Methods for rna analysis

Patent metadata

Field	Value
Publication number	US-2022073962-A1
Application number	US-201917416681-A
Country	US
Kind code	A1
Filing date	Dec 20, 2019
Priority date	Dec 21, 2018
Publication date	Mar 10, 2022
Grant date	—

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

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.

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 1 or 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, preferably by HPLC or by an affinity chromatography including an oligo-dT based capturing column chromatography.

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

6 . The method of any one of claims 2 - 5 , 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,
preferably by analytical HPCL and/or mass-spectrometry.

8 . The method of claim 6 or 7 , wherein the 5′ fragment has a length of about 1 to about 100 nucleotides, preferably about 1 to about 50 nucleotides, more preferably about 1 to about 25 nucleotides and most preferably about 10-15 nucleotides.

9 . The method of any one of claims 2 - 6 , wherein the 3′ fragment is analyzed.

10 . The method of claim 9 , wherein the 3′ fragment comprises a homopolymeric sequence, preferably a polyA and/or polyC sequence.

11 . The method of claim 9 or 10 , wherein the 3′ fragment is analyzed for its nucleotide composition and/or length, preferably 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 any one of claims 9 - 11 , wherein the 3′ fragment has a length of about 10 to about 500 nucleotides, preferably about 50 to about 500 nucleotides and more preferably about 50 to about 250 nucleotides.

13 . The method of any one of claims 1 - 6 , wherein the analysis of the fragments involves mass spectroscopy, preferably analytical mass spectrometry, HPLC, preferably analytical HPLC and/or sequencing.

14 . The method of any one of claims 1 - 13 , wherein the RNA molecule is single stranded RNA, preferably a therapeutic mRNA molecule.

15 . The method of any one of claims 1 - 14 , wherein the RNA molecule is generated by RNA in vitro transcription.

16 . The method of any one of claims 1 - 15 , wherein the RNA molecule comprises a 5′ cap structure and/or a 3′ homopolymeric sequence.

17 . The method of any one of claims 1 - 16 , wherein at least two conjugates are provided and contacted at the same time with the RNA molecule to simultaneously cleave the RNA molecule, wherein the at least two conjugates comprise oligonucleotides with sequences complementary to different target sequences of the RNA molecule.

18 . The method of any one of claims 1 - 16 , wherein at least two conjugates are provided and contacted one after the other with the RNA molecule to sequentially cleave the RNA molecule, wherein the at least two conjugates comprise oligonucleotides with sequences complementary to different target sequences of the RNA molecule.

19 . The method of any one of claims 1 - 18 , wherein the target sequence is present once in the RNA molecule.

20 . The method of any one of claims 1 - 18 , wherein the target sequence is present more than once in the RNA molecule.

21 . 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.

22 . 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.

23 . The method of claim 21 or 22 , wherein cleaving the population of RNA molecules results in 5′ fragments, 3′ fragments and optionally central fragments.

24 . The method of any one of claims 21 - 23 , wherein the fragments are separated from each other before analyzing the RNA fragments in step (iv).

25 . The method of claim 24 , wherein the fragments are separated by chromatography, preferably by HPLC or by an affinity chromatography including an oligo-dT based capturing column chromatography.

26 . The method of claim 24 , wherein the fragments are separated by electrophoresis.

27 . The method of any one of claims 21 - 26 , wherein the 5′ fragments are analyzed and/or the 3′ fragments are analyzed.

28 . The method of claim 27 , wherein the 5′ fragments are analyzed for one or more of
(i) presence and/or integrity of the cap structure,
(ii) methylation pattern; and
(iii) orientation,
preferably by analytical HPCL and/or mass-spectrometry.

29 . The method of claim 27 , wherein the 3′ fragments are analyzed.

30 . The method of

Assignees

• Curevac Ag

Inventors

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

Classifications

• C12Q1/6813 Primary

  Hybridisation assays · CPC title

• C12Q2521/30

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

• C12Q2523/107

  Chemical cleaving agents · CPC title

• C12Q1/6806 Primary

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

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title