamid:bisacrylamid 19:1 (AppliChem), 1TBE, 1% APS (AppliChem), 0.1% TEMED (AppliChem); 180 V, 2 hours, Mini-PROTEAN Tetra Cell (BioRad)). Gels were stained for 10 minutes in 1:10,000 SYBR Gold (Invitrogen) in TBE and documented on a E-BOX VX2 gel documentation system with 312 nm-UV Transilluminator (Peqlab) (excitation maximum for SYBR Gold: 300 nm, emission: 537 nm).

[0345] To determine the capped proportion in the mRNA preparations, bands of the respective 13-mer (derived from the non-capped fraction) or 14-mer (derived from the capped fraction) cleavage products can be quantified using Quantity One 1-D Analysis Software (BioRad).

[0346] The degrees of capped and non-capped RNA, respectively, can be calculated according to:

[00003]$.Math.\mathrm{capped}.Math..Math.\mathrm{RNA}\left(\%\right)=\frac{\text{signalintensity}.Math..Math.14.Math.\mathrm{mer}}{.Math.\text{signalintensities}.Math..Math.\left(13.Math.\mathrm{mer}+14.Math.\mathrm{mer}\right)}100$$\mathrm{non}.Math.\text{-}.Math.\mathrm{capped}.Math..Math.\mathrm{RNA}\left(\%\right)=\frac{\text{signalintensity}.Math..Math.14.Math.\mathrm{mer}}{.Math.\text{signalintensities}.Math..Math.\left(13.Math.\mathrm{mer}+14.Math.\mathrm{mer}\right)}100$

[0347] As can be seen in FIG. 7, the capped and uncapped RNA fragments produced by ribozyme cleavage of long mRNA molecules can be resolved by denaturing PAGE.

[0348] 4. HPLC Separation, Quantification of Cleavage Products and Calculation of Capping Degree

[0349] For the experiment shown in FIG. 8 an mRNA sample was prepared by mixing 60% enzymatically capped mRNA coding for Photinus pyralis Luciferase (PpLuc) and 40% non-capped mRNA coding for Photinus pyralis Luciferase (PpLuc). Subsequently this sample was incubated with the hammerhead (HH) ribozyme HHNU2d as described above and analysed by HPLC.

[0350] Analysis was performed via ion-pair, reversed-phase chromatography on a Dionex Parallel-HPLC U3000 CV-P-1247, equipped with analytical pump (DPG-3600SD), column oven (TCC-3000SD) and UV/Vis-4-channel-detectors (2VWD-3400R5) with analytical SST measuring cell (11 L, 10 mm, for VWD-3x00 detector). An AQUITY UPLC OST C18 column (2.150 mm, 1.7 m particle size; Waters Corporation, Milford, Mass., USA) was used. Column temperature was set to 60 C. Buffer A contained 0.1 M triethylammonium acetate (TEAA), pH 6.8, buffer B 0.1 M TEAA, pH 7.3, 25% acetonitrile. The column was equilibrated with 14% buffer B.

[0351] For sample preparation, HPLC equilibration buffer (86% buffer A, 14% buffer B) was added to the stopped hammerhead ribozyme reactions to obtain a final volume of 1700 l.

[0352] 1650 l of the RNA solution were loaded using a SEMIPREP-Autosampler (WPS-3000SL, Dionex) and run with a stepped gradient beginning with 14% buffer B for 3 minutes, increasing to 19% buffer B over 2 minutes, to 21% buffer B over 9 minutes. 21% buffer B was held for 1 minute, then increased to 100% B over 5 minutes, held for 3.5 minutes, then decreased to 14% buffer B over 1.5 minutes.

[0353] Signal integration was done using Chromeleon software 6.80 SR11 Build 3161 (Dionex). The relative peak areas of capped 5 RNA fragment (Peak 1, FIG. 8B) and non-capped 5 RNA fragments (Peak 2, FIG. 8B) were determined. The degree of capped RNA was calculated by dividing the relative peak area of Peak 1 by the sum of peak areas 1 and 2. Deviation from the expected capping degree was determined by dividing the calculated capping degree by the expected capping degree.

TABLE-US-00007 TABLE 3 Determination of capping degree after HPLC separation of cleavage products Relative Relative % peak area peak area % % Deviation capped Peak 1 Peak 2 capped calculated/ expected capped non-capped calculated expected 60 67.6 32.4 67.6 12.7

[0354] As can be seen from FIG. 8, the capped 5 mRNA fragment, non-capped 5 mRNA fragment, 3 mRNA fragment and hammerhead ribozyme can be separated by HPLC and the capping degree was calculated as explained above.

