RNA analysis by total hydrolysis and quantification of released nucleosides

1. A method for analyzing a sample comprising RNA molecules transcribed from a template DNA with an expected sequence and length, comprising the steps of:

a) providing a sample comprising RNA molecules, wherein the RNA molecules comprise RNA molecules that are capped and polyadenylated;

b) completely hydrolyzing the RNA molecules, thereby releasing nucleosides;

c) separating and quantifying the released nucleosides by HPLC, capillary electrophoresis or mass spectrometry;

d) determining at least one of:

(i) the capping degree of the RNA molecules within the sample by using formula 4:

% cap= n ( CA )/ n (RNA)*100%,

 wherein % cap is the percentage of capped RNA molecules, n(CA) is the content of a cap analogue in the sample, and n(RNA) is the number of copies of the RNA molecules in the sample;

(ii) the number of copies of the RNA (n(RNA)) molecules in the sample by using formula 1.1:

n (RNA)=⅓*( n ( C )/ u ( C )+ n ( G )/ u ( G )+ n ( U )/ u ( U )),

 wherein n(C), n(U), and n(G) is the measured content of the corresponding nucleoside and any modifications thereof in the sample, and u(C), u(U), and u(G) is the number of the corresponding nucleoside and any modifications thereof in the expected sequence of the RNA molecules; and/or

(iii) the length of a homopolymeric element in the RNA molecules by using formula 5:

l x m=n ( X )/ n (RNA)−[ u ( X )− l x e],

wherein l x m is the calculated average length of the poly(X) stretch, n(X) is the measured content of a nucleoside X forming the homopolymeric element in the sample, n(RNA) is the number of copies of the RNA molecules in the sample, u(X) is the number of X in the expected sequence of the RNA molecules, l x e is the expected average length of the poly(X) stretch in the RNA molecules.

2. The method of claim 1 , wherein step d) further comprises determining at least one of:

the incorporation and amount of modified nucleotides into the RNA molecules, and

the oxidation status of the RNA molecules.

3. The method of claim 1 , wherein step d) comprises determining:

the number of copies of the RNA molecules in the sample,

the matching of the sequence of the RNA molecules with the expected sequence,

the matching of the length of the RNA molecules with the expected length,

the capping degree of the RNA molecules within the sample,

the length of homopolymeric elements in the RNA molecules,

the incorporation and amount of modified nucleotides into the RNA molecules, and

the oxidation status of the RNA molecules.

4. The method of claim 1 , further comprising transcribing the RNA molecules from a DNA template by in vitro transcription.

5. The method of claim 4 , further comprising a step a1) of purifying the RNA molecules by HPLC.

6. The method of claim 1 , wherein step b) comprises completely hydrolyzing the RNA molecules by treatment with a nuclease, a phosphatase, and a phosphodiesterase.

7. The method of claim 1 , wherein step c) comprises separating and quantifying the released nucleosides by HPLC.

8. The method of claim 1 , wherein step d) comprises determining the number of copies of the RNA (n(RNA)) molecules in the sample by using formula 1.1:

n (RNA)=⅓*( n ( C )/ u ( C )+ n ( G )/ u ( G )+ n ( U )/ u ( U )),

wherein n(C), n(U), and n(G) is the measured content of the corresponding nucleoside and any modifications thereof in the sample, and u(C), u(U), and u(G) is the number of the corresponding nucleoside and any modifications thereof in the expected sequence of the RNA molecules.

9. The method of claim 1 , wherein step d) comprises determining the matching of the sequence of the RNA molecules with the expected sequence and/or matching of the length of the RNA molecules with the expected length by comparing the measured ratio of a specific nucleoside in the sample with the expected ratio of said nucleoside.

10. The method of claim 9 , wherein the measured ratio of a specific nucleoside in the sample is determined using formula 2.1:

rm ( X )= n ( X )/[ n ( C )+ n ( G )+ n ( U )]

and the expected ratio of said nucleoside is determined using formula 3.1:

re ( X )= u ( X )/[ u ( C )+ u ( G )+ u ( U )],

wherein rm is the measured ratio of a specific nucleoside in the sample, re is the expected ratio of the corresponding nucleoside in the RNA molecules, n(C), n(U), and n(G) is the measured content of the corresponding nucleoside and any modifications thereof in the sample, u(C), u(U), and u(G) is the number of the corresponding nucleoside and any modifications thereof in the expected sequence of the RNA molecules, and X is a nucleotide selected from C, G and U and is the same nucleotide for both rm(X) and re(X), wherein a mismatch between the values for rm and re indicates that the sequence of the RNA molecules does not match with the expected sequence and/or that the length of the RNA molecules does not match with the expected length.

11. The method of claim 1 , wherein step d) comprises determining the capping degree of the RNA molecules within the sample by using formula 4:

% cap= n ( CA )/ n (RNA)*100%,

wherein % cap is the percentage of capped RNA molecules, n(CA) is the content of a cap analogue in the sample, and n(RNA) is the number of copies of the RNA molecules in the sample.

12. The method of claim 1 , wherein step d) comprises determining the length of a homopolymeric element in the RNA molecules by using formula 5:

l x m=n ( X )/ n (RNA)−[ u ( X )− l x e ],

wherein l x m is the calculated average length of the poly(X) stretch, n(X) is the measured content of a nucleoside X forming the homopolymeric element in the sample, n(RNA) is the number of copies of the RNA molecules in the sample, u(X) is the number of X in the expected sequence of the RNA molecules, l x e is the expected average length of the poly(X) stretch in the RNA molecules.

13. The method of claim 12 , wherein the homopolymeric element is a poly(A) stretch and wherein the length of the poly (A) stretch is determined by using formula 5.1:

l A m=n ( A )/ n (RNA)−[ u ( A )− l A e ];

wherein l A m is the calculated average length of the poly(A) stretch, n(A) is the measured content of the nucleoside A in the sample, n(RNA) is the number of copies of the RNA molecules in the sample, u(A) is the number of nucleoside A in the expected sequence of the RNA molecules, and l A e is the expected average length of the poly(A) stretch in the RNA molecules.

14. The method of claim 2 , wherein step d) further comprises determining the incorporation of modified nucleosides into the RNA molecules by using formula 6:

% mN=n ( mN )/[ n ( uN )+ n ( mN )]*100%,

wherein % mN is the percent incorporation of a modified nucleoside in the RNA molecules, n(mN) is the measured content of the modified nucleoside in the RNA molecules, and n(uN) is the measured content of the unmodified nucleoside in the RNA molecules.

15. The method of claim 14 , wherein the modified nucleosides are oxidized nucleosides, and wherein % mN indicates the oxidation status of the RNA molecules.

16. The method of claim 1 , wherein the RNA molecules are mRNA molecules.

17. The method of claim 1 , additionally comprising step al) of modifying the RNA molecules.

18. The method of claim 7 , wherein an octadecyl capped silica column is used for separating the released nucleosides by HPLC.

19. The method of claim 1 , wherein step c) comprises separating and quantifying the released nucleosides by capillary electrophoresis.

20. The method of claim 1 , wherein step c) comprises separating and quantifying the released nucleosides by mass spectrometry.