U.S. patent application number 16/809375 was filed with the patent office on 2020-07-09 for methods for rna analysis.
This patent application is currently assigned to CureVac Real Estate GmbH. The applicant listed for this patent is CureVac Real Estate GmbH. Invention is credited to Aniela WOCHNER.
Application Number | 20200216878 16/809375 |
Document ID | / |
Family ID | 49998192 |
Filed Date | 2020-07-09 |
View All Diagrams
United States Patent
Application |
20200216878 |
Kind Code |
A1 |
WOCHNER; Aniela |
July 9, 2020 |
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.
Inventors: |
WOCHNER; Aniela; (Tubingen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CureVac Real Estate GmbH |
Tubingen |
|
DE |
|
|
Assignee: |
CureVac Real Estate GmbH
Tubingen
DE
|
Family ID: |
49998192 |
Appl. No.: |
16/809375 |
Filed: |
March 4, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15195901 |
Jun 28, 2016 |
10648017 |
|
|
16809375 |
|
|
|
|
PCT/EP2014/003482 |
Dec 30, 2014 |
|
|
|
15195901 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 30/88 20130101;
G01N 2030/8827 20130101; B01D 15/325 20130101; B01D 15/163
20130101; C07H 21/02 20130101; C12Q 1/6806 20130101; C12Q 1/6806
20130101; C12Q 2521/337 20130101; C12Q 2565/137 20130101; C12Q
1/6806 20130101; C12Q 2521/337 20130101; C12Q 2565/125
20130101 |
International
Class: |
C12Q 1/6806 20060101
C12Q001/6806; B01D 15/16 20060101 B01D015/16; B01D 15/32 20060101
B01D015/32; C07H 21/02 20060101 C07H021/02; G01N 30/88 20060101
G01N030/88 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 30, 2013 |
EP |
PCT/EP2013/003947 |
Claims
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.sup.7G[5]ppp[5]G, m.sub.3.sup.2,2,7G[5]ppp[5]G,
m.sub.2.sup.7,3'-OG[5]ppp[5]G (3'-ARCA), m.sub.2.sup.7,2'-OGpppG
(2'-ARCA), m.sub.2.sup.7,2'-OGppspG D1 (.beta.-S-ARCA D1) and
m.sub.2.sup.7,2'-OGppspG D2 (.beta.-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.sup.7G[5]ppp[5]G, m.sub.3.sup.2,2,7G[5]ppp[5]G,
m.sub.2.sup.7,3'-OG[5]ppp[5]G (3'-ARCA), m.sub.2.sup.7,2'-OGpppG
(2'-ARCA), m.sub.2.sup.7,2'-OGppspG D1 (.beta.-S-ARCA D1) and
m.sub.2.sup.7,2'-OGppspG D2 (.beta.-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.sub.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.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/195,901, filed Jun. 28, 2016, which is a continuation of
International Application No. PCT/EP2014/003482, filed Dec. 30,
2014, which claims priority to European Application No.
PCT/EP2013/003947, filed Dec. 30, 2013, the entirety of each of
which is incorporated herein by reference.
[0002] The sequence listing that is contained in the file named
"CRVCP0153USC1.txt", which is 6 KB (as measured in Microsoft
Windows.RTM.) and was created on Mar. 4, 2020, is filed herewith by
electronic submission and is incorporated by reference herein.
[0003] 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. In one
aspect, 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. In particular,
the invention relates to the use of a catalytic nucleic acid
molecule in a method for determining the presence of a cap
structure in an RNA molecule, the use of a catalytic nucleic acid
molecule in a method for determining the capping degree of a
population of RNA molecules, the use of a catalytic nucleic acid
molecule in a method for determining the orientation of the cap
structure in a capped RNA molecule and in a method for determining
the relative amounts of correctly capped RNA molecules and
reverse-capped RNA molecules in a population of RNA molecules.
Furthermore, the present invention provides a 5' terminal RNA
fragment obtainable by the methods according to the invention. In
addition, an RNA molecule is provided, which comprises a cap
structure at its 5' terminus and the sequence motif NUH as defined
herein at its 3' terminus. Further, the invention also relates to
uses of the 5' terminal RNA fragment and the RNA molecule.
[0004] The present invention relates inter alia to a method of
determining the capping degree of a population of RNA molecules
having a cleavage site for a catalytic nucleic acid molecule,
comprising the steps of: [0005] a) providing a sample containing
the population of RNA molecules, [0006] 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, [0007]
c) separating the RNA fragments obtained in step b), [0008] d)
determining a measure for the amount of the capped and non-capped
5' terminal RNA fragments separated in step c) of said population
of RNA molecules, and [0009] 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.
[0010] Furthermore the invention provides the use of a catalytic
nucleic acid molecule for determining the capping degree of a
population of RNA molecules, particularly in the quality control of
the production process of nucleic acids, particularly of RNA.
[0011] Therapeutic RNA molecules represent an emerging class of
drugs. RNA-based therapeutics include mRNA molecules encoding
antigens for use as vaccines. mRNA vaccines combine desirable
immunological properties with the flexibility of genetic vaccines.
In addition, mRNA is considered to be a safer vector than DNA-based
vectors because RNA cannot integrate into genomic DNA possibly
leading to insertional mutagenesis. In addition, it is envisioned
to use mRNA therapeutics for replacement therapies, e.g. providing
missing proteins such as growth factors or enzymes to patients
(Schlake et al., 2012. RNA Biol. 9(11):1319-30).
[0012] Successful protein expression from transfected RNA depends
on transfection efficiency, RNA stability and translation
efficiency. The 5' cap structure and the 3' poly(A) tail are
important features for the efficient translation of mRNA and
protein synthesis in eukaryotic cells. Newly synthesized mRNAs are
usually modified within the producing cell with a 5' cap structure
when the transcript reaches a length of 20 to 30 nucleotides.
First, the 5' terminal nucleotide pppN is converted to 5' GpppN by
a bi-functional capping enzyme containing both RNA
5'-triphosphatase and guanylyltransferase activities. Then the
GpppN part is methylated by a second enzyme with
(guanine-7)-methyltransferase activity to form the monomethylated
m7GpppN type 0 cap structure. The type 0 cap is then converted to
an m7GpppN type 1 structure in the nucleus by 2'-O-methylation
(Tcherepanova et al., 2008. BMC Mol. Biol. 9:90).
[0013] Short RNA molecules can be synthesized by chemical methods,
whereas long RNAs are typically produced by in vitro transcription
using suitable DNA templates with a promoter and RNA polymerases,
for example bacteriophage T3 or T7 RNA polymerases. In principle,
5' cap structures can be introduced into in vitro transcribed RNA
by using one of two protocols.
[0014] In the first protocol, capping occurs concurrently with the
initiation of transcription (co-transcriptional capping). In this
approach, a dinucleotide cap analog such as m7G(5')ppp(5')G (m7G)
is added to the reaction mixture. The DNA template is usually
designed in such a way that the first nucleotide transcribed is a
guanosine. The cap analog directly competes with GTP for
incorporation as initial nucleotide and is incorporated as readily
as any other nucleotide (WO2006/004648). A molar excess of the cap
analog relative to GTP facilitates the incorporation of the cap
dinucleotide at the first position of the transcript. However, this
approach always yields a mixture of capped and uncapped RNAs.
Uncapped mRNAs can usually not be translated after transfection
into eukaryotic cells, thus reducing the efficacy of the RNA
therapeutic.
[0015] The effective concentration of co-transcriptionally capped
mRNAs with the standard cap analog (m7GpppG) is further reduced
because the analog can be incorporated in the reverse orientation
(Gpppm7G), which is less competent for translation (Stepinski et
al., 2001. RNA 7(10):1486-95). The issue of cap analog orientation
can be solved by using anti-reverse cap analogs (ARCA) such as
(3'-O-methyl)GpppG which cannot be incorporated in the reverse
orientation (Grudzien et al., 2004. RNA 10(9):1479-87).
[0016] In the second protocol, capping is done in a separate
enzymatic reaction after in vitro transcription
(post-transcriptional or enzymatic capping). Vaccinia Virus Capping
Enzyme (VCE) possesses all three enzymatic activities necessary to
synthesize a m7G cap structure (RNA 5'-triphosphatase,
ganylyltransferase, and guanine-7-methyltransferase). Using GTP as
substrate the VCE reaction yields RNA caps in the correct
orientation. In addition, a type 1 cap can be created by adding a
second Vaccinia enzyme, 2' O methyltransferase, to the capping
reaction (Tcherepanova et al., 2008. BMC Mol. Biol. 9:90).
[0017] Accordingly, the 5' cap structure is an important feature
for the efficient translation of RNA molecules and protein
synthesis in eukaryotic cells. The presence of non-capped RNA
molecules may reduce the translation efficiency of a population of
RNA molecules and should therefore be avoided or at least reduced.
Therefore it is important to determine the capping degree of a
population of RNA molecules. Since also the orientation of the cap
structure at the 5' terminus of an RNA may influence, for example,
its translation, it is also necessary to determine the orientation
of the cap structure in an RNA molecule.
[0018] For the therapeutic use of RNA in patients a rigorous
quality control of the synthetic RNA is mandatory. For example, the
capping degree needs to be monitored for each production batch
because the capping degree influences the stability and
translational efficiency and thus the pharmacokinetic and
pharmacodynamic properties of the RNA therapeutic. Several
approaches were described for the determination of capping degrees
including gel shift assays and RNaseH cleavage assays.
[0019] For the characterization of novel cap analogs and the
determination of the capping degree of short in vitro transcripts a
gel shift assay was reported (Kore et al., 2008. Bioorg. Med. Chem.
Lett. 18(3):880-4). In the in vitro transcription reaction only ATP
(including .alpha.-.sup.32P ATP for radioactive labeling) and GTP
were used whereas CTP and UTP were omitted. Therefore, only six
nucleotides at the 5' end were transcribed by T7 RNA polymerase.
This setup produces a transcript of short enough length to
distinguish via denaturing gel electrophoresis whether the cap or
regular guanosine was incorporated. Capped RNAs migrate more slowly
than uncapped RNAs, allowing the determination of the relative
incorporation of cap versus unmodified G. As the gel shift assay
requires single nucleotide resolution to distinguish capped from
non-capped RNA, this method is limited to the analysis of short RNA
molecules.
[0020] To measure the percentage of capped RNA in a population of
long RNA molecules, an oligonucleotide-directed RNaseH cleavage
assay was described (Tcherepanova et al., 2008. BMC Mol. Biol.
9:90). A DNA oligonucleotide was annealed in proximity to the 5'
end of the RNA molecule such that the size of the digested products
was 19 nucleotides long for non-capped and 20 nucleotides long for
capped RNA molecules. The digested fragments were radio-labeled and
separated by polyacrylamide gel electrophoresis (PAGE) and
visualized by autoradiography. The interpretation of the resulting
band pattern was complicated by the presence of an additional band
in the uncapped RNA lane possibly resulting from "RNA-oligo hybrid
breathing" or altered conformation due to the absence of a cap
structure. Thus it needs to be assured that the RNaseH cleaves
precisely at the intended position and that the cleavage reaction
proceeds completely which may require further optimization of the
reaction conditions for individual oligonucleotide-RNA pairs.
[0021] In view of the above, there is a continued need for novel
analytical methods to assess the quality of RNA, and especially the
capping degree of RNA, particularly of long RNA molecules.
[0022] It is thus one of the objectives of the present invention to
provide a method for analyzing RNA. In particular, a method shall
be provided, which is suitable for use in quality control during or
following production of RNA, especially of RNA, which is intended
to be used in diagnostic or therapeutic environments. Furthermore,
it is an objective of the present invention to provide a method for
analyzing a mixture of RNA molecules or an RNA population.
SUMMARY OF THE INVENTION
[0023] The present invention relates, inter alia, to a method for
analyzing an RNA molecule having a cleavage site for a catalytic
nucleic acid molecule, the method comprising the steps of: [0024]
a) providing an RNA molecule having a cleavage site for a catalytic
nucleic acid molecule, [0025] 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, [0026] c) determining a physical
property of the RNA molecule by analyzing the 5' terminal RNA
fragment.
[0027] In a preferred embodiment, the method according to the
invention comprises analyzing the 5' terminus, a 5' terminal
modification or a 5' terminal fragment of an RNA molecule.
Preferably, the method for analyzing an RNA molecule according to
the invention comprises determining the presence or absence of a 5'
cap structure at the 5' terminus of an RNA molecule. Further, the
method may comprise determining the orientation of a cap structure
at the 5' terminus of an RNA molecule.
[0028] In another preferred embodiment, the method according to the
invention comprises the analysis of a population of RNA molecules.
Therein, the method preferably comprises determining the relative
amounts of RNA molecules having distinct physical properties, such
as the relative amount of capped RNA molecules, the relative amount
of correctly capped RNA molecules or the relative amount of RNA
molecules having a specific structural feature at the 5'
terminus.
[0029] In a preferred embodiment, the present invention relates to
a method of determining the capping degree of a population of RNA
molecules having a cleavage site for a catalytic nucleic acid
molecule, comprising the steps of: [0030] a) providing a sample
containing the population of RNA molecules, [0031] 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, [0032]
c) separating the RNA fragments obtained in step b), [0033] d)
determining a measure for the amount of the capped and non-capped
5' terminal RNA fragments separated in step c) of said population
of RNA molecules, and [0034] 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.
[0035] In another aspect, the present invention further provides a
novel use of a catalytic nucleic acid molecule for analyzing an RNA
molecule as further defined herein.
[0036] In addition, the invention provides an 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. A 5' terminal fragment is further
provided, which is obtainable by the methods described herein. The
invention also relates to the uses of the RNA molecule or the 5'
terminal fragment as defined herein.
Definitions
[0037] For the sake of clarity and readability the following
definitions are provided. Any technical feature mentioned for these
definitions may be read on each and every embodiment of the
invention. Additional definitions and explanations may be
specifically provided in the context of these embodiments as
discussed and explained further below.
[0038] Population of RNA molecules: In the context of the present
invention, the phrases "population of RNA molecules" or "RNA
population" refers to a plurality of RNA molecules comprising at
least one RNA molecule having a cleavage site for a catalytic
nucleic acid molecule. Preferably, the at least one RNA molecule
having a cleavage site for a catalytic nucleic acid molecule is
characterized by a distinct property or a structural feature, which
may be determined by the method according to the invention. In
addition to the at least one RNA molecule having a cleavage site
for a catalytic nucleic acid molecule, the population may
optionally further comprise at least one other RNA molecule that
does not have such a cleavage site for a catalytic nucleic acid
molecule. In one embodiment, a population of RNA molecules may be a
plurality of identical RNA molecules having a cleavage site for a
catalytic nucleic acid molecule. In another embodiment, a
population of RNA molecules comprises at least two distinct RNA
molecules having a cleavage site for a catalytic nucleic acid
molecule. In that embodiment, the two distinct RNA molecules are
distinct from each other with regard to at least one distinct
physical property or structural feature as defined herein. In a
preferred embodiment, a "population of RNA molecules" in the
context of the present invention, comprises at least two distinct
RNA molecules having a cleavage site for a catalytic nucleic acid
molecule, wherein the at least two distinct RNA molecules differ
from each other only in one physical property or only in one
structural feature, which is preferably located close to the 5'
terminus of the RNA molecules, and wherein the distinct physical
property or the structural feature as defined herein may be
determined by the method according to the invention.
[0039] In a preferred embodiment, the phrase "population of RNA
molecules" refers to a plurality of RNA molecules, which have,
apart from the cap molecule present on some RNA molecules, the same
nucleotide sequence. In other words, the population of RNA
molecules comprises a plurality of capped and non-capped RNA
molecules having the identical nucleotide sequence with the
exception of the presence of a cap structure at the 5' end of
capped RNA molecules.
[0040] According to the invention, said RNA molecules of the
population contain a cleavage site for a catalytic nucleic acid
molecule, allowing the cleavage of the RNA molecules into
fragments, which can then be separated and detected. In this
context, said RNA molecules can be isolated RNA molecules.
