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.

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 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). 
     
     
         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 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). 
     
     
         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 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 .