Example 4: Determination of Cap Orientation

[0355] For the experiment shown in FIG. 9, non-capped, enzymatically capped and co-transcriptionally capped RNA samples encoding Photinus pyralis luciferase (PpLuc) were prepared as described in Example 2. Subsequently, these samples were incubated with the hammerhead (HH) ribozyme HHNU2d as described in Example 3 and analysed in parallel by denaturing polyacrylamide gel electrophoresis (dPAGE) and HPLC. On the dPAGE gel, two bands are detected for co-transcriptionally capped RNA (FIG. 9A), whereas five peaks are detected in the HPLC chromatogram for the same sample (FIG. 9D).

[0356] In order to characterize the peaks in the HPLC chromatogram shown in FIG. 9, they were separated by HPLC according to the protocol described above and collected (fraction collection by time: 12-17 min, 20 sec/fraction) (FIG. 10). Double peaks at 13.30-13.80 min and 14.30-14.80 min could not be separated and were thus pooled prior to Matrix-assisted laser desorption/ionization (MALDI-TOF) analysis (sample S1=fractions 12-14, S2=21-24, S3=28-30). MALDI-TOF mass spectrometry was performed by using an AnchorChip target at the service provider PANAteqs (Heilbronn, Germany).

[0357] Whereas two double peaks were detected by HPLC (FIG. 10), mass spectrometry only revealed a single mass (in addition to minor salt adducts) for each double peak (FIG. 11), corresponding to the expected capped 5 fragment (FIG. 10 Peak S2, FIG. 11B) and a further double peak (FIG. 10 Peak S1, FIG. 11A), respectively. Double peak S1 indicated a capped RNA population lacking one nucleotide in the 5 terminal RNA fragment. It was speculated that in vitro transcription from the template used in Example 2 (SEQ ID NO: 2, see FIG. 2) does not only yield the desired full-length transcript, but also an aberrant transcript lacking the 5 terminal guanine nucleotide of SEQ ID NO: 2. It was further speculated that double peak S1 in the chromatogram of FIG. 9 was derived from said aberrant transcripts.

[0358] To verify the mass spectrometry results indicating a capped 5 terminal RNA fragment derived from an RNA population lacking one guanosine phosphate, enzymatically capped Photinus pyralis luciferase (PpLuc) RNAs lacking the 5 terminal guanosine (Ecap-G1) were synthesized as described above. The RNAs were incubated with hammerhead (HH) ribozyme HHNU2d as described in Example 3 and analysed by HPLC.

[0359] Overlay of the chromatograms of co-transcriptionally capped RNA and the control construct Ecap(-G1) confirmed the MALDI results (FIG. 12), identifying double peak S1 in the chromatogram of FIG. 9 as RNA shortened by one guanosine at the 5 end, i.e. lacking the first G nucleotide in SEQ ID NO: 2. Therein, the first peak of the first double peak (S1) co-eluted with the enzymatically capped shortened control molecule (Ecap(-G1)). Likewise, the first peak of the second double peak (S2) eluted simultaneously with the enzymatically capped control (Ecap), again confirming the MALDI results.

[0360] In enzymatically capped RNA, the cap is present in the correct orientation (correctly capped). As confirmed by the simultaneous elution of the first peak of the double peaks with the capped controls (Ecap and Ecap(-G1), respectively), the respective first peaks thus correspond to correctly capped RNA. The respective second peaks correspond to those RNA molecules, which have, in contrast, incorporated the cap in the reverse orientation (reverse-capped). The reverse-capped fractions displayed delayed elution due to higher hydrophobicity at the 5 end (Dickman, 2011. Ion Pair Reverse-Phase Chromatography: A Versatile Platform for the Analysis of RNA. Chromatography Today, March 2011, p. 22-26). The capping degree for the different populations was calculated as explained above (FIG. 13).

TABLE-US-00008 TABLE 4 Determination of the relative amounts of correctly capped, reverse- capped and non-capped RNA populations after HPLC separation of cleavage products of co-transcriptionally produced mRNA RNA population % of total RNA Correct cap minusG1 13.0 Reverse cap minusG1 14.2 Correct cap full-length 28.8 Reverse cap full-length 38.0 No cap 6.1