[0041] In a further preferred embodiment, the phrase "population of
RNA molecules" refers to a plurality of RNA molecules, wherein at
least RNA molecule is capped and has a cleavage site for a
catalytic nucleic acid molecule and wherein the orientation of the
cap may be determined by the method according to the invention.
[0042] Catalytic nucleic acid molecule: By "catalytic nucleic acid
molecule" it is meant a nucleic acid molecule capable of catalyzing
reactions including, but not limited to, site-specific cleavage of
other nucleic acid molecules.
[0043] In a preferred embodiment, the term "catalytic nucleic acid
molecule" means a nucleic acid molecule with endonuclease activity.
Such a molecule with endonuclease activity may have complementarity
in a substrate binding region to a specified binding site in a
nucleic acid target, and also has an enzymatic activity that
specifically cleaves RNA or DNA in that target at a specific
cleavage site. Therefore, the nucleic acid molecule with
endonuclease activity is able to intramolecularly (in cis) or
intermolecularly (in trans) cleave RNA or DNA. This complementarity
functions to allow sufficient hybridization of the catalytic
nucleic acid molecule to the target RNA or DNA and thereby allowing
the cleavage of the target RNA or DNA at a specific cleavage site.
In this context, 100% complementarity in the substrate binding
region of the catalytic nucleic acid molecule to the binding site
of the nucleic acid target is preferred, but complementarity of at
least 50%, of at least 60%, of at least 70%, more preferably of at
least 80 or 90% and most preferably of at least 95% may also be
useful in this invention.
[0044] The catalytic nucleic acid molecule may contain modified
nucleotides, which may be modified at the base, sugar, and/or
phosphate groups. The term catalytic nucleic acid is used
interchangeably with phrases such as enzymatic nucleic acid or
nucleic acid enzyme. All of these terminologies describe nucleic
acid molecules with enzymatic activity. The specific enzymatic
nucleic acid molecules described in the instant application are not
limiting in the invention and those skilled in the art will
recognize that all that is important in an enzymatic nucleic acid
molecule is that it has a specific substrate binding region which
is complementary to one or more binding sites of the target nucleic
acid, and that it has nucleotide sequences within or surrounding
that substrate binding region which impart a nucleic acid cleaving
activity to the molecule. The term "catalytic nucleic acid
molecule" includes ribozymes and DNAzymes as defined below.
[0045] Ribozyme: A ribozyme is a catalytic nucleic acid molecule
which is an RNA molecule capable of catalyzing reactions including,
but not limited to, site-specific cleavage of other nucleic acid
molecules such as RNA molecules. The term ribozyme is used
interchangeably with phrases such as catalytic RNA, enzymatic RNA,
or RNA enzyme.
[0046] In the early 80s natural RNA molecules were discovered which
are capable of catalyzing reactions in the absence of any protein
component and these molecules were named ribozymes. Several classes
of ribozymes occurring in natural systems have been discovered,
most of which catalyse intramolecular splicing or cleavage
reactions (reactions `in cis`). Since most of the naturally
occurring ribozymes catalyse self-splicing or self-cleavage
reactions, it was necessary to convert them into RNA enzymes which
can cleave or modify target RNAs without becoming altered
themselves (reactions `in trans`).
[0047] Ribozymes are broadly grouped into two classes based on
their size and reaction mechanisms: large and small ribozymes. The
first group consists of the self-splicing group I and group II
introns as well as the RNA component of RNase P, whereas the latter
group includes the hammerhead,
hairpin, hepatitis delta ribozymes and varkud satellite (VS) RNA as
well as artificially selected nucleic acids. Large ribozymes
consist of several hundreds up to 3000 nucleotides and they
generate reaction products with a free 3'-hydroxyl and 5'-phosphate
group. In contrast, small catalytically active nucleic acids from
30 to .about.150 nucleotides in length generate products with a
2'-3'-cyclic phosphate and a 5'-hydroxyl group (Schubert and
Kurreck, 2004. Curr. Drug Targets 5(8):667-681).
[0048] Group I introns include the self-splicing intron in the
pre-ribosomal RNA of the ciliate Tetrahymena thermophilia. Further
examples of group I introns interrupt genes for rRNAs, tRNAs and
mRNAs in a wide range of organelles and organisms. Group I introns
perform a splicing reaction by a two-step transesterification
mechanism: The reaction is initiated by a nucleophilic attack of
the 3'-hydroxyl group of an exogenous guanosine cofactor on the
5'-splice site. Subsequently, the free 3'-hydroxyl of the upstream
exon performs a second nucleophilic attack on the 3'-splice site to
ligate both exons and release the intron. Substrate specificity of
group I introns is achieved by an Internal Guide Sequence (IGS).
The catalytically active site for the transesterification reaction
resides in the intron, which can be re-engineered to catalyse
reactions in trans.
[0049] Group II introns are found in bacteria and in organellar
genes of eukaryotic cells. They catalyse a self-splicing reaction
that is mechanistically distinct from group I introns because they
do not require a guanosine cofactor. Instead, the 2'-hydroxyl of a
specific adenosine at the so-called branch site of the intron
initiates the reaction by a nucleophilic attack on the splice-site
to form a lariat-type structure.
[0050] RNase P was the first example of a catalytic RNA that acts
in trans on multiple substrates. RNase P can be considered to be
the only true naturally occurring trans-cleaving RNA enzyme known
to date. However, for full enzymatic activity under in vivo
conditions the protein component is essential.
[0051] The hammerhead ribozyme is found in several plant virus
satellite RNAs, viroids and transcripts of a nuclear satellite DNA
of newt. This ribozyme is the smallest of the naturally occurring
ribozymes and processes the linear concatamers that are generated
during the rolling circle replication of circular RNA plant
pathogens. The development of hammerhead variants that cleave
target RNA molecules in trans was a major advancement that made
possible the use of ribozyme technology for practical applications.
The hammerhead ribozyme motif that has widely been applied since
then comprises three helical sections connected via a three-way
helical junction.
[0052] In hairpin ribozymes the catalytic entity is part of a
four-helix junction. A minimal catalytic motif containing
approximately 50 nucleotides has been identified that can be used
for metal-ion dependent cleavage reactions in trans. It consists of
two domains, each harbouring two helical regions separated by an
internal loop, connected by a hinge region. One of these domains
results from the association of 14 nucleotides of a substrate RNA
with the ribozyme via base-pairing.
[0053] The hepatitis delta virus (HDV) ribozyme is found in a
satellite virus of hepatitis B virus. Both the genomic and the
antigenomic strand express cis-cleaving ribozymes of .about.85
nucleotides that differ in sequence but fold into similar secondary
structures. The crystal structure of the ribozyme reveals five
helical regions are organized by two pseudoknot structures. The
catalytic mechanism of the hepatitis delta virus ribozyme appears
to involve the action of a cytosine base within the catalytic
centre as a general acid-base catalyst. The hepatitis delta
ribozyme displays high resistance to denaturing agents like urea or
formamide. Trans-cleaving derivatives of this ribozyme have been
developed.
[0054] The Varkud Satellite (VS) ribozyme is a 154 nucleotide long
and is transcribed from a plasmid discovered in the mitochondria of
certain strains of Neurospora. The VS ribozyme is the largest of
the known nucleolytic ribozymes.
[0055] DNAzyme: A DNAzyme is a catalytic nucleic acid molecule
which is a DNA molecule capable of catalyzing reactions including,
but not limited to, site-specific cleavage of other nucleic acid
molecules such as RNA molecules. The term DNAzyme is used
interchangeably with phrases such as catalytic DNA, enzymatic DNA,
or DNA enzyme.
[0056] DNAzymes are intrinsically more stable than ribozymes made
of RNA. Although DNAzymes have not been found in nature, artificial
DNAzymes such as "10-23" DNAzymes have been obtained by using in
vitro selection methods (Schubert and Kurreck, 2004. Curr. Drug
Targets 5(8):667-681).
[0057] One of the most active DNAzymes is the RNA-cleaving "10-23"
DNAzyme which was generated by an in vitro selection method
(Santoro et al., 1997. Proc. Natl. Acad. Sci. USA 94(9):4262-6).
10-23 DNAzymes consist of a catalytic core of about 15 nucleotides
and two substrate binding arms of variable length and sequence. The
10-23 DNAzyme cleaves its RNA substrate using divalent ions to
yield a 2'-3'-cyclo phosphate and a free 5'-hydroxyl group.
[0058] 10-23 DNAzymes can be designed and used to cleave almost any
target RNA in a sequence-specific manner. Consisting of a catalytic
core of 15 nucleotides and two substrate-binding arms of variable
length and sequence, they bind the target RNA in a
sequence-specific manner and cleave it between a paired pyrimidine
base and a free purine base (Schubert et al., 2003. Nucleic Acids
Res. 31(20):5982-92). For example, the DNAzyme cleavage reaction
can be performed by incubating the DNAzyme and the substrate RNA in
cleavage buffer (10 mM MgCl.sub.2, 50 mM Tris-HCl, pH7.5) at
37.degree. C. Prior to mixing the enzyme and the substrate RNA,
both solutions are denatured separately for 5 minutes at 85.degree.
C. Methods for the production of DNAzymes are known in the art. For
example, DNAzymes can be chemically synthesized using standard DNA
synthesis methods (Schubert et al., 2003. Nucleic Acids Res.
31(20):5982-92).
[0059] 5'-Cap structure: A 5' cap is typically a modified
nucleotide, particularly a guanine nucleotide, added to the 5' end
of an RNA molecule. Preferably, the 5' cap is added using a
5'-5'-triphosphate linkage. A 5' cap may be methylated, e.g.
m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic
acid carrying the 5' cap, typically the 5'-end of an RNA. The
naturally occurring 5' cap is m7GpppN.
[0060] Further examples of 5'cap structures include glyceryl,
inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide,
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide,
carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,
L-nucleotides, alpha-nucleotide, modified base nucleotide,
threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide,
acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl
nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic
moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic
moiety, 1,4-butanediol phosphate, 3'-phosphoramidate,
hexylphosphate, aminohexyl phosphate, 3'-phosphate,
3'phosphorothioate, phosphorodithioate, or bridging or non-bridging
methylphosphonate moiety.
[0061] Particularly preferred 5' cap structures are CAP1
(methylation of the ribose of the adjacent nucleotide of m7G), CAP2
(methylation of the ribose of the 2.sup.nd nucleotide downstream of
the m7G), CAP3 (methylation of the ribose of the 3.sup.rd
nucleotide downstream of the m7G), CAP4 (methylation of the ribose
of the 4.sup.th nucleotide downstream of the m7G), A 5' cap
structure may be formed by a Cap analog.
[0062] Cap analog: A cap analog refers to a non-extendable
di-nucleotide that has cap functionality which means that it
facilitates translation or localization, and/or prevents
degradation of the RNA molecule when incorporated at the 5' end of
the RNA molecule. Non-extendable means that the cap analog will be
incorporated only at the 5'terminus because it does not have a 5'
triphosphate and therefore cannot be extended in the 3' direction
by a template-dependent RNA polymerase. Cap analogs include, but
are not limited to, a chemical structure selected from the group
consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs
(e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG),
trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated
symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap
analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG,
m7,3'dGpppG and their tetraphosphate derivatives) (Stepinski et
al., 2001. RNA 7(10):1486-95).
[0063] Examples of cap analogs are shown in Table 1.
TABLE-US-00001 TABLE 1 Cap analogs (D1 and D2 denote counterpart
diastereoisomers) Triphosphate cap analog Tetraphosphate cap analog
m.sup.7Gp.sub.3G m.sup.7Gp.sub.4G m.sub.2.sup.7,3'-OGp.sub.3G
b.sup.7Gp.sub.4G b.sup.7Gp.sub.3G b.sup.7m.sup.3'-OGp.sub.4G
e.sup.7Gp.sub.3G m.sub.2.sup.2,7Gp.sub.4G m.sub.2.sup.2,7Gp.sub.3G
m.sub.3.sup.2,2,7Gp.sub.4G m.sub.3.sup.2,2,7Gp.sub.3G
b.sup.7m.sup.2Gp.sub.4G m.sup.7Gp.sub.32'dG m7Gp.sup.4m.sup.7G
m.sup.7Gp.sub.3m.sup.2-OG m.sup.7Gp.sub.3m.sup.7G
m.sub.2.sup.7,2'-OGp.sub.3G m.sub.2.sup.7,2'-OGpppsG (D1)
m.sub.2.sup.7,2'-OGpppsG (D2) m.sub.2.sup.7,2'-OGppspG (D1)
m.sub.2.sup.7,2'-OGppspG (D2) m.sub.2.sup.7,2'-OGpsppG (D1)
m.sub.2.sup.7,2'-OGpsppG (D2)
[0064] Further cap analogs have been described previously (U.S.
Pat. No. 7,074,596, WO2008/016473, WO2008/157688, WO2009/149253,
WO2011/015347, and WO2013/059475). The synthesis of
N.sup.7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs
has been described recently (Kore et al., 2013. Bioorg. Med. Chem.
21(15):4570-4).
[0065] Particularly preferred cap analogs are G[5]ppp[5]G,
m.sup.7G[5]ppp[5]G, m.sub.3.sup.2,2,7G[5]ppp[5]G,
m.sub.2.sup.7,3'-OG[5]ppp[5]G (3'-ARCA), m.sub.2.sup.7,2'-OGpppG
(2'-ARCA), m.sub.2.sup.7,2'-OGppspG D1 (.beta.-S-ARCA D1) and
m.sub.2.sup.7,2'-OGppspG D2 (.beta.-S-ARCA D2).
[0066] Nucleic acid: The term nucleic acid means any DNA- or
RNA-molecule and is used synonymous with polynucleotide.
Furthermore, modifications or derivatives of the nucleic acid as
defined herein are explicitly included in the general term "nucleic
acid". For example, peptide nucleic acid (PNA) is also included in
the term "nucleic acid".
[0067] Monocistronic RNA: A monocistronic RNA may typically be an
RNA, preferably an mRNA, that comprises only one open reading
frame. An open reading frame in this context is a sequence of
several nucleotide triplets (codons) that can be translated into a
peptide or protein.
[0068] Bi-/multicistronic RNA: RNA, preferably mRNA, that typically
may have two (bicistronic) or more (multicistronic) open reading
frames (ORF). An open reading frame in this context is a sequence
of several nucleotide triplets (codons) that can be translated into
a peptide or protein.
[0069] Nucleotide analogs: Nucleotide analogs are nucleotides
structurally similar (analog) to naturally occurring nucleotides
which include phosphate backbone modifications, sugar
modifications, or modifications of the nucleobase.
[0070] Nucleic acid synthesis: Nucleic acid molecules used
according to the invention as defined herein may be prepared using
any method known in the art, including synthetic methods such as
e.g. solid phase synthesis, in vivo propagation (e.g. in vivo
propagation of viruses), as well as in vitro methods, such as in
vitro transcription reactions.
[0071] For preparation of a nucleic acid molecule, especially if
the nucleic acid is in the form of an
[0072] RNA or mRNA, a corresponding DNA molecule may e.g. be
transcribed in vitro. This DNA template preferably comprises a
suitable promoter, e.g. a T7 or SP6 promoter, for in vitro
transcription, which is followed by the desired nucleotide sequence
coding for the nucleic acid molecule, e.g. mRNA, to be prepared and
a termination signal for in vitro transcription. The DNA molecule,
which forms the template of the at least one RNA of interest, may
be prepared by fermentative proliferation and subsequent isolation
as part of a plasmid which can be replicated in bacteria. Plasmids
which may be mentioned as suitable for the present invention are
e.g. the plasmids pT7 Ts (GenBank accession number U26404; Lai et
al., Development 1995, 121: 2349 to 2360), pGEM.RTM. series, e.g.
pGEM.RTM.-1 (GenBank accession number X65300; from Promega) and
pSP64 (GenBank accession number X65327); cf. also Mezei and Storts,
Purification of PCR Products, in: Griffin and Griffin (ed.), PCR
Technology: Current Innovation, CRC Press, Boca Raton, Fla.,
2001.
