Methods for RNA analysis

Patent metadata

Field	Value
Publication number	US-12492425-B2
Application number	US-201917416681-A
Country	US
Kind code	B2
Filing date	Dec 20, 2019
Priority date	Dec 21, 2018
Publication date	Dec 9, 2025
Grant date	Dec 9, 2025

Abstract

The present invention is concerned with methods for analyzing RNA molecules. The provided methods involve conjugates for RNA cleavage comprising a chemical moiety with RNA cleaving activity and an oligonucleotide. The oligonucleotide is designed based on a target sequence present in an RNA molecule, and the cleavage of the RNA molecule is inter alia carried out at conditions allowing the hybridization of the oligonucleotide to the target 5 sequence. Thereby, the method is easily applicable to RNA molecules of any sequence. The method further involves the analysis of the RNA fragments obtained after cleavage to obtain information on the physical properties of the RNA molecule.

First claim

The invention claimed is:

1 . A method for analyzing an RNA molecule comprising the following steps:
(i) providing an RNA molecule; (ii) providing at least one conjugate comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the sequence of said oligonucleotide is complementary to a target sequence of the RNA molecule; (iii) cleaving the RNA molecule provided in step (i) to obtain RNA fragments by contacting the RNA molecule with the at least one conjugate provided in step (ii) under conditions allowing the hybridization of said oligonucleotide to said target sequence and the cleavage of the RNA molecule; and (iv) determining a physical property of the RNA molecule by analyzing one or more of the RNA fragments obtained in step (iii), wherein the RNA molecule is an mRNA molecule and comprises a 5′ cap structure and a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecule comprise one or more temperature shifts, wherein step (iii) of the method is as follows: (iii) cleaving the RNA molecule provided in step (i) to obtain RNA fragments by contacting the RNA molecule with the conjugate provided in step (ii) at a first temperature between about 5° C. and about 50° C., and at a second temperature between about 70° C. and about 90° C.; followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).

2 . The method of claim 1 , wherein cleaving the RNA molecule results in a 5′ fragment, a 3′ fragment and optionally one or more central fragments.

3 . The method of claim 2 , wherein the fragments are separated from each other before analyzing the one or more of the RNA fragments in step (iv).

4 . The method of claim 3 , wherein the fragments are separated by chromatography.

5 . The method of claim 3 , wherein the fragments are separated by electrophoresis.

6 . The method of claim 3 , wherein the 5′ fragment is analyzed and/or the 3′ fragment is analyzed.

7 . The method of claim 6 , wherein the 5′ fragment is analyzed for one or more of
(i) presence and/or integrity of the cap structure,
(ii) methylation pattern; and
(iii) orientation,
by analytical HPLC and/or mass-spectrometry.

8 . The method of claim 6 , wherein the 5′ fragment has a length of 1 to 100 nucleotides.

9 . The method of claim 3 , wherein the 3′ fragment is analyzed.

10 . The method of claim 9 , wherein the 3′ fragment comprises the sequence.

11 . The method of claim 10 , wherein the 3′ fragment is analyzed for its nucleotide composition and/or length by complete hydrolysis of the 3′ fragment followed by analysis of the individual nucleotides gained thereby by analytical HPLC and/or mass spectrometry.

12 . The method of claim 10 , wherein the 3′ fragment has a length of 10 to 500 nucleotides.

13 . The method of claim 6 , wherein the RNA molecule is 300 to 9,000 nucleotides in length.

14 . The method of claim 1 , wherein the target sequence is present only once in the RNA molecule.

15 . A method for analyzing a population of RNA molecules comprising the following steps:
(i) providing a population of RNA molecules, wherein the population of RNA molecules comprises at least two different types of RNA molecules, wherein the different types of RNA molecules comprise an identical target sequence; (ii) providing a conjugate comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the sequence of said oligonucleotide is complementary to the target sequence; (iii) cleaving the population of RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugate provided in step (ii) under conditions allowing the hybridization of said oligonucleotide to said target sequence and the cleavage of the RNA molecules; and (iv) determining a physical property of the RNA molecules in the population by analyzing one or more of the RNA fragments obtained in step (iii), wherein the RNA molecules are mRNA molecules and each comprise a 5′ cap structure and/or a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecules comprises one or more temperature shifts, wherein step (iii) of the method is as follows: (iii) cleaving the RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugates provided in step (ii) at a first temperature between about 5° C. and about 50° C., and at a second temperature between about 70° C. and about 90° C.; followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).

16 . A method for analyzing a population of RNA molecules comprising the following steps:
(i) providing a population of RNA molecules, wherein the population of RNA molecules comprises at least two different types of RNA molecules, wherein the different types of RNA molecules comprise different target sequences; (ii) providing at least two conjugates comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the oligonucleotide sequence of each conjugate is complementary to one of the different target sequences; (iii) cleaving the population of RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the at least two conjugates provided in step (ii) under conditions allowing the hybridization of said oligonucleotides to said target sequences and the cleavage of the RNA molecules; and (iv) determining a physical property of the RNA molecules in the population by analyzing one or more of the RNA fragments obtained in step (iii), wherein the RNA molecules are mRNA molecules and each comprise a 5′ cap structure and/or a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecules comprises one or more temperature shifts, wherein step (iii) of the method is as follows: (iii) cleaving the RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugates provided in step (ii) at a first temperature between about 5° C. and about 50° C., and at a second temperature between about 70° C. and about 90° C.; followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).

17 . The method of claim 4 , wherein the chromatography is HPLC.

18 . The method of claim 4 , wherein the chromatography is affinity chromatography.

19 . The method of claim 6 , wherein the sequence of said oligonucleotide is complementary to the target sequence of the RNA molecule over a length of 5-25 nucleotides.

20 . The method of claim 12 , wherein the wherein the 3′ fragment has a length 50 to 250 nucleotides.

21 . The method of claim 20 , wherein the RNA molecule is 300 to 9,000 nucleotides in length.

22 . The method of claim 21 , wherein the sequence of said oligonucleotide is complementary to the target sequence of the RNA molecule over a length of 5-25 nucleotides.

23 . The method of claim 22 , wherein the 3′ fragment is analyzed to determine PolyA length.

24 . The method of claim 23 , wherein the chemical moiety with RNA cleaving activity is tris (2-aminobenzimidazol), 1H-Imidazo[1,2-a]imidazole, 5H-Benzimidazo[1,2-a]benzimidazol, Hexahydro-2H-pyrimido[1,2a]pyrimidin-2,8-dion, 2-Aminobenzimidazol, Imidazo[1,2-a]benzimidazol, or 2-Aminochinolin.

Assignees

• CureVac SE

Inventors

• Schwenger Alexander
• Heinz Stefan
• Roos Tilmann
• Yazdan Panah Benyamin
• Wagner Veronika

Classifications

• C12Q2521/30

  Phosphoric diester hydrolysing, i.e. nuclease · CPC title

• C12Q2523/107

  Chemical cleaving agents · CPC title

• C12Q1/6813 Primary

  Hybridisation assays · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title