Rna analysis by total hydrolysis
AssigneeCureVac AG
InventorsFabian Johannes EBER, Aniela WOCHNER, Tilmann ROOS
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Abstract
The present invention relates to analysis of an RNA molecule. It further relates to the use of this method for the quality control of an RNA molecule produced by in vitro transcription or for the quality control of an RNA molecule produced by chemical synthesis.
Claims51
Method for analyzing a sample comprising RNA molecules transcribed from a template DNA with known sequence and length or chemically synthesized with known sequence and length, comprising the steps of:
- a) providing a sample comprising RNA molecules;
- b) completely hydrolyzing the RNA molecules, thereby releasing nucleosides;
- c) separating and quantifying the released nucleosides by HPLC;
- d) determining at least one of:
- the number of copies of the RNA molecules in the sample,
- the matching of the sequence of the RNA molecules with the known sequence,
- the matching of the length of the RNA molecules with the known length,
- the capping degree of the RNA molecules within the sample, and
- the length of homopolymeric elements in the RNA molecules.
Method according to any one of the preceding claims, 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 known sequence,
- the matching of the length of the RNA molecules with the known 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.
Method according to any one of the preceding claims, wherein the RNA molecules are provided by chemical synthesis.
Method according to any one of the preceding claims, wherein step b) comprises completely hydrolyzing the RNA molecules by enzyme treatment.
Method according to any one of the preceding claims, wherein step b) comprises completely hydrolyzing the RNA molecules by treatment with a nuclease, a phosphatase and a phosphodiesterase.
Method according to any one of the preceding claims, wherein in the HPLC of step c) an octadecyl capped silica column is used for separating the released nucleosides.
Method according to any one of the preceding claims, wherein in the HPLC a mobile phase comprising an aqueous solvent is used.
Method according to any one of the preceding claims, wherein step c) further comprises detecting the released nucleosides and identifying the released nucleosides by comparison to a standard for each nucleoside.
Method according to any one of the preceding claims, wherein the released nucleosides are quantified by determining the peak area for each of the nucleosides and comparing it to a standard curve.
Method according to any one of the preceding claims, wherein the RNA molecules are polyadenylated and 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 known sequence of the RNA molecules.
Method according to any one of the preceding claims, wherein step d) comprises determining the matching of the sequence of the RNA molecules with the known sequence and/or matching of the length of the RNA molecules with the known length by comparing the measured ratio of a specific nucleoside in the sample with the expected ratio of said nucleoside.
Method according to any one of the preceding claims, 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.
Method according to any one of the preceding claims, wherein step d) comprises determining the length of a homopolymeric element in the RNA molecules by using formula 5:
- lxm=n(X)/n(RNA)−[u(X)−lxe],
- wherein lxm 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 known sequence of the RNA molecules, lxe is the expected average length of the poly(X) stretch in the RNA molecules.
Method according to any one of the preceding claims, wherein the RNA molecules are mRNA molecules.
Method according to any one of the preceding claims, additionally comprising step al) of modifying the RNA molecules.
Dependencies are inferred from the claim text.