[0073] RNA: RNA is the usual abbreviation for ribonucleic acid. It
is a nucleic acid molecule, i.e. a polymer consisting of
nucleotides. These nucleotides are usually adenosine-monophosphate,
uridine-monophosphate, guanosine-monophosphate and
cytidine-monophosphate monomers which are connected to each other
along a so-called backbone. The backbone is formed by
phosphodiester bonds between the sugar, i.e. ribose, of a first and
a phosphate moiety of a second, adjacent monomer. The specific
succession of the monomers is called the RNA-sequence.
[0074] Messenger RNA (mRNA): In eukaryotic cells, transcription is
typically performed inside the nucleus or the mitochondria. In
vivo, transcription of DNA usually results in the so-called
premature RNA which has to be processed into so-called messenger
RNA, usually abbreviated as mRNA. Processing of the premature RNA,
e.g. in eukaryotic organisms, comprises a variety of different
posttranscriptional modifications such as splicing, 5'-capping,
polyadenylation, export from the nucleus or the mitochondria and
the like. The sum of these processes is also called maturation of
mRNA. The mature messenger RNA usually provides the nucleotide
sequence that may be translated into an amino acid sequence of a
particular peptide or protein. Typically, a mature mRNA comprises a
5' cap, a 5'UTR, an open reading frame, a 3'UTR and a poly(A)
sequence. In the context of the present invention, an mRNA may also
be an artificial molecule, i.e. a molecule not occurring in nature.
This means that the mRNA in the context of the present invention
may, e.g., comprise a combination of a 5'UTR, open reading frame,
3'UTR and poly(A) sequence, which does not occur in this
combination in nature.
[0075] Self-replicating RNA (Replicons): Self-replicating RNA are
delivery vectors based on alphaviruses which have been developed
from Semliki Forest virus (SFV), Sindbis (SIN) virus, and
Venezuelan equine encephalitis (VEE) virus. Alphaviruses are single
stranded RNA viruses in which heterologous genes of interest may
substitute for the alphavirus' structural genes. By providing the
structural genes in trans, the replicon RNA is packaged into
replicon particles (RP) which may be used for gene therapy purposes
or genetic vaccination (see for example Vander Veen et al., 2012.
Alphavirus replicon vaccines. Animal Health Research Reviews, p.
1-9). After entry into the host cell, the genomic viral RNA
initially serves as an mRNA for translation of the viral
nonstructural proteins (nsPs) required for initiation of viral RNA
amplification. RNA replication occurs via synthesis of a
full-length minusstrand intermediate that is used as the template
for synthesis of additional genome-length RNAs and for
transcription of a plus-strand subgenomic RNA from an internal
promoter. Such RNA may then be considered as self-replicating RNA,
since the non-structural proteins responsible for replication (and
transcription of the heterologous genes) are still present in such
replicon. Such alphavirus vectors are referred to as
"replicons."
[0076] Replicon particle: A replicon particle consist of two or
three parts: i) the genetic material (=the replicon) (comprising
viral genes and optional substituted heterologous genes) made from
either DNA or RNA; ii) a protein coat that protects these genes;
and in some cases iii) an envelope of lipids that surrounds the
protein coat when they are outside a cell.
[0077] Sequence of a nucleic acid molecule: The sequence of a
nucleic acid molecule is typically understood to be the particular
and individual order, i.e. the succession of its nucleotides.
[0078] Sequence identity: Two or more sequences are identical if
they exhibit the same length and order of nucleotides or amino
acids. The percentage of identity typically describes the extent to
which two sequences are identical, i.e. it typically describes the
percentage of nucleotides that correspond in their sequence
position with identical nucleotides of a reference-sequence. For
determination of the degree of identity, the sequences to be
compared are considered to exhibit the same length, i.e. the length
of the longest sequence of the sequences to be compared. This means
that a first sequence consisting of 8 nucleotides is 80% identical
to a second sequence consisting of 10 nucleotides comprising the
first sequence. In other words, in the context of the present
invention, identity of sequences preferably relates to the
percentage of nucleotides of a sequence which have the same
position in two or more sequences having the same length. Gaps are
usually regarded as non-identical positions, irrespective of their
actual position in an alignment.
[0079] Fragment of a sequence: A fragment of a sequence is
typically a shorter portion of a full-length sequence of e.g. a
nucleic acid sequence or an amino acid sequence. Accordingly, a
fragment of a sequence, typically, consists of a sequence that is
identical to the corresponding stretch or corresponding stretches
within the full-length sequence. A preferred fragment of a sequence
in the context of the present invention, consists of a continuous
stretch of entities, such as nucleotides or amino acids,
corresponding to a continuous stretch of entities in the molecule
the fragment is derived from, which represents at least 5%,
preferably at least 20%, preferably at least 30%, more preferably
at least 40%, more preferably at least 50%, even more preferably at
least 60%, even more preferably at least 70%, and most preferably
at least 80% of the total (i.e. full-length) molecule from which
the fragment is derived. It is particularly preferred that the
fragment of a sequence is a functional fragment, i.e. that the
fragment fulfils one or more of the functions fulfilled by the
sequence the fragment is derived from.
[0080] Fragments of nucleic acids: "Fragments" of nucleic acid
sequences in the context of the present invention may comprise a
sequence of a nucleic acid as defined herein, which is, with regard
to its nucleic acid molecule 5'-, 3'- and/or intrasequentially
truncated compared to the nucleic acid molecule of the original
(native) nucleic acid molecule. A sequence identity with respect to
such a fragment as defined herein may therefore preferably refer to
the entire nucleic acid as defined herein.
[0081] Transfection: The term `transfection` refers to the
introduction of nucleic acid molecules, such as DNA or RNA (e.g.
mRNA) molecules, into cells, preferably into eukaryotic cells. In
the context of the present invention, the term `transfection`
encompasses any method known to the skilled person for introducing
nucleic acid molecules, preferably RNA molecules, into cells,
preferably into eukaryotic cells, such as into mammalian cells.
Such methods encompass, for example, electroporation, lipofection,
e.g. based on cationic lipids and/or liposomes, calcium phosphate
precipitation, nanoparticle based transfection, virus based
transfection, or transfection based on cationic polymers, such as
DEAE-dextran or polyethylenimine etc.
[0082] Open reading frame: An open reading frame (ORF) in the
context of the invention may typically be a sequence of several
nucleotide triplets which may be translated into a peptide or
protein. An open reading frame preferably contains a start codon,
i.e. a combination of three subsequent nucleotides coding usually
for the amino acid methionine (ATG or AUG), at its 5'-end and a
subsequent region which usually exhibits a length which is a
multiple of 3 nucleotides. An ORF is preferably terminated by a
stop codon (e.g., TAA, TAG, TGA). Typically, this is the only stop
codon of the open reading frame. Thus, an open reading frame in the
context of the present invention is preferably a nucleotide
sequence, consisting of a number of nucleotides that may be divided
by three, which starts with a start codon (e.g. ATG or AUG) and
which preferably terminates with a stop codon (e.g., TAA, TGA, or
TAG or UAA, UAG, UGA, respectively). The open reading frame may be
isolated or it may be incorporated in a longer nucleic acid
sequence, for example in a vector or an mRNA. An open reading frame
may also be termed "protein coding region" or "coding region".
[0083] 5'-untranslated region (5'-UTR): A 5'-UTR is typically
understood to be a particular section of messenger RNA (mRNA). It
is located 5' of the open reading frame of the mRNA. Typically, the
5'-UTR starts with the transcriptional start site and ends one
nucleotide before the start codon of the open reading frame. The
5'-UTR may comprise elements for controlling gene expression, also
called regulatory elements. Such regulatory elements may be, for
example, ribosomal binding sites. The 5'-UTR may be
post-transcriptionally modified, for example by addition of a 5'
cap structure. In the context of the present invention, the term
"5'-UTR" typically refers to the sequence of an mRNA, which is
located between the 5' cap structure and the start codon.
Preferably, the 5'-UTR is the sequence, which extends from a
nucleotide located 3' to the 5' cap structure, preferably from the
nucleotide located immediately 3' to the 5' cap structure, to a
nucleotide located 5' to the start codon of the protein coding
region (or ORF), preferably to the nucleotide located immediately
5' to the start codon of the protein coding region.
DETAILED DESCRIPTION OF THE INVENTION
[0084] In a first aspect, the present invention relates to a method
for analyzing an RNA molecule having a cleavage site for a
catalytic nucleic acid molecule. In particular, the invention
relates to 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.
[0085] It has been found by the inventors that the generation of 5'
terminal fragments by using a catalytic nucleic acid molecule and
subsequent determination of a physical property of said fragment is
particularly useful in methods typically employed in quality
control of RNA having a cleavage site for the catalytic nucleic
acid molecule. Advantageously, the method according to the
invention allows specific and rapid analysis of RNA molecules
during or following RNA production, preferably RNA production by in
vitro transcription.
[0086] In general, the method according to the invention is not
limited with respect to the type of RNA molecule to be analyzed.
Preferably, the RNA molecule having a cleavage site for a catalytic
nucleic acid molecule is an RNA molecule as defined herein. For
example, the RNA molecule to be analyzed may be an single-stranded
or a double-stranded RNA, preferably, without being limited
thereto, an RNA oligonucleotide (oligoribonucleotide), preferably a
short oligonucleotide, a coding RNA, a messenger RNA (mRNA), an
immunostimulatory RNA, a ribosomal RNA (rRNA), a transfer RNA
(tRNA), a viral RNA (vRNA), a self-replicating RNA (replicon), a
small interfering RNA (siRNA), a microRNA, a small nuclear RNA
(snRNA), a small-hairpin (sh) RNA or riboswitch, a ribozyme, or an
aptamer. Preferably the RNA molecule is a primary microRNA
(pri-miRNA) molecule. It is known that miRNAs are first transcribed
as a largely unstructured precursor, termed a primary miRNA
(pri-miRNA), which is sequentially processed in the nucleus, to
give the approximately 65-nt pre-miRNA hairpin intermediate, and
then in the cytoplasm, to give the mature miRNA. These pre-miRNA
molecules can be capped and polyadenylated (Cal et al., 2004. RNA
10(12):1957-66).
[0087] Further preferably, the RNA molecule having a cleavage site
for the catalytic nucleic acid molecule comprises at least one open
reading frame (ORF) encoding at least one peptide or protein. More
preferably, the RNA molecule is a (linear) single-stranded RNA,
even more preferably an mRNA or an immunostimulatory RNA. In the
context of the present invention, an mRNA is typically an RNA,
which is composed of several structural elements, e.g. an optional
5' terminal cap structure, an optional 5'-UTR region, an upstream
positioned ribosomal binding site followed by a coding region (open
reading frame, ORF), an optional 3'-UTR region, which may be
followed by a poly-A tail, a poly-C-tail, and/or a histone
stem-loop sequence. An mRNA may occur as a mono-, di-, or even
multicistronic RNA, i.e. an RNA, which carries the coding sequences
of one, two or more proteins or peptides. Such coding sequences in
di-, or even multicistronic mRNA may be separated by at least one
IRES sequence, e.g. as defined herein.
[0088] In a preferred embodiment of the invention, the inventive
method is for analyzing an RNA molecule having a cleavage site for
a catalytic nucleic acid molecule, wherein the RNA molecule
comprises at least one modification. In the context of the
invention, an RNA molecule having at least one modification is also
referred to as "modified RNA molecule". Therein, the modification
is not limited to any particular structure. Preferably, the
structural modification is a structural feature that is typically
not found in the respective naturally occurring RNA, but is
preferably introduced in an artificial RNA molecule, preferably in
an artificial mRNA molecule. Several RNA modifications are known in
the art, which can be applied to a given RNA in the context of the
present invention. In the following, some exemplary modifications
are described.
[0089] Chemical Modifications:
[0090] The term "RNA modification" as used herein may refer to
chemical modifications comprising backbone modifications as well as
sugar modifications or base modifications.
[0091] In this context, the modified RNA molecule as defined herein
may contain nucleotide analogues/modifications, e.g. backbone
modifications, sugar modifications or base modifications. A
backbone modification in connection with the present invention is a
modification, in which phosphates of the backbone of the
nucleotides contained in an RNA molecule as defined herein are
chemically modified. A sugar modification in connection with the
present invention is a chemical modification of the sugar of the
nucleotides of the RNA molecule as defined herein. Furthermore, a
base modification in connection with the present invention is a
chemical modification of the base moiety of the nucleotides of the
RNA molecule. In this context, nucleotide analogues or
modifications are preferably selected from nucleotide analogues
which are applicable for transcription and/or translation.
[0092] Sugar Modifications:
[0093] The modified nucleosides and nucleotides, which may be
incorporated into the modified RNA as described herein, can be
modified in the sugar moiety. For example, the 2' hydroxyl group
(OH) can be modified or replaced with a number of different "oxy"
or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group
modifications include, but are not limited to, alkoxy or aryloxy
(--OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or
sugar); polyethyleneglycols (PEG), -0(CH2CH2o)nCH2CH2OR; "locked"
nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by
a methylene bridge, to the 4' carbon of the same ribose sugar; and
amino groups (--O-amino, wherein the amino group, e.g., NRR, can be
alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,
heteroarylamino, or diheteroaryl amino, ethylene diamine,
polyamino) or aminoalkoxy.
[0094] "Deoxy" modifications include hydrogen, amino (e.g. NH2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid); or the amino
group can be attached to the sugar through a linker, wherein the
linker comprises one or more of the atoms C, N, and O.
[0095] The sugar group can also contain one or more carbons that
possess the opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, a modified RNA can include
nucleotides containing, for instance, arabinose as the sugar.
[0096] Backbone Modifications:
[0097] The phosphate backbone may further be modified in the
modified nucleosides and nucleotides, which may be incorporated
into the modified RNA, as described herein. The phosphate groups of
the backbone can be modified by replacing one or more of the oxygen
atoms with a different substituent. Further, the modified
nucleosides and nucleotides can include the full replacement of an
unmodified phosphate moiety with a modified phosphate as described
herein. Examples of modified phosphate groups include, but are not
limited to, phosphorothioate, phosphoroselenates, borano
phosphates, borano phosphate esters, hydrogen phosphonates,
phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
Phosphorodithioates have both non-linking oxygens replaced by
sulfur. The phosphate linker can also be modified by the
replacement of a linking oxygen with nitrogen (bridged
phosphoroamidates), sulfur (bridged phosphorothioates) and carbon
(bridged methylene-phosphonates).
[0098] Base Modifications:
[0099] The modified nucleosides and nucleotides, which may be
incorporated into the modified RNA, as described herein, can
further be modified in the nucleobase moiety. Examples of
nucleobases found in RNA include, but are not limited to, adenine,
guanine, cytosine and uracil. For example, the nucleosides and
nucleotides described herein can be chemically modified on the
major groove face. In some embodiments, the major groove chemical
modifications can include an amino group, a thiol group, an alkyl
group, or a halo group.
[0100] In particularly preferred embodiments of the present
invention, the nucleotide analogues/modifications are selected from
base modifications, which are preferably selected from
2-amino-6-chloropurineriboside-5'-triphosphate,
2-Aminopurine-riboside-5'-triphosphate;
2-aminoadenosine-5'-triphosphate,
2'-Amino-2'-deoxycytidine-triphosphate,
2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate,
2'-Fluorothymidine-5'-triphosphate, 2'-O-Methyl
inosine-5'-triphosphate 4-thiouridine-5'-triphosphate,
5-aminoallylcytidine-5'-triphosphate,
5-aminoallyluridine-5'-triphosphate,
5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate,
5-Bromo-2'-deoxycytidine-5'-triphosphate,
5-Bromo-2'-deoxyuridine-5'-triphosphate,
5-iodocytidine-5'-triphosphate,
5-Iodo-2'-deoxycytidine-5'-triphosphate,
5-iodouridine-5'-triphosphate,
5-Iodo-2'-deoxyuridine-5'-triphosphate,
5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate,
5-Propynyl-2'-deoxycytidine-5'-triphosphate,
5-Propynyl-2'-deoxyuridine-5'-triphosphate,
6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate,
6-chloropurineriboside-5'-triphosphate,
7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate,
8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate,
benzimidazole-riboside-5'-triphosphate,
N1-methyladenosine-5'-triphosphate,
N1-methylguanosine-5'-triphosphate,
N6-methyladenosine-5'-triphosphate,
O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate,
or puromycin-5'-triphosphate, xanthosine-5'-triphosphate.
