Methods for RNA analysis
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.
1. A method for analyzing the cap structure at the 5′ terminus of 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) denaturing the RNA molecule, annealing the denatured RNA molecule with the catalytic nucleic acid molecule under conditions allowing cleavage of the denatured RNA molecule to produce a 5′ terminal RNA fragment and at least one 3′ RNA fragment, and
c) determining the presence a cap structure at the 5′ terminus of the RNA molecule.
2. The method of claim 1 , further comprising separating the RNA fragments obtained in step b).
3. The method of claim 2 , wherein the RNA fragments are separated by denaturing gel electrophoresis or liquid chromatography.
4. The method of claim 3 , wherein liquid chromatography comprises high performance liquid chromatography, fast protein liquid chromatography, or reverse phase liquid chromatography.
5. The method of claim 1 , wherein the catalytic nucleic acid molecule has a specific cleavage site.
6. The method of claim 1 , wherein the cleavage site for the catalytic nucleic acid molecule is located within 50 nucleotides of the 5′ terminus of the RNA molecule.
7. The method of claim 1 , wherein the catalytic nucleic acid molecule is a ribozyme.
8. The method of claim 7 , wherein the ribozyme is a hammerhead ribozyme, a hairpin ribozyme, or an hepatitis delta virus ribozyme.
9. The method of claim 1 , wherein the RNA molecule is generated by in vitro transcription.
10. The method of claim 9 , wherein the in vitro transcription is carried out in the presence of a cap analog.
11. The method of claim 10 , wherein the cap analog is selected from the group consisting of G[5′]ppp[5′]G, m 7 G[5′]ppp[5′]G, m 3 2,2,7 G[5′]ppp[5′]G, m 2 7,3′-O G[5′]ppp[5′]G (3′-ARCA), m 2 7,2′-O GpppG (2′-ARCA), m 2 7,2′-O GppspG D1 (β-S-ARCA D1) and m 2 7,2′-O GppspG D2 (β-S-ARCA D2).
12. The method of claim 9 , wherein the in vitro transcribed RNA molecule is enzymatically capped.
13. The method of claim 1 , wherein the RNA molecule is an mRNA molecule.
14. The method of claim 1 , wherein the RNA molecule comprises at least one modification.
15. The method of claim 1 , wherein determining comprises comparing a structural feature or a physical parameter of the cap structure with that of a reference RNA fragment.
16. The method of claim 1 , wherein step c) comprises spectroscopic analysis, quantitative mass spectrometry, or sequencing.
17. The method of claim 1 , wherein determining comprises determining the orientation of a cap on the 5′ terminus of the RNA molecule.