Particular preference is given to nucleotides for base
modifications selected from the group of base-modified nucleotides
consisting of 5-methylcytidine-5'-triphosphate,
7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate,
and pseudouridine-5'-triphosphate.
[0101] In some embodiments, modified nucleosides include
pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,
2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,
5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,
1-carboxymethyl-pseudouridine, 5-propynyl-uridine,
1-propynyl-pseudouridine, 5-taurinomethyluridine,
1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,
1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,
1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine.
[0102] In some embodiments, modified nucleosides include
5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine,
N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine,
5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,
pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine.
[0103] In other embodiments, modified nucleosides include
2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine.
[0104] In other embodiments, modified nucleosides include inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
[0105] In some embodiments, the nucleotide can be modified on the
major groove face and can include replacing hydrogen on C-5 of
uracil with a methyl group or a halo group.
[0106] In specific embodiments, a modified nucleoside is
5'-O-(1-Thiophosphate)-Adenosine, 5'-O-(1-Thiophosphate)-Cytidine,
5'-0-(1-Thiophosphate)-Guanosine, 5'-0-(1-Thiophosphate)-Uridine or
5'-0-(1-Thiophosphate)-Pseudouridine.
[0107] In further specific embodiments the modified RNA may
comprise nucleoside modifications selected from 6-aza-cytidine,
2-thio-cytidine, .alpha.-thio-cytidine, Pseudo-iso-cytidine,
5-aminoallyluridine, 5-iodo-uridine, N1-methyl-pseudouridine,
5,6-dihydrouridine, .alpha.-thio-uridine, 4-thio-uridine,
6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine,
5-methyl-uridine, Pyrrolo-cytidine, inosine,
.alpha.-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,
8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,
2-amino-6-Chloro-purine, N6-methyl-2-amino-purine,
Pseudo-iso-cytidine, 6-Chloro-purine, N6-methyl-adenosine,
.alpha.-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.
[0108] Lipid Modification:
[0109] According to a further embodiment, the modified RNA as
defined herein can contain a lipid modification. Such a
lipid-modified RNA typically comprises an RNA as defined herein.
Such a lipid-modified RNA molecule as defined herein typically
further comprises at least one linker covalently linked with that
RNA molecule, and at least one lipid covalently linked with the
respective linker. Alternatively, the lipid-modified RNA molecule
comprises at least one RNA molecule as defined herein and at least
one (bifunctional) lipid covalently linked (without a linker) with
that RNA molecule. According to a third alternative, the
lipid-modified RNA molecule comprises an RNA molecule as defined
herein, at least one linker covalently linked with that RNA
molecule, and at least one lipid covalently linked with the
respective linker, and also at least one (bifunctional) lipid
covalently linked (without a linker) with that RNA molecule. In
this context, it is particularly preferred that the lipid
modification is present at the terminal ends of a linear RNA
sequence.
[0110] Modification of the 5'-End of the Modified RNA:
[0111] According to another preferred embodiment of the invention,
the modified RNA molecule as defined herein, can be modified by the
addition of a so-called "5' CAP" structure.
[0112] A 5'-cap is an entity, typically a modified nucleotide
entity, which generally "caps" the 5'-end of a mature mRNA. A
5'-cap may typically be formed by a modified nucleotide,
particularly by a derivative of a guanine nucleotide. Preferably,
the 5'-cap is linked to the 5'-terminus via a 5'-5'-triphosphate
linkage. A 5'-cap may be methylated, e.g. m7GpppN, wherein N is the
terminal 5' nucleotide of the nucleic acid carrying the 5'-cap,
typically the 5'-end of an RNA. m7Gppp(N) (wherein "N" is the first
transcribed nucleotide) is the 5'-cap structure, which naturally
occurs in mRNA transcribed by polymerase II and is therefore not
considered as modification comprised in the modified RNA according
to the invention. This means the modified RNA according to the
present invention may comprise a m7Gppp(N) as 5'-cap, but
additionally the modified RNA comprises at least one further
modification as defined herein.
[0113] Further examples of 5'cap structures include glyceryl,
inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide,
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide,
carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,
L-nucleotides, alpha-nucleotide, modified base nucleotide,
threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide,
acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl
nucleotide, 3'-3'-inverted nucleotide moiety, 3'3'-inverted abasic
moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic
moiety, 1,4-butanediol phosphate, 3'-phosphoramidate,
hexylphosphate, aminohexyl phosphate, 3'-phosphate,
3'phosphorothioate, phosphorodithioate, or bridging or non-bridging
methylphosphonate moiety. These modified 5'-cap structures are
regarded as at least one modification comprised in the modified RNA
according to the present invention.
[0114] Particularly preferred modified 5'-cap structures are CAP1
(methylation of the ribose of the adjacent nucleotide of m7G), CAP2
(methylation of the ribose of the 2.sup.nd nucleotide downstream of
the m7G), CAP3 (methylation of the ribose of the 3.sup.rd
nucleotide downstream of the m7G), CAP4 (methylation of the ribose
of the 4.sup.th nucleotide downstream of the m7G), ARCA
(anti-reverse CAP analogue, modified ARCA (e.g. phosphothioate
modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
LNA-guanosine, and 2-azido-guanosine.
[0115] Sequence Modification of the Open Reading Frame:
[0116] Modification of the G/C Content:
[0117] In a particularly preferred embodiment of the present
invention, the G/C content of the coding region, encoding at least
one peptide or protein of the modified RNA as defined herein, is
modified, particularly increased, compared to the G/C content of
its particular wild type coding region, i.e. the unmodified coding
region. The encoded amino acid sequence of the coding region is
preferably not modified compared to the coded amino acid sequence
of the particular wild type coding region.
[0118] The modification of the G/C-content of the coding region of
the modified RNA as defined herein is based on the fact that the
sequence of any mRNA region to be translated is important for
efficient translation of that mRNA. Thus, the composition and the
sequence of various nucleotides are important. In particular, mRNA
sequences having an increased G (guanosine)/C (cytosine) content
are more stable than mRNA sequences having an increased A
(adenosine)/U (uracil) content. According to the invention, the
codons of the coding region are therefore varied compared to its
wild type coding region, while retaining the translated amino acid
sequence, such that they include an increased amount of G/C
nucleotides. In respect to the fact that several codons code for
one and the same amino acid (so-called degeneration of the genetic
code), the most favourable codons for the stability can be
determined (so-called alternative codon usage).
[0119] Depending on the amino acid to be encoded by the coding
region of the modified RNA as defined herein, there are various
possibilities for modification of the RNA sequence, e.g. the coding
region, compared to its wild type coding region. In the case of
amino acids, which are encoded by codons, which contain exclusively
G or C nucleotides, no modification of the codon is necessary.
Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC
or GCG) and Gly (GGC or GGG) require no modification, since no A or
U is present.
[0120] In contrast, codons which contain A and/or U nucleotides can
be modified by substitution of other codons which code for the same
amino acids but contain no A and/or U. Examples of these are:
the codons for Pro can be modified from CCU or CCA to CCC or CCG;
the codons for Arg can be modified from CGU or CGA or AGA or AGG to
CGC or CGG; the codons for Ala can be modified from GCU or GCA to
GCC or GCG; the codons for Gly can be modified from GGU or GGA to
GGC or GGG.
[0121] In other cases, although A or U nucleotides cannot be
eliminated from the codons, it is however possible to decrease the
A and U content by using codons, which contain a lower content of A
and/or U nucleotides. Examples of these are:
the codons for Phe can be modified from UUU to UUC; the codons for
Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG; the
codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG
or AGC; the codon for Tyr can be modified from UAU to UAC; the
codon for Cys can be modified from UGU to UGC; the codon for His
can be modified from CAU to CAC; the codon for Gln can be modified
from CAA to CAG; the codons for Ile can be modified from AUU or AUA
to AUC; the codons for Thr can be modified from ACU or ACA to ACC
or ACG; the codon for Asn can be modified from AAU to AAC; the
codon for Lys can be modified from AAA to AAG; the codons for Val
can be modified from GUU or GUA to GUC or GUG; the codon for Asp
can be modified from GAU to GAC; the codon for Glu can be modified
from GAA to GAG; the stop codon UAA can be modified to UAG or
UGA.
[0122] In the case of the codons for Met (AUG) and Trp (UGG), on
the other hand, there is no possibility of sequence
modification.
[0123] The substitutions listed above can be used either
individually or in any possible combination to increase the G/C
content of the coding region of the modified RNA as defined herein,
compared to its particular wild type coding region (i.e. the
original sequence). Thus, for example, all codons for Thr occurring
in the wild type sequence can be modified to ACC (or ACG).
[0124] Preferably, the G/C content of the coding region of the
modified RNA as defined herein is increased by at least 7%, more
preferably by at least 15%, particularly preferably by at least
20%, compared to the G/C content of the wild type coding region.
According to a specific embodiment at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, more preferably at least 70%, even more preferably at
least 80% and most preferably at least 90%, 95% or even 100% of the
substitutable codons in the coding region encoding at least one
peptide or protein, which comprises a pathogenic antigen or a
fragment, variant or derivative thereof, are substituted, thereby
increasing the G/C content of said coding region.
[0125] In this context, it is particularly preferable to increase
the G/C content of the coding region of the modified RNA as defined
herein, to the maximum (i.e. 100% of the substitutable codons),
compared to the wild type coding region.
[0126] Codon Optimization:
[0127] According to the invention, a further preferred modification
of the coding region encoding at least one peptide or protein of
the modified RNA as defined herein, is based on the finding that
the translation efficiency is also determined by a different
frequency in the occurrence of tRNAs in cells. Thus, if so-called
"rare codons" are present in the coding region of the wild type RNA
sequence, to an increased extent, the mRNA is translated to a
significantly poorer degree than in the case where codons coding
for relatively "frequent" tRNAs are present.
[0128] In this context, the coding region of the modified RNA is
preferably modified compared to the corresponding wild type coding
region such that at least one codon of the wild type sequence,
which codes for a tRNA which is relatively rare in the cell, is
exchanged for a codon, which codes for a tRNA which is relatively
frequent in the cell and carries the same amino acid as the
relatively rare tRNA. By this modification, the coding region of
the modified RNA as defined herein, is modified such that codons,
for which frequently occurring tRNAs are available, are inserted.
In other words, according to the invention, by this modification
all codons of the wild type coding region, which code for a tRNA
which is relatively rare in the cell, can in each case be exchanged
for a codon, which codes for a tRNA which is relatively frequent in
the cell and which, in each case, carries the same amino acid as
the relatively rare tRNA.
[0129] Which tRNAs occur relatively frequently in the cell and
which, in contrast, occur relatively rarely is known to a person
skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001,
11(6): 660-666. The codons which use for the particular amino acid
the tRNA which occurs the most frequently, e.g. the Gly codon,
which uses the tRNA which occurs the most frequently in the (human)
cell, are particularly preferred.
[0130] According to the invention, it is particularly preferable to
link the sequential G/C content, which is increased, in particular
maximized, in the coding region of the modified RNA as defined
herein, with the "frequent" codons without modifying the amino acid
sequence of the peptide or protein encoded by the coding region of
the RNA sequence. This preferred embodiment allows provision of a
particularly efficiently translated and stabilized (modified) RNA
sequence as defined herein.
[0131] In one embodiment, the RNA molecule having a cleavage site
for a catalytic nucleic acid molecule is produced by non-enzymatic
chemical RNA synthesis (e.g. Marshall and Kaiser, 2004. Curr. Opin.
Chem. Biol. 8(3):222-229). That method is preferably employed in
the case of an RNA molecule having a length of about 100
nucleotides or less. In a particularly preferred embodiment, the
RNA molecule having a cleavage site for a catalytic nucleic acid
molecule is synthesized in an in vitro transcription reaction.
[0132] In particularly preferred embodiments, the RNA molecule
having a cleavage site for the catalytic nucleic acid molecule is a
long RNA molecule comprising at least 100, 150, 200 or more
preferably at least 500 nucleotides in length. Preferably, the RNA
molecule has a length of from 5 to 30000 nucleotides, 10 to 25000
nucleotides, 50 to 20000 nucleotides, 100 to 18000 nucleotides, 300
to 15000 nucleotides or 500 to 12000 nucleotides.
[0133] The RNA molecule, which is analyzed by the method according
to the invention, comprises a cleavage site for a catalytic nucleic
acid molecule. Typically, the RNA molecule is cleaved at the
cleavage site by the catalytic nucleic acid molecule, which yields
a 5' terminal RNA fragment and at least one 3' RNA fragment. In
general, the RNA molecule to be analyzed may comprise a cleavage
site for any catalytic nucleic acid molecule, wherein the method is
not limited with respect to a certain catalytic nucleic acid
molecule. Typically, the cleavage site is specifically recognized
by the respective catalytic nucleic acid molecule, preferably as
defined herein, which is employed in the method according to the
invention. As used herein, the cleavage site for the catalytic
nucleic acid molecule may be comprised in the RNA molecule, e.g.
because it is part of a naturally occurring coding sequence or a
naturally occurring 5' UTR comprised in the RNA molecule.
Preferably, the sequence of the RNA molecule has been designed or
artificially modified in order to comprise a cleavage site for a
catalytic nucleic acid molecule. Methods for changing or
introducing nucleotides into DNA molecules to produce specific
sites are known in the art. That DNA template can then be used to
produce an RNA molecule, e.g. by in vitro transcription. These
methods are known in the art. Preferably, the RNA molecule to be
analyzed comprises a sequence, which is at least 30%, 40%, 50%,
60%, 70%, 80%, 90% or 95% identical to the consensus sequence of a
cleavage site for a particular catalytic nucleic acid molecule.
[0134] For example, hairpin ribozymes cleave 5' of the guanosine in
NGUC sequences, wherein N is any nucleotide. Furthermore, for
example, a hammerhead ribozyme can be directed to cleave 3' of any
NUH sequence, wherein N is any nucleotide, U is conserved, and H
can be any nucleotide except G (N=G,A,C,U; H=A,C,U) (Haseloff and
Gerlach, 1988. Nature 334: 585-591; McCall et al., 2000. Molecular
Biotechnology 14: 5-17).
[0135] The RNA molecule to be analyzed comprises at least one
cleavage site for the catalytic nucleic acid molecule. The RNA
molecule may comprise any number of cleavage sites for the
catalytic nucleic acid molecule, wherein the location of the most
5' cleavage site (i.e. the cleavage site, which is located closest
to the 5' terminus of the RNA molecule) is preferably selected in
order to allow separation and detection of the resulting 5'
terminal RNA fragment.
[0136] Preferably, the location of the most 5' cleavage site is
chosen such that cleavage of the RNA molecule at that site
generates a 5' terminal RNA fragment that has a suitable size (i.e.
number of nucleotides) in order to be separated by methods known in
the art. Preferably, the most 5' cleavage site is located in a
position between nucleotide positions 1 to 500 in 5'-3' direction
of the RNA molecule, so that the resulting 5' RNA fragment has a
size equal to or smaller than 500 nucleotides. More preferably, the
most 5'cleavage site is located between nucleotide positions 1 and
400, 1 and 300, 1 and 200, 1 and 100 or 1 and 50 in 5'-3' direction
of the RNA molecule, wherein "position 1" corresponds the 5'
terminal nucleotide of the RNA molecule, "position 2" corresponds
to the second nucleotide starting from the 5' terminus, and so
forth. Most preferably, the cleavage site is located between
nucleotide positions 1 and 5, 1 and 10, 1 and 20, 1 and 30, 1 and
40, 1 and 50, 1 and 60, 1 and 70, 1 and 80, 1 and 90 or 1 and 100
in 5'-3' direction of the RNA molecule. Even more preferably, the
RNA molecule is cleaved by the catalytic nucleic acid molecule (in
5' to 3' direction) after nucleotide position 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25. In a particularly preferred embodiment, cleavage occurs between
nucleotide position 5 and 15 or between position 8 and 20.
[0137] It is further preferred that the RNA molecule comprises an
open reading frame encoding at least one protein or peptide,
wherein the most 5' cleavage site for a catalytic nucleic acid
molecule is located between the 5' terminus of the RNA molecule and
the first nucleotide of the open reading frame. More preferably,
the RNA molecule having a cleavage site is an mRNA molecule and
comprises a 5'-UTR as defined herein. Preferably, the most 5'
cleavage site is positioned in the 5'-UTR of said mRNA
molecule.
[0138] Generally, the length of the 5' terminal RNA fragment
resulting from the cleavage of the RNA molecule with a catalytic
nucleic acid molecule is not limited in any way. In particular,
according to the invention, the 5' terminal RNA fragment may have
any length that allows separation and resolution of the fragment,
preferably separation from a 3' RNA fragment.
[0139] Depending, amongst other factors, on the physical property
to be determined and depending on the means of separation that are
envisaged, the skilled person may adapt the length of the 5'
terminal RNA fragment by choosing the respective position of the
most 5' cleavage site in the RNA molecule to be analyzed.
Preferably, the most 5' terminal cleavage site in the RNA molecule
is chosen such that cleavage with a catalytic nucleic acid molecule
results in a 5' terminal RNA fragment, which comprises at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 nucleotides. Alternatively, the length of the 5' terminal RNA
fragment is from 1 to 500, from 1 to 400, from 1 to 300, from 1 to
200, from 1 to 100, from 1 to 50 or from 1 to 30 nucleotides. In a
particularly preferred embodiment, the location of the most 5'
cleavage site in the RNA molecule is chosen such that the length of
the 5' terminal RNA fragment resulting from the cleavage is from 5
to 20, from 8 to 25, from 10 to 20 or from 12 to 19
nucleotides.
[0140] The skilled person knows that one option to distinguish the
5' RNA fragments of interest from other nucleic acid molecules or
fragments may be the choice of an appropriate size of the 5' RNA
fragments by choosing an appropriate cleavage site, in particular
by choosing an appropriate most 5' cleavage site. Alternatively,
the 5' terminal RNA fragments are labelled with an appropriate
marker so that the 5' terminal RNA fragments may be detected and
distinguished from non-labelled fragments, e.g. 3' RNA
fragments.
[0141] As used herein, the term "labelled" refers to an RNA
molecule that is either directly or indirectly labelled with a
molecule, which provides a detectable signal, e.g. radioisotope,
fluorescent tag, chemiluminescent tag, a peptide or specific
binding molecules. Specific binding molecules include pairs, such
as biotin and streptavidin, digoxin and antidigoxin. The label can
directly or indirectly provide a detectable signal. Radioisotopes
(e.g. .sup.18F, .sup.125I, .sup.35S, .sup.14C, .sup.3H, or
.sup.99mTc) are commonly used in biological applications for the
detection of a variety of nucleic acids such as RNA. Methods for
the synthesis and labelling of RNA in vitro are known in the art
(e.g. Huang and Yu, 2013. Synthesis and Labelling of RNA In Vitro.
Current Protocols in Molecular Biology. 102:4.15.1-4.15.14).
[0142] For example, the synthesis and use of biotin labeled cap
analogs has been described (Jemielity et al., 2012. Org. Biomol.
Chem. 10(43):8570-4; WO2013/059475). These cap analogs can be
incorporated into RNA molecules to produce 5'-capped and
biotinylated RNAs, which retain their biological functionality and
can be used for biotin-streptavidin technologies.
[0143] In a preferred embodiment, the method according to the
invention uses a catalytic nucleic acid molecule that has been
designed to be able to cleave the RNA molecule at a specific
cleavage site, preferably at the most 5' cleavage site as described
herein. Methods for designing catalytic nucleic acid molecules, in
particular ribozymes that cleave RNA substrate molecules at a
defined site, are known in the art.
[0144] For example, hairpin ribozymes cleave 5' of the guanosine in
NGUC sequences, wherein N is any nucleotide. Furthermore, for
example, a hammerhead ribozyme can be directed to cleave 3' of any
NUH sequence, wherein N is any nucleotide, U is conserved, and H
can be any nucleotide except G (N=G,A,C,U; H=A,C,U) (Haseloff and
Gerlach, 1988. Nature 334: 585-591; McCall et al., 2000. Molecular
Biotechnology 14: 5-17).
[0145] According to the substrate requirements of a catalytic
nucleic acid molecule described above, an RNA molecule can--in
principle--be expected to contain a number of possible sites for
sequence-specific cleavage by a catalytic nucleic acid molecule. In
addition to the target site, the number of base pairs to be formed
between the catalytic nucleic acid molecule and the substrate are
preferably chosen (substrate binding region). The affinity of a
catalytic nucleic acid molecule towards its substrate can be
adjusted by altering the length of the substrate binding region of
the catalytic nucleic acid molecule. Although high affinity is
usually desirable, an extended substrate binding region may cause
problems regarding specificity and catalytic activity. Multiple
turnover catalysis may be severely impaired if product release is
slow due to strong binding of the target nucleic acid molecule to
the catalytic nucleic acid molecule. Catalytic nucleic acid
molecules with short binding arms (substrate binding region),
however, may lack specificity.
[0146] Therefore, catalytic activity on the one hand and
specificity on the other hand are preferably balanced when
designing a catalytic nucleic acid molecule. Catalytic nucleic acid
molecules, which form a larger number of base pairs with the
substrate RNA, are less likely to dissociate from the cleaved
substrate, and are thus not available for further cleavage.
Therefore, the number of base pairs is preferably selected in such
a way that the catalytic nucleic acid molecule-substrate complex
formed is relatively stable under the conditions allowing the
cleavage of the RNA molecule, but is able to dissociate once
cleavage of the substrate has occurred. This typically requires 11
to 17 base pairs. Depending on the actual requirements in the
specific case, that number may vary considerably. As a general
rule, for specificity, the number of base pairs formed between the
catalytic nucleic acid molecule and the substrate RNA should be
high enough to make the target sequence unique, but not so high
that imperfectly matched substrates would form stable complexes.
Statistically, about 13 nucleotides are required to uniquely define
a particular site in an RNA pool.
[0147] Methods for the production of catalytic nucleic acid
molecules are known in the art. For example, a ribozyme can be
chemically synthesized using the standard procedure for RNA
synthesis as described (Wincott et al., 1995. Nucleic Acids Res.
23(14):2677-84). Ribozymes can also be synthesized by in vitro
transcription of suitable DNA templates using e.g. bacteriophage T7
RNA polymerase (Haseloff and Gerlach, 1988. Nature 334:
585-591).
[0148] In this context, it is particularly preferred that the
catalytic nucleic acid molecule is provided in trans. This means
that the RNA molecule having a cleavage site for the catalytic
nucleic acid molecule and the catalytic nucleic acid molecule are
not part of the same molecule. However, the present invention also
comprises the use of the catalytic nucleic acid molecule in cis,
i.e. a situation, where the RNA molecule having a cleavage site and
the catalytic nucleic acid molecule are part of the same
molecule.
[0149] In a particularly preferred embodiment of the present
invention, the catalytic nucleic acid molecule is a ribozyme. In
this context it is particularly preferred that the ribozyme is
selected from the group consisting of hammerhead ribozymes, hairpin
ribozymes, and HDV ribozymes. In an even more preferred embodiment,
the ribozyme is a hammerhead ribozyme.
[0150] Particularly preferred in this context is a hammerhead
ribozyme comprising an RNA sequence according to SEQ ID NO: 1. Most
preferably, the ribozyme comprising an RNA sequence according to
SEQ ID NO: 1 specifically cleaves an RNA molecule 3' of the
sequence motif NUH as shown in FIG. 5, wherein N is G, A, C, or U,
and H is A, C, or U (Haseloff and Gerlach, 1988. Nature 334:
585-591; McCall et al., 2000. Molecular Biotechnology, 14:
5-17).
[0151] In an even more preferred embodiment a hammerhead ribozyme
HHNUH2d according to SEQ ID NO: 2 is used in the method according
to the invention. The ribozyme according to SEQ ID NO:2
specifically targets the 5' region of the RNA sequences according
to SEQ ID NO: 3-5 and shown in FIGS. 1 to 3, forming helix III with
mRNA positions 1-12, and helix I with mRNA positions 14-18 (FIG. 6)
of the RNA sequences according to SEQ ID No: 3-5. The 5' region of
the target RNA sequence contains two possible recognition sites,
NUH1 (positions 10-12) and NUH2 (positions 11-13), of which NUH2 is
the preferred target site.
[0152] Sequence of the trans-acting hammerhead ribozyme HHNUH2d
TABLE-US-00002 (SEQ ID NO: 2):
5'-GCAUGGCUGAUGAGGCCUCGACCGAUAGGUCGAGGCCGAAAAGCU UUCUCCC-3'
[0153] In another particularly preferred embodiment, the catalytic
nucleic acid molecule is a DNAzyme, e.g. a''10-23'' DNAzyme.
[0154] By the cleavage with the catalytic nucleic acid molecule,
the RNA molecule having at least one cleavage site for the
catalytic nucleic acid molecule is specifically cleaved at that (at
least one) defined site so that a 5' terminal and at least one 3'
RNA fragment is produced.
[0155] Step b) of the methods as defined above comprises cleavage
of the RNA molecule having a cleavage site for the catalytic
nucleic acid molecule with the catalytic nucleic acid molecule.
Therein, the RNA molecule is contacted with the catalytic nucleic
acid molecule under conditions allowing the cleavage of the RNA
molecule. Preferably, such conditions allow the specific
interaction of the catalytic nucleic acid molecule and the RNA
molecule having a cleavage site for the catalytic nucleic acid
molecule, and the cleavage of the RNA molecule having a cleavage
site. Such conditions may vary depending on the RNA molecule to be
analyzed and the catalytic nucleic acid molecule that is employed.
Nevertheless, methods are known in the art to select suitable
conditions once a selection has been made concerning the RNA
molecule to be analyzed and/or the catalytic nucleic acid molecule.
The skilled person knows how to adjust the parameters, such as
magnesium ion concentration, buffer composition, pH, temperature
and incubation times.
[0156] Preferably, step b) of the method according to the invention
comprises denaturing the nucleic acid molecules, preferably by
heating, annealing the RNA molecule to be analyzed and the
catalytic nucleic acid molecule and cleavage of the RNA molecule to
be analyzed, wherein the annealing and the cleavage preferably take
place at a lower temperature than the denaturing. Typically, the
nucleic acid molecules (i.e. the RNA to be analyzed and the
catalytic nucleic acid molecule) are heated either together (i.e.
in a mixture) or separately in a suitable buffer that does
preferably not contain magnesium ions (Mg.sup.++). Subsequently,
the nucleic acid molecules are cooled to cleavage reaction
temperature, either together or separately. Preferably, the heating
step involves heating of the buffer containing the nucleic acid
molecules to a temperature of at least 70.degree. C., more
preferably at least 80.degree. C., 85.degree. C., 90.degree. C.,
95.degree. C. or at least 96.degree. C., preferably for at least 30
seconds, 60 seconds, 90 seconds or at least 120 seconds. After the
heating step, the nucleic acid molecules are typically cooled down
to the cleavage reaction temperature, which is typically lower than
the temperature in the initial heating step. Preferably, the
nucleic acid molecules are cooled in a controlled manner, for
instance at a rate of 0.1.degree. C. per second. The cleavage
reaction preferably takes place at a temperature from 20.degree. C.
to 50.degree. C., more preferably from 20.degree. C. to 40.degree.
C., 24.degree. C. to 38.degree. C. or 25.degree. C. to 37.degree.
C., most preferably at 25.degree. C. or 37.degree. C., for a period
of preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 30, or 60
minutes. After cooling of the heated nucleic acid molecules and
before starting the cleavage reaction (e.g. by addition of
magnesium ions (Mg')), an optional annealing step is employed,
wherein the temperature is preferably equal to the cleavage
reaction temperature and which is typically carried out in absence
of magnesium ions, preferably for at least 1, 2, 3, 4, 5, 6, 7, 8,
9 or at least 10 minutes.
[0157] Preferably, the RNA molecule to be analyzed and the
catalytic nucleic acid molecule, preferably a ribozyme, are
provided in about the same molar amounts.
[0158] In one embodiment, the catalytic nucleic acid molecule,
preferably a ribozyme, and the RNA molecule to be analyzed are
heated together at, for example, 95.degree. C., preferably for 1 to
2 minutes, in the presence of buffer without magnesium ions, and
subsequently cooled, preferably at a controlled cooling rate, to
the reaction temperature of 20-37.degree. C., preferably 25.degree.
C., in order to promote annealing. Subsequently, Mg.sup.++ (e.g.
MgCl.sub.2) is added to initiate the cleavage reaction. In another
embodiment, the catalytic nucleic acid molecule, preferably an
ribozyme, and the RNA molecule to be analyzed are heated separately
at, for example, 95.degree. C. without Mg.sup.++, preferably for
one to two minutes, and are then cooled to the reaction
temperature. Mg.sup.++ is added to both the catalytic nucleic acid
molecule and the RNA to be analyzed and the cleavage reaction is
started by mixing both. In a preferred embodiment of the method
according to the invention, the cleaving in step b) takes place in
the presence of at least 10, 20 or 30 mM Mg', most preferably in
presence of 30 mM MgCl.sub.2.
[0159] In order to achieve a sufficient degree of cleavage of the
RNA molecule to be analyzed, the Mg.sup.++ concentration, buffer
composition, pH value, temperature and reaction time may need to be
adjusted. As used herein, the phrase "conditions allowing the
cleavage of the RNA molecule" refers to conditions, which--at
suitable incubation time--preferably allow cleavage of at least
50%, preferably at least 75%, 80%, 85%, 90%, 95% or 98% of the RNA
molecules in a population, which have a cleavage site for a
catalytic nucleic acid molecule. For example, "conditions allowing
the cleavage of the RNA molecule" may comprise 50-200 mM NaCl or
KCl, 0.1-200 mM Mg.sup.++, 5-100 mM Tris-HCl, pH 6.5-8.5,
20-37.degree. C. for 5 minutes to 2 hours. A non-ionic detergent
(Tween, NP-40, Triton-X 100) is preferably present, usually at
about 0.001 to 2%, typically 0.05-0.2% (volume/volume).
[0160] The cleavage of the RNA molecule having at least one
cleavage site for a catalytic nucleic acid molecule with the
catalytic nucleic acid molecule, leads to the generation of a 5'
terminal RNA fragment and at least one 3' RNA fragment. The number
of 3' RNA fragments depends on the number of cleavage sites for the
catalytic nucleic acid molecule. For example, cleavage of an RNA
molecule having one cleavage site typically leads to a 5' terminal
RNA fragment and one 3' RNA fragment. On the other hand, cleavage
of an RNA molecule having two cleavage sites typically results in
three RNA fragments, i.e. a 5' terminal RNA fragments and two 3'
RNA fragments.
[0161] Preferably, the 5' terminal RNA fragment obtained after
cleavage of the RNA molecule to be analyzed with the catalytic
nucleic acid molecule preferably comprises at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
nucleotides. Alternatively, the length of the 5' terminal RNA
fragment is from 1 to 500, from 1 to 400, from 1 to 300, from 1 to
200, from 1 to 100, from 1 to 50 or from 1 to 30 nucleotides. In a
particularly preferred embodiment, the length of the 5' terminal
RNA fragment resulting from the cleavage of the RNA molecule having
a cleavage site is from 5 to 20, from 8 to 25, from 10 to 20 or
from 12 to 19 nucleotides.
[0162] Step c) of the method according to the invention comprises
determining a physical property of the RNA molecule by analyzing
the 5' terminal RNA fragment.
[0163] In the context of the present invention, the expression "a
physical property" (or "physical properties") typically refers to a
physical property or to a structural feature of an RNA molecule,
preferably a modified RNA molecule as defined herein. Where the
plural ("physical properties") is used, it may likewise refer to a
single property or single feature. Preferably, the expression as
used herein refers to a physical property or a structural feature
of the RNA molecule, which distinguishes the RNA molecule from
other, preferably structurally related, RNA molecules. Preferably,
a physical property or a structural feature is capable of
distinguishing the RNA molecule from a similar, preferably
structurally related, RNA molecule lacking the physical property or
a structural feature, more preferably from an RNA molecule, which
is identical apart from the lacking physical property or the
lacking structural feature. Typically, the distinct physical
property reflects a structural feature, such as e.g. a distinct
molecular weight, charge, or specific nucleotide composition. As
used herein, a physical property or a structural feature may
preferably be determined by standard analytical methods known in
the art. Preferably, a physical property or a structural feature
can be determined after cleavage of the RNA molecule having a
cleavage site for a catalytic nucleic acid molecule. According to
the invention, a distinct physical property or a distinct
structural feature of the RNA molecule having a cleavage site for a
catalytic nucleic acid molecule is determined by analysis of the 5'
terminal RNA fragment obtained after cleavage of the RNA molecule
with the catalytic nucleic acid molecule. In other words, the 5'
terminal RNA fragment obtained by cleavage of the RNA molecule
having a cleavage site for a catalytic nucleic acid molecule with
the catalytic nucleic acid molecule reflects a physical property or
a structural feature of the RNA molecule. Thus, by analyzing the 5'
terminal RNA fragment, preferably with respect to a distinct
physical property or a structural feature as defined herein, a
distinct physical property of the RNA molecule, from which the 5'
terminal RNA fragment is derived, is determined. In a preferred
embodiment, the physical property or structural feature is selected
from the molecular weight, the charge, the nucleotide sequence, and
the presence or absence, respectively, of a nucleotide, preferably
a modified nucleotide, a 5' terminal modification as defined
herein, or a specific moiety of a nucleotide, preferably of a
modified nucleotide, such as a modified base, in the 5' terminal
RNA fragment.
[0164] In a preferred embodiment, step c) involves separating or
resolving the 5' terminal RNA fragment from the at least one 3' RNA
fragment. In order to determine the physical property of the 5'
terminal RNA fragment--or the respective RNA molecule, from which
it is derived--it is typically sufficient to resolve the fragment
in any manner, i.e. to employ an analytic technique that allows to
determine the presence or absence of an RNA fragment with certain
physical properties. By determining the presence or absence of said
fragment with a certain physical property, the skilled person is
capable of determining the physical property of the RNA molecule,
from which the fragment is derived. To this end, the fragment does
not necessarily need to be physically separated or isolated from
another fragment or other fragments that may be present. The
resolution of a fragment with a certain physical property may also
be achieved in mixture, e.g. by using labelling techniques or
molecular markers and relevant methods for detection.
[0165] In one embodiment, the 5' terminal RNA fragment is separated
from another fragment, preferably from the at least one 3' RNA
fragment. Any suitable method for separating RNA fragments can be
used, including, but not limited to, denaturing gel electrophoresis
or liquid chromatography. In general, the separation technique is
used according to the characteristics, e.g. the size, of the
fragments to be separated. The skilled person can thus select a
suitable separation technology on the basis of the characteristics
of the expected fragment.
[0166] In a particularly preferred embodiment of the first aspect
of the present invention, the RNA fragments are separated in step
c) by denaturing gel electrophoresis or liquid chromatography,
preferably HPLC, FPLC or RPLC. Separation of RNA molecules by
denaturing gel electrophoresis has been described (Maniatis et al.,
1975. Biochemistry 14(17):3787-3794). For example, polyacrylamide
gels that contain a high concentration of a denaturing agent such
as urea are capable of resolving short (<500 nucleotides)
single-stranded RNA fragments that differ in length by as little as
one nucleotide. In this context, polyacrylamide gels comprising
urea, preferably 8 M urea, are particularly preferred.
[0167] The RNA fragments obtained by cleavage of the RNA molecule
having a cleavage site for a catalytic nucleic acid molecule can
also be separated by liquid chromatography. As used herein, the
term "liquid chromatography" (LC) preferably refers to a process of
selective retardation of one or more components of a fluid solution
as the fluid uniformly percolates through a column of a finely
divided, preferably porous, substance, or through capillary
passageways. The retardation results from the distribution of the
components of the mixture between one or more stationary phases and
the bulk fluid (i.e. the mobile phase), as this fluid moves
relative to the stationary phase(s). LC includes reverse phase
liquid chromatography (RPLC), high performance liquid
chromatography (HPLC), high turbulence liquid chromatography (HTLC)
and fast performance liquid chromatography (FPLC). In contrast to
HPLC, the buffer pressure used in FPLC is relatively low, typically
less than 5 bar, but the flow rate is relatively high, typically
1-5 ml/min.
[0168] Stationary phases for the use in liquid chromatography are
known in the art. Preferably, the stationary phase is selected from
the group consisting of a porous polystyrene, a porous
non-alkylated polystyrene, a polystyrenedi-vinylbenzene, a porous
non-alkylated polystyrenedivinylbenzene, a porous silica gel, a
porous silica gel modified with non-polar residues, a porous silica
gel modified with alkyl containing residues, selected from butyl-,
octyl and/or octadecyl containing residues, a porous silica gel
modified with phenylic residues, and a porous polymethacrylate (see
also WO2008077592, the disclosure of which is incorporated herewith
by reference).
[0169] In this context, ethylene-bridged hybrid organic/inorganic
stationary phases are particularly preferred (see also Wyndham et
al., 2003. Anal. Chem. 75(24):6781-8 and WO2003014450, the
disclosure of which is incorporated herewith by reference).
[0170] For example, the separation process of RNA molecules by HPLC
has been described (Weissman et al., 2013. Methods Mol. Biol.
969:43-54).
[0171] In a preferred embodiment, the separation of the 5' terminal
RNA fragment in itself already reveals the distinct property of the
RNA molecule, from which it is derived and which is to be analyzed.
For example, if the presence of an extra nucleotide or a
modification at the 5' terminal RNA fragment is investigated, then
it is typically enough to separate the fragments in order to obtain
the result.
[0172] Preferably, 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. For example, a 5' terminal RNA fragment may be compared
to a reference RNA fragment, which is known to exhibit a certain
property, in order to confirm that property in the 5' terminal RNA
fragment obtained in step b). Preferably, this comparison is
carried out after separation of the 5' terminal RNA fragment
obtained in step b).
[0173] In another preferred embodiment, the separated fragment is
further analyzed by further analytical methods in order to
determine the distinct physical property of the fragment.
[0174] In a preferred embodiment, the physical property of the 5'
terminal RNA fragment is determined in step c) by spectroscopic
methods, quantitative mass spectrometry, or sequencing.
[0175] Spectroscopic methods for RNA analysis include traditional
absorbance measurements at 260 nm and more sensitive fluorescence
techniques using fluorescent dyes such as ethidium bromide and a
fluorometer with an excitation wavelength of 302 or 546 nm
(Gallagher, 2011. Quantitation of DNA and RNA with Absorption and
Fluorescence Spectroscopy. Current Protocols in Molecular Biology.
93:A.3D.1-A.3D.14).
[0176] A mass spectrometer (MS) is a gas phase spectrometer that
measures a parameter that can be translated into mass-to-charge
ratio of gas phase ions. Examples of mass spectrometers are
time-of-flight, magnetic sector, quadrupole filter, ion trap, ion
cyclotron resonance, electrostatic sector analyser and hybrids of
these. Methods for the application of MS methods to the
characterization of nucleic acids are known in the art.
[0177] For example, Matrix-Assisted Laser Desorption/Ionization
Mass Spectrometry (MALDI-MS) can be used to analyse
oligonucleotides at the 120-mer level and below (Castleberry et
al., 2008. Matrix-Assisted Laser Desorption/Ionization
Time-of-Flight Mass Spectrometry of Oligonucleotides. Current
Protocols in Nucleic Acid Chemistry. 33:10.1.1-10.1.21).
[0178] Electrospray Ionization Mass Spectrometry (ESI-MS) allows
the analysis of high-molecular-weight compounds through the
generation of multiply charged ions in the gas phase and can be
applied to molecular weight determination, sequencing and analysis
of oligonucleotide mixtures (Castleberry et al., 2008. Electrospray
Ionization Mass Spectrometry of Oligonucleotides. Current Protocols
in Nucleic Acid Chemistry. 35:10.2.1-10.2.19). Preferably, the mass
spectrometry analysis is conducted in a quantitative manner to
determine the amount of RNA.
[0179] Methods for sequencing of RNA are known in the art. A
recently developed technique called RNA Sequencing (RNA-Seq) uses
massively parallel sequencing to allow for example transcriptome
analyses of genomes at a far higher resolution than is available
with Sanger sequencing- and microarray-based methods. In the
RNA-Seq method, complementary DNAs (cDNAs) generated from the RNA
of interest are directly sequenced using next-generation sequencing
technologies. RNA-Seq has been used successfully to precisely
quantify transcript levels, confirm or revise previously annotated
5' and 3' ends of genes, and map exon/intron boundaries (Eminaga et
al., 2013. Quantification of microRNA Expression with
Next-Generation Sequencing. Current Protocols in Molecular Biology.
103:4.17.1-4.17.14). Consequently, the amount of the RNA fragments
can be determined also by RNA sequencing.
[0180] In a preferred embodiment, step c) comprises analyzing the
5' terminal RNA fragment by comparison to a reference fragment. In
particular, 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.
Preferably, at least one reference 5' terminal RNA fragment is used
as reference. The 5' terminal RNA fragment obtained in step b) of
the method according to the invention is thus compared to one or
more reference fragments. For example, a 5' terminal fragment
having a physical property of interest (e.g. the presence of a
certain modification, such as a 5' cap structure) may be analyzed
in parallel with the 5' terminal RNA fragment derived from an RNA
molecule, which is to be analyzed.
[0181] In a preferred embodiment, the method according to the
invention is used for controlling the quality of RNA, preferably
for controlling the quality of in vitro produced RNA. Preferably,
the method is employed for controlling the quality of artificial
RNA, preferably an mRNA, which is preferably synthesized by in
vitro transcription.
[0182] According to one embodiment, the method is used for
determining a structural feature in an RNA molecule, preferably a
modified RNA molecule, having a cleavage site for a catalytic
nucleic acid molecule, wherein the structural feature is located
between the 5' terminus of the RNA molecule and the cleavage site
for a catalytic nucleic acid molecule. In one embodiment, the
method is used for determining the presence of a 5' terminal
modification as defined herein. Preferably, the method is used for
determining a structural feature selected from the presence or
absence of a cap structure, the orientation of a cap structure, the
presence of a modified cap structure, e.g. a cap analog as
described herein, or any other modification, such as a base
modification.
[0183] In a particularly preferred embodiment, step c) of the
method according to the invention comprises determining the
presence or the absence of a cap structure at the 5' terminus of
the RNA molecule having a cleavage site for the catalytic nucleic
acid molecule, wherein the RNA molecule preferably comprises at
least one modification. The 5' terminal RNA fragment of a capped
RNA differs from the 5' terminal RNA fragment of an uncapped
RNA--which is otherwise identical--by one nucleotide, i.e. the 5'
cap structure. That distinct property is exploited in order to
determine the capping status of the RNA molecule to be analyzed by
analyzing the 5' terminal RNA fragment.
[0184] In this context, the RNA molecule, preferably an mRNA
molecule, having a cleavage site for the catalytic nucleic acid
molecule may be produced by in vitro transcription in the presence
of a cap analog (co-transcriptional capping). Capped in vitro
transcripts can be synthesized by substituting a cap analog such as
a m7G(5')ppp(5')G (m7G) for a portion of the GTP in the
transcription reaction, typically the cap analog is used at a
four-fold excess compared to GTP. Methods for in vitro
transcription are known in the art (Geall et al., 2013. Semin.
Immunol. 25(2): 152-159) and typically include:
1) a linearized DNA template with a promoter sequence that has a
high binding affinity for its respective RNA polymerase such as
bacteriophage-encoded RNA polymerases, 2) ribonucleotide
triphosphates (NTPs) for the four bases (adenine, cytosine, guanine
and uracil); 3) a cap analog as defined above (e.g. m7G(5')ppp(5')G
(m7G)); 4) a DNA-dependent RNA polymerase (e.g. T7, T3 or SP6 RNA
polymerase); 5) a ribonuclease (RNase) inhibitor to inactivate any
contaminating RNase; 6) a pyrophosphatase to degrade pyrophosphate,
which may inhibit transcription; 7) MgCl.sub.2, which supplies
Mg.sup.2+ as a co-factor for the polymerase; 8) a buffer to
maintain a suitable pH value, which can also contain antioxidants
and polyamines such as spermidine at optimal concentrations.
[0185] In a preferred embodiment, the cap analog is selected from
the group consisting of G[5']ppp[5']G, m.sup.7G[5']ppp[5']G,
m.sub.3.sup.2,2,7G[5]ppp[5']G, m.sub.2.sup.7,3'-OG[5']ppp[5']G
(3'-ARCA), m.sub.2.sup.7,2'-OGpppG (2'-ARCA),
m.sub.2.sup.7,2'-OGppspG D1 (.beta.-S-ARCA D1) and
m.sub.2.sup.7,2'-OGppspG D2 (.beta.-S-ARCA D2).
[0186] In another preferred embodiment, the RNA molecule,
preferably the mRNA molecule, to be analyzed is produced by in
vitro transcription and subsequent enzymatic capping (e.g.
post-transcriptional capping). Vaccinia Virus Capping Enzyme (VCE)
possesses all three enzymatic activities necessary to synthesize an
m7G cap structure (RNA 5'-triphosphatase, guanylyltransferase, and
guanine-7-methyltransferase). In vitro transcripts can be capped in
the presence of the capping enzyme, reaction buffer, GTP, and the
methyl donor S-adenosylmethionine (SAM). Using GTP as substrate the
VCE reaction yields RNA caps in the correct orientation. In
addition, a type 1 cap can be created by adding a second Vaccinia
enzyme, 2' O methyltransferase, to the capping reaction. RNA
carrying type I caps are reported to have enhanced translational
activity compared to type 0 caps (Tcherepanova et al., 2008. BMC
Mol. Biol. 9:90).
[0187] In a preferred embodiment, the cleavage site in the RNA
molecule is chosen in such a way that the resulting 5' terminal RNA
fragment can be separated or resolved, as described herein. Any
size is possible for the 5' terminal RNA fragment, as long as the
produced capped or non-capped 5' terminal RNA fragment, which
typically differ in length by one nucleotide--can be identified.
The skilled person will understand that one option to distinguish
the 5' terminal RNA fragment from other nucleic acid molecules may
be the selection of an appropriate size of the 5' terminal RNA
fragment by choosing an appropriate cleavage site. Alternatively or
in addition to the aforementioned, the 5' terminal RNA fragment may
also be labeled, preferably as described herein, with an
appropriate marker allowing specific detection of the 5' terminal
RNA fragment. In addition or alternatively to the separation
methods mentioned above, any suitable further analytical method,
preferably as described herein, may be employed in order to
determine whether the obtained 5' terminal RNA fragment is capped
or not.
[0188] Preferably, a reference RNA fragment (i.e. a fragment
sharing the same RNA sequence and having a known capping status) is
analyzed in parallel to the 5' terminal RNA fragment, which is
derived from the RNA molecule to be analyzed. For example, a capped
reference fragment is used in parallel as a control in step c) of
the method. The skilled person knows how to synthesize such
fragments, e.g. chemically or by enzymatic capping of an RNA
molecule.
[0189] In another embodiment, the method of the invention concerns
a method, wherein the RNA molecule, preferably an mRNA molecule,
more preferably a modified mRNA 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.
[0190] As mentioned above, a capped RNA molecule may comprise a 5'
terminal cap structure having, for example, the general structure
mGpppG, wherein "mG" is the cap structure (modified guanine
nucleotide, for instance, methylated at carbon 7), "ppp" is a 5' to
5' triphosphate linkage and "G" is a guanine nucleotide, wherein
"G" represents position 1 of an RNA molecule as defined herein and
is thus linked to position 2 of that RNA molecule. "G" is
preferably non-methylated, whereas "mG" is preferably methylated,
e.g. on carbon 7 and/or carbon 2. Thus, an RNA molecule having a 5'
terminus comprising a structure such as "5'-mG-ppp-G-N-3'", wherein
"N" is the nucleotide at position 2, as defined herein, of the RNA
molecule, is referred to herein as "correctly capped" RNA molecule.
On the other hand, a capped RNA molecule may--as an
alternative--comprise a 5' terminal structure, such as
"5'-G-ppp-Gm-N-3'", wherein the modified guanine Gm, e.g. a guanine
nucleotide methylated at carbon 7, is positioned closer to the
nucleotide at position 2 of the RNA moecule. Such an RNA molecule
is referred to herein as "reverse-capped" RNA molecule.
[0191] For example, reverse-capped RNA molecules are
synthesized--as a side product--by in vitro transcription,
preferably by in vitro transcription, which is carried out in
presence of a dinucleotide cap analog (such as m7G(5')ppp(5')G
(m7G)), also known as co-transcriptional capping.
[0192] The orientation is preferably determined by using a suitable
analytical method as described above. The 5' terminal RNA fragment
of a correctly capped RNA molecule and the 5' terminal RNA fragment
of a reverse-capped RNA molecule, wherein the only difference
between the correctly capped and the reverse-capped RNA molecule is
the orientation of the cap, typically have the same mass.
Nevertheless, the distinct orientation is associated with a
physical property that is determined by using the method according
to the invention. For example, fragments having the same mass may
interact in a distinct manner with the stationary and/or the mobile
phase in a chromatography assay, thus allowing the resolution of
two 5' terminal RNA fragments, which differ only by the orientation
of the cap structure, by means of chromatography.
[0193] In a preferred embodiment, the invention provides a method
for analyzing a capped RNA, preferably a capped modified RNA as
defined herein, having a cleavage site for a catalytic nucleic acid
molecule, wherein step c) comprises separating a 5' terminal RNA
fragment from the at least one 3' RNA fragment, preferably by the
means described herein, and determining the orientation of the 5'
cap structure at the 5' terminus of the fragment. Preferably, the
5' terminal RNA fragment is separated by a chromatographic
technique, preferably reversed-phase chromatography. In one
embodiment, the fragment is analyzed in a HPLC system
[0194] In a preferred embodiment, step c) comprises determining the
orientation of a cap structure in a 5' terminal RNA fragment, which
comprises comparison with a reference fragment. Preferably, a
capped reference fragment is used, of which the capping orientation
is known. More preferably, an enzymatically capped RNA is used for
synthesizing the reference fragment.
[0195] In addition, it has been found that the method according to
the invention is useful for characterizing a population of RNA
molecules, preferably as defined herein. Preferably, the method is
for analyzing a modified RNA molecule as defined herein.
Specifically, the invention provides 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: [0196]
a) providing a sample containing the population of RNA molecules,
[0197] 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, [0198] 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 [0199]
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.
[0200] While steps a), b) and c) are typically as defined herein
for the method for analyzing an RNA molecule having a cleavage site
for a catalytic nucleic acid molecule, step d) of the method for
analyzing a population of RNA molecules is specific for the latter.
Hence, all the features described above for steps a), b) and c)
applies in analogous manner to the method for analyzing an RNA
population. The method for analyzing an RNA population, however,
additionally comprises step d), which comprises 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.
[0201] As used herein, the population of RNA molecules typically
comprises at least one RNA molecule, preferably a modified RNA
molecule, having a cleavage site for a catalytic nucleic acid
molecule, wherein the at least one RNA molecule is characterized by
a distinct physical property or a distinct structural feature,
which may preferably be determined by analyzing the 5' terminal RNA
fragment obtained in step b) of the method for analyzing the RNA
population. Preferably, a population of RNA molecules comprises at
least one first RNA molecule having a cleavage site for a catalytic
nucleic acid molecule, and further comprises at least one second
RNA molecule having a cleavage site for a catalytic nucleic acid
molecule, wherein the first RNA molecule and the second RNA
molecule differ in a physical property or a structural feature i5
that may be determined by analyzing the respective 5' terminal RNA
fragments. By measuring the relative amounts of those 5' terminal
RNA fragments, the relative amounts of the respective RNA molecules
in the population of RNA molecules are determined. Therein, the
relative amounts of the 5' terminal RNA fragments are measured by
using any suitable technique for nucleic acid molecule
quantitation, preferably by using the techniques described herein.
In a preferred embodiment, the amounts of the 5' terminal RNA
fragments are measured in step c) by spectroscopic methods,
quantitative mass spectrometry, or sequencing. Step d) preferably
comprises calculating the ratio of the amount of an RNA molecule
with a distinct physical property to the amount of another RNA
molecule in the population or to the total amount of RNA molecules
in the population.
[0202] In a preferred embodiment of the method for analyzing an RNA
population, the population comprises at least one capped RNA
molecule having a cleavage site for the catalytic nucleic acid
molecule. Therein, step d) preferably comprises determining the
relative amount of capped RNA molecules in the population,
preferably by measuring the total amount of 5' terminal RNA
fragments and the amount of capped 5' terminal RNA fragments.
[0203] Preferably, 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. In that embodiment,
step c) comprises separating capped 5' terminal RNA fragments and
non-capped 5' terminal RNA fragments. Preferably, the amounts of
capped and non-capped 5' terminal RNA fragments are measured and
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. The relative amount (in percent) of capped RNA
molecules with respect to the total amount of RNA molecules in the
RNA population is also referred to herein as "capping degree".
[0204] In a preferred embodiment, the relative amounts (in percent)
of capped and non-capped 5' terminal RNA fragments--or capped and
non-capped RNA molecules to be analyzed--is calculated. In this
context, reference is also made to Example 3.
capped RNA ( % ) = amount of capped RNA amount ( non - capped RNA +
capped RNA ) .times. 100 ##EQU00001## non - capped RNA ( % ) =
amount non-capped RNA amount ( non - capped RNA + capped RNA )
.times. 100 ##EQU00001.2##
[0205] In another embodiment, the population comprises at least one
correctly capped RNA molecule as defined herein having a cleavage
site for the catalytic nucleic acid molecule and 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, preferably by measuring the total amount of 5'
terminal RNA fragments and the amount of correctly capped 5'
terminal RNA fragments.
[0206] Preferably, 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. In
that embodiment, step c) comprises separating correctly capped 5'
terminal RNA fragments and reverse-capped 5' terminal RNA
fragments. Preferably, the amounts of correctly capped and
reverse-capped 5' terminal RNA fragments are measured and 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.
[0207] In a particularly preferred embodiment, the method for
analyzing an RNA population comprises both, determining the
relative amount of capped RNA molecules in the RNA population and
determining the relative amount of correctly capped RNA molecules.
The population comprises at least one RNA molecule having a
cleavage site for a catalytic nucleic acid molecule, wherein the
RNA molecule is preferably modified as defined herein. The method
is not limited as to which property is determined first. In a
preferred embodiment, a technique is selected in step c) which
allows both, determining the presence of a cap and determining the
orientation of a cap structure. Preferably, step c) comprises a
chromatography technique, more preferably a liquid chromatography
technique as described herein or most preferably a HPLC technique.
In a particularly preferred embodiment, step c) comprises a
chromatography technique and a spectrometry technique, such as a
mass spectrometry technique.
[0208] In a further aspect, the present invention relates to a
method of determining the capping degree of a population of RNA
molecules having a cleavage site for a catalytic nucleic acid
molecule, comprising the steps of: [0209] a) providing a sample
containing the population of RNA molecules, [0210] 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, [0211]
c) separating the RNA fragments obtained in step b), [0212] d)
determining a measure for the amount of the capped and non-capped
5' terminal RNA fragments separated in step c) of said population
of RNA molecules, and [0213] 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.
[0214] In the context of the present invention, it has been found
that the method of the present invention is suitable for the
determination of the capping degree of a population of RNA
molecules. The method of the invention is especially suitable
because it allows the characterization of the capping degree of a
population of RNA molecules of any length, including very long RNA
molecules. Very long RNA molecules comprise at least 1000
nucleotides in length. This is achieved by cleaving the RNA
molecules with a catalytic nucleic acid molecule at a known site
resulting in 5'-terminal RNA fragments of the RNA molecules which
can be used to differentiate between capped and non-capped RNA
molecules.
[0215] According to the invention, the term "capped RNA molecule"
or "capped RNA fragment" means that the RNA molecule or the RNA
fragment bears at its 5'-terminus a 5' cap structure as defined
above.
[0216] The capped and non-capped RNA molecules or 5'-RNA fragments
of the population only differ by one nucleotide in size, because
typically the size of a 5' cap structure corresponds to the length
of one nucleotide.
[0217] According to the invention, the term "capping degree"
indicates how many of the RNA molecules of the population have a
cap at their 5' end, in particular, this term indicates the
percentage of capped RNA molecules of the population.
[0218] In particularly preferred embodiments the RNA molecules are
long RNA molecules comprising at least 100, 150, 200 or more
preferably at least 500 nucleotides in length.
[0219] Preferably the RNA molecules of the population used
according to the invention comprise at least one open reading frame
coding for at least one peptide or protein.
[0220] In this context the RNA molecules of the population can
comprise one (monocistronic), two (bicistronic) or more
(multicistronic) open reading frames (ORF). The RNA molecules of
the population can be messenger RNA (mRNA) molecules, viral RNA
molecules or self-replicating RNA molecules (replicons).
[0221] Preferably the RNA molecules of the population are mRNA
molecules.
[0222] Preferably the RNA molecules of the population are primary
microRNA (pri-miRNA) molecules.
[0223] It is known that miRNAs are first transcribed as a largely
unstructured precursor, termed a primary miRNA (pri-miRNA), which
is sequentially processed in the nucleus, to give the approximately
65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to
give the mature miRNA. These pre-miRNA molecules can be capped and
polyadenylated (Cai et al., 2004. RNA 10(12):1957-66).
[0224] According to the invention, the RNA molecules of the
population comprise a cleavage site for a catalytic nucleic acid
molecule allowing the cleavage of the RNA molecules of the
population into a 5' RNA fragment and at least one 3' RNA fragment.
In a preferred embodiment, the cleavage site in the RNA molecule is
chosen in such a way that short enough capped and non-capped 5' RNA
fragments are produced that can be separated. In general, and as
known by the person skilled in the art, the choice of the cleavage
site (and thereby the length of the obtained RNA fragments) will
also depend on the separation method used and its resolution
capacity to discriminate between RNA fragments of different length.
Since the capped 5' RNA fragment originating from a capped RNA
molecule of the population is usually about only one nucleotide
longer than the non-capped 5' RNA fragment originating from a
non-capped RNA molecule of the population, single-nucleotide
resolution is necessary. Preferably, the defined cleavage site is
located between nucleotide positions 1 to 500 in 5'-3' direction of
the RNA molecule, so that the resulting 5' RNA fragment has a size
equal to or smaller than 500 nucleotides. More preferred, the
defined cleavage site is located between nucleotide positions 1 to
400, 1 to 300, 1 to 200 or 1 to 100 in 5'-3' direction of the RNA
molecule. Most preferred, the defined cleavage site is located
between nucleotide positions 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1
to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90 or 1 to 100 in
5'-3' direction of the RNA molecule.
[0225] Any of the above sizes are possible as long as the produced
capped and non-capped 5' RNA fragments can be distinguished from
each other, from the catalytic nucleic acid molecule, and from
other nucleic acid molecules, especially from the 3' RNA fragments.
The skilled person will understand that one option to distinguish
the 5' RNA fragments of interest from other nucleic acid molecules
may be the choice of an appropriate size of the 5' RNA fragments by
choosing an appropriate cleavage site. Another option is to provide
the 5' RNA fragments with an appropriate marker so that the 5' RNA
fragments are labeled which allows the specific detection of the
cleaved 5' RNA fragments.
[0226] By "labeled" is meant that the RNA molecule is either
directly or indirectly labeled with a molecule which provides a
detection signal, e.g. radioisotope, fluorescent tag,
chemiluminescent tag, a peptide or specific binding molecules.
Specific binding molecules include pairs, such as biotin and
streptavidin, digoxin and antidigoxin. The label can directly or
indirectly provide a detectable signal. Radioisotopes (e.g.
.sup.18F, .sup.125I, .sup.35S, .sup.14C, .sup.3H, or .sup.99mTc)
are commonly used in biological applications for the detection of a
variety of nucleic acids such as RNA. Methods for the synthesis and
labelling of RNA in vitro are known in the art (e.g. Huang and Yu,
2013. Synthesis and Labelling of RNA In Vitro. Current Protocols in
Molecular Biology. 102:4.15.1-4.15.14).
[0227] For example, the synthesis and use of biotin labeled cap
analogs has been described (Jemielity et al., 2012. Org. Biomol.
Chem. 10(43):8570-4; WO2013/059475). These cap analogs can be
incorporated into RNA molecules to produce 5'-capped and
biotinylated RNAs which retain their biological functionality and
can be used for biotin-streptavidin technologies.
[0228] In the first step of the method according to the invention,
a sample containing the population of RNA molecules is
provided.
[0229] The method according to the present invention can be
performed with any RNA preparation as a starting material, as long
as the respective population of RNA molecules of interest is
present in the preparation. The RNA preparation can be derived from
a cell endogenously expressing said population of RNA molecules of
interest or a cell that is transfected with a nucleic acid such as
DNA or RNA or infected by a virus and therefore expressing said
population of RNA molecules. For example, said RNA preparation can
be derived from a cell, tissue, organ, organism, bacterial cell or
virus. Methods for the isolation of RNA from these sources are
known in the art (Liu and Harada, 2013. RNA Isolation from
Mammalian Samples. Current Protocols in Molecular Biology.
103:4.16.1-4.16.16).
[0230] In the context of the present invention, the term
"endogenously" means that the respective cell expresses said
population of RNA molecules without being transfected with an
RNA-encoding nucleic acid.
[0231] The sample containing the population of RNA molecules
provided in the first step of the method according to the present
invention may, apart from the population of RNA molecules of
interest, also contain other nucleic acid molecules, especially
other RNA molecules.
[0232] In a preferred embodiment of the first aspect of the present
invention, 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.
[0233] In this context, the population of RNA molecules may be
produced by in vitro transcription in the presence of a cap analog
(co-transcriptional capping). Capped in vitro transcripts can be
synthesized by substituting a cap analog such as a m7G(5')ppp(5')G
(m7G) for a portion of the GTP in the transcription reaction,
typically the cap analog is used at a four-fold excess compared to
GTP. Methods for in vitro transcription are known in the art (Geall
et al., 2013. Semin. Immunol. 25(2): 152-159) and typically
include:
1) a linearized DNA template with a promoter sequence that has a
high binding affinity for its respective RNA polymerase such as
bacteriophage-encoded RNA polymerases, 2) ribonucleotide
triphosphates (NTPs) for the four bases (adenine, cytosine, guanine
and uracil); 3) a cap analog as defined above (e.g. m7G(5')ppp(5')G
(m7G)); 4) a DNA-dependent RNA polymerase (e.g. T7, T3 or SP6 RNA
polymerase); 5) a ribonuclease (RNase) inhibitor to inactivate any
contaminating RNase; 6) a pyrophosphatase to degrade pyrophosphate,
which may inhibit transcription; 7) MgCl.sub.2, which supplies
Mg.sup.2+ as a co-factor for the polymerase; 8) a buffer to
maintain a suitable pH value, which can also contain antioxidants
and polyamines such as spermidine at optimal concentrations.
[0234] In a preferred embodiment, the cap analog is selected from
the group consisting of G[5']ppp[5']G, m.sup.7G[5']ppp[5']G,
m.sub.3.sup.2,2,7G[5]ppp[5']G, m.sub.2.sup.7,3'-OG[5']ppp[5']G
(3'-ARCA), m.sub.2.sup.7,2'-OGpppG (2'-ARCA),
m.sub.2.sup.7,2'-OGppspG D1 (.beta.-S-ARCA D1) and
m.sub.2.sup.7,2'-OGppspG D2 (.beta.-S-ARCA D2).
[0235] In another preferred embodiment, the population of RNA
molecules is produced by in vitro transcription and subsequent
enzymatic capping (e.g. post-transcriptional capping). Vaccinia
Virus Capping Enzyme (VCE) possesses all three enzymatic activities
necessary to synthesize an m7G cap structure (RNA
5'-triphosphatase, guanylyltransferase, and
guanine-7-methyltransferase). In vitro transcripts can be capped in
the presence of the capping enzyme, reaction buffer, GTP, and the
methyl donor S-adenosylmethionine (SAM). Using GTP as substrate the
VCE reaction yields RNA caps in the correct orientation. In
addition, a type 1 cap can be created by adding a second Vaccinia
enzyme, 2' O methyltransferase, to the capping reaction. RNA
carrying type I caps are reported to have enhanced translational
activity compared to type 0 caps (Tcherepanova et al., 2008. BMC
Mol. Biol. 9:90).
[0236] In a further preferred embodiment, the population of RNA
molecules is produced by non-enzymatic chemical RNA synthesis (e.g.
Marshall and Kaiser, 2004. Curr. Opin. Chem. Biol. 8(3):222-229).
Currently, the length of RNA molecules synthesized by chemical
methods is limited to about 100 nucleotides.
[0237] In a further step of the method according to the present
invention, the RNA molecules of the population are cleaved by the
catalytic nucleic acid molecule into RNA fragments (a 5' RNA
fragment and at least one 3' RNA fragment) by contacting the sample
resulting from the first step of the method according to the
present invention with the catalytic nucleic acid molecule under
conditions allowing the cleavage of the RNA molecule.
[0238] In a preferred embodiment, the catalytic nucleic acid
molecule has been designed to be able to cleave the RNA molecules
of the population at a specific cleavage site.
[0239] Methods to design catalytic nucleic acid molecules, in
particular ribozymes that cleave RNA substrate molecules at a
defined site, are known in the art.
[0240] For example, hairpin ribozymes cleave 5' of the guanosine in
NGUC sequences, wherein N is any nucleotide. Furthermore, for
example, a hammerhead ribozyme can be directed to cleave 3' of any
NUH sequence, wherein N is any nucleotide, U is conserved, and H
can be any nucleotide except G (N=G,A,C,U; H=A,C,U) (Haseloff and
Gerlach, 1988. Nature 334: 585-591; McCall et al., 2000. Molecular
Biotechnology 14: 5-17).
[0241] According to the substrate requirements described above, any
RNA molecule can be expected to contain a number possible sites for
sequence-specific cleavage by a catalytic nucleic acid molecule. In
addition to the target site, the number of base pairs to be formed
between the catalytic nucleic acid molecule and substrate must be
chosen (substrate binding region). The affinity of a catalytic
nucleic acid molecule towards its substrate can be adjusted by
altering the length of the substrate binding region of the
catalytic nucleic acid molecule. Although high affinity is usually
desirable, an extended substrate binding region may cause problems
regarding specificity and catalytic activity. Multiple turnover
catalysis may be severely impaired if product release is slow due
to strong binding of the target nucleic acid molecule to the
catalytic nucleic acid molecule. Catalytic nucleic acid molecules
with short binding arms (substrate binding region), however, may
lack specificity.
[0242] The aspects to consider are catalytic activity and
specificity. Catalytic nucleic acid molecules which form a larger
number of base pairs with the substrate RNA are less likely to
dissociate from the cleaved substrate, and therefore are not
available for further cleavage. Therefore, the number of base pairs
should be selected in such a way that the catalytic nucleic acid
molecule-substrate complex formed is relatively stable under the
conditions of the experiment, but is able to dissociate once
cleavage of the substrate occurs. This typically requires 11 to 17
base pairs. For specificity, the number of base pairs formed
between the catalytic nucleic acid molecule and the substrate RNA
should be large enough to make the target sequence unique, but not
so large that imperfectly matched substrates form stable complexes.
Statistically, about 13 nucleotides are required to uniquely define
a particular site in an RNA pool.
[0243] Methods for the production of catalytic nucleic acid
molecules are known in the art. For example, ribozymes can be
chemically synthesized using the procedure for normal RNA synthesis
as described (Wincott et al., 1995. Nucleic Acids Res.
23(14):2677-84). Ribozymes can also be synthesized by in vitro
transcription of suitable DNA templates using e.g. bacteriophage T7
RNA polymerase (Haseloff and Gerlach, 1988. Nature 334:
585-591).
[0244] Alternatively, in the context of the present invention, it
is also envisaged that the RNA molecules of the population has been
designed to have a cleavage site for the catalytic nucleic acid
molecule. Methods for changing or introducing nucleotides into DNA
molecules to produce specific sites are known in the art. This DNA
template can then be used to produce an RNA molecule by in vitro
transcription methods as explained above. These methods are known
in the art.
[0245] In this context it is particularly preferred that the
catalytic nucleic acid molecule is provided in trans. This means
that the RNA molecules of the population and the catalytic nucleic
acid molecule are not part of the same molecule. However, it is
also included within the present invention that they are included
in cis, i.e. the RNA molecules of the population and the catalytic
nucleic acid molecule are part of the same molecule.
[0246] In a particularly preferred embodiment of the first aspect
of the present invention the catalytic nucleic acid molecule is a
ribozyme.
[0247] In this context it is particularly preferred that the
ribozyme is selected from the group consisting of hammerhead
ribozymes, hairpin ribozymes, and HDV ribozymes.
[0248] In an even more preferred embodiment, the ribozyme is a
hammerhead ribozyme.
[0249] Particularly preferred in this context is a hammerhead
ribozyme comprising an RNA sequence according to SEQ ID NO. 1 which
can be directed to cleave 3' of any NUH sequence as shown in FIG. 5
(N=G,A,C,U; H=A,C,U) (Haseloff and Gerlach, 1988. Nature 334:
585-591; McCall et al., 2000. Molecular Biotechnology, 14:
5-17).
[0250] In an even more particularly preferred embodiment a
hammerhead ribozyme HHNUH2d according to SEQ ID NO. 2 can be used
which was designed to target the 5' region of the RNA sequences
according to SEQ ID NO. 3-5 and shown in FIGS. 1 to 3, forming
helix III with mRNA positions 1-12, and helix I with mRNA positions
14-18 (FIG. 6) of the RNA sequences according to SEQ ID No. 3-5.
The 5' region of the target RNA sequence contains two possible
recognition sites, NUH1 (positions 10-12) and NUH2 (positions
11-13), of which NUH2 is the preferred target site.
[0251] Sequence of the trans-acting hammerhead ribozyme HHNUH2d
TABLE-US-00003 (SEQ ID NO: 2):
5'-GCAUGGCUGAUGAGGCCUCGACCGAUAGGUCGAGGCCGAAAAGCU UUCUCCC-3'
[0252] In another particularly preferred embodiment, the catalytic
nucleic acid molecule is a DNAzyme, e.g. a "10-23" DNAzyme.
[0253] By the cleavage with the catalytic nucleic acid molecule,
the RNA molecules of the population are cleaved at a defined site
so that a 5' terminal and at least one 3' terminal RNA fragment is
produced.
[0254] In the context of the present invention, the term "under
conditions allowing the cleavage of the RNA molecule" means that
the reaction conditions are chosen in a way to allow the binding of
the catalytic nucleic acid molecule to the substrate RNA molecule
and subsequent cleavage of the substrate RNA molecule. The skilled
person will know which conditions can be applied in order to enable
the cleavage of the substrate RNA molecule.
[0255] Two protocols are commonly used to perform the cleavage
reaction. In one protocol the ribozyme and the substrate RNA are
heated together at 95.degree. C. (preferably 1 to 2 minutes) in the
presence of buffer without magnesium, and subsequently cooled to
the reaction temperature (20-37.degree. C., preferably 25.degree.
C.) to promote annealing. Then MgCl.sub.2 is added to initiate the
cleavage reaction. In another protocol the ribozyme and substrate
RNA are heated separately at 95.degree. C. without MgCl.sub.2 for
one to two minutes and then cooled to the reaction temperature.
MgCl.sub.2 is added to both the ribozyme and the substrate RNA and
the cleavage reaction is started by mixing both.
[0256] To achieve complete cleavage the Mg' concentration, buffer
composition, pH value, temperature and reaction time may need to be
adjusted.
[0257] For example, "conditions allowing the cleavage of the RNA
molecule" may comprise 50-200 mM NaCl or KCl, 0.1-200 mM Mg', 5-100
mM Tris-HCl, pH 6.5-8.5, 20-37.degree. C. for 5 minutes to 2
hours.
[0258] A non-ionic detergent (Tween, NP-40, Triton-X 100) can often
be present, usually at about 0.001 to 2%, typically 0.05-0.2%
(volume/volume).
[0259] Preferably, "conditions allowing the cleavage of the RNA
molecule" mean a pH of from 6.5-7.5, preferably from 7.0-7.5,
and/or a buffer concentration of from 5-100 mM, preferably from 25
to 50 mM, and/or a concentration of monovalent salts (e.g. Na or K)
of from 120-170 mM, preferably 150 mM, and/or Mg.sup.++ at a
concentration of from 0.1-200 mM, preferably 20-40 mM, wherein more
preferably the buffer is selected from the group consisting of
Tris-HCl or HEPES.
[0260] In a further step of the method according to the present
invention, the RNA fragments obtained from the cleaving step b)
described above are separated.
[0261] Any suitable method for separating RNA fragments can be
used, including, but not limited to denaturing gel electrophoresis
or liquid chromatography.
[0262] In a particularly preferred embodiment of the first aspect
of the present invention the RNA fragments are separated in step c)
by denaturing gel electrophoresis or liquid chromatography,
preferably HPLC, FPLC or RPLC.
[0263] Separation of RNA molecules by denaturing gel
electrophoresis has been described (Maniatis et al., 1975.
Biochemistry 14(17):3787-3794). For example, polyacrylamide gels
that contain a high concentration of a denaturing agent such as
urea are capable of resolving short (<500 nucleotides)
single-stranded RNA fragments that differ in length by as little as
one nucleotide. In this context polyacrylamide gels comprising 8 M
urea are particularly preferred.
[0264] The RNA molecules can also be separated by liquid
chromatography methods. As used herein, the term "liquid
chromatography" (LC) means a process of selective retardation of
one or more components of a fluid solution as the fluid uniformly
percolates through a column of a finely divided substance, or
through capillary passageways. The retardation results from the
distribution of the components of the mixture between one or more
stationary phases and the bulk fluid, (i.e., the mobile phase), as
this fluid moves relative to the stationary phase(s). LC includes
reverse phase liquid chromatography (RPLC), high performance liquid
chromatography (HPLC), high turbulence liquid chromatography (HTLC)
and fast performance liquid chromatography (FPLC). In contrast to
HPLC, the buffer pressure used in FPLC is relatively low, typically
less than 5 bar, but the flow rate is relatively high, typically
1-5 ml/min.
[0265] Stationary phases for the use in liquid chromatography are
known in the art. For example, the stationary phase can be a porous
polystyrene, a porous non-alkylated polystyrene, a
polystyrenedi-vinylbenzene, a porous non-alkylated
polystyrenedivinylbenzene, a porous silica gel, a porous silica gel
modified with non-polar residues, a porous silica gel modified with
alkyl containing residues, selected from butyl-, octyl and/or
octadecyl containing residues, a porous silica gel modified with
phenylic residues, or a porous polymethacrylate (WO2008077592, the
disclosure is incorporated herewith by reference).
[0266] In this context, ethylene-bridged hybrid organic/inorganic
stationary phases are particularly preferred (Wyndham et al., 2003.
Anal. Chem. 75(24):6781-8 and WO2003014450, the disclosure is
incorporated herewith by reference).
[0267] For example, the separation process of RNA molecules by HPLC
has been described (Weissman et al., 2013. Methods Mol. Biol.
969:43-54).
[0268] In a further step of the method of the present invention, a
measure for the amount of the capped and non-capped 5' terminal RNA
fragments separated in step c) described above of said population
of RNA molecules is determined.
[0269] In the context of the present invention, it is necessary to
determine and compare measures for the amount of the obtained
capped and non-capped 5'-terminal RNA fragments. Any suitable
measure can be taken including but not limited to the signal
intensity of the RNA fragments.
[0270] In a preferred embodiment, 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.
[0271] The signal intensity is particularly preferred, because it
can be detected directly e.g. in denaturing gel electrophoresis
using appropriate dyes for the staining of the RNA fragments like
ethidium bromide. As explained above, it is also possible to
provide the RNA fragments with an appropriate marker like a
fluorescence marker or a radioactive marker and then detect the
signal intensity of the marker molecule.
[0272] Alternatively, it is also included within the present
invention to detect and determine the amount of the RNA fragments
itself, e.g. by determining the number of RNA fragments or the mass
of the RNA fragments.
[0273] In a preferred embodiment, 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.
[0274] Spectroscopic methods for RNA quantification include
traditional absorbance measurements at 260 nm and more sensitive
fluorescence techniques using fluorescent dyes such as ethidium
bromide and a fluorometer with an excitation wavelength of 302 or
546 nm (Gallagher, 2011. Quantitation of DNA and RNA with
Absorption and Fluorescence Spectroscopy. Current Protocols in
Molecular Biology. 93:A.3D.1-A.3D.14).
[0275] A mass spectrometer (MS) is a gas phase spectrometer that
measures a parameter that can be translated into mass-to-charge
ratio of gas phase ions. Examples of mass spectrometers are
time-of-flight, magnetic sector, quadrupole filter, ion trap, ion
cyclotron resonance, electrostatic sector analyser and hybrids of
these. Methods for the application of MS methods to the
characterization of nucleic acids are known in the art.
[0276] For example, Matrix-Assisted Laser Desorption/Ionization
Mass Spectrometry (MALDI-MS) can be used to analyse
oligonucleotides at the 120-mer level and below (Castleberry et
al., 2008. Matrix-Assisted Laser Desorption/Ionization
Time-of-Flight Mass Spectrometry of Oligonucleotides. Current
Protocols in Nucleic Acid Chemistry. 33:10.1.1-10.1.21).
[0277] Electrospray Ionization Mass Spectrometry (ESI-MS) allows
the analysis of high-molecular-weight compounds through the
generation of multiply charged ions in the gas phase and can be
applied to molecular weight determination, sequencing and analysis
of oligonucleotide mixtures (Castleberry et al., 2008. Electrospray
Ionization Mass Spectrometry of Oligonucleotides. Current Protocols
in Nucleic Acid Chemistry. 35:10.2.1-10.2.19). Preferably, the mass
spectrometry analysis is conducted in a quantitative manner to
determine the amount of RNA.
[0278] Methods for sequencing of RNA are known in the art. A
recently developed technique called RNA Sequencing (RNA-Seq) uses
massively parallel sequencing to allow for example transcriptome
analyses of genomes at a far higher resolution than is available
with Sanger sequencing- and microarray-based methods. In the
RNA-Seq method, complementary DNAs (cDNAs) generated from the RNA
of interest are directly sequenced using next-generation sequencing
technologies. RNA-Seq has been used successfully to precisely
quantify transcript levels, confirm or revise previously annotated
5' and 3' ends of genes, and map exon/intron boundaries (Eminaga et
al., 2013. Quantification of microRNA Expression with
Next-Generation Sequencing. Current Protocols in Molecular Biology.
103:4.17.1-4.17.14). Consequently, the amount of the RNA fragments
can be determined also by RNA sequencing.
[0279] In a further step of the method of the present invention,
said measures of capped and non-capped 5' terminal RNA fragments
determined as described above in step d) are compared, thereby
determining the capping degree of said population of RNA
molecules.
[0280] Said determination and comparison can be performed by eye or
with the help of technical systems, e.g. by using computer
software.
[0281] In a preferred embodiment, the ratio of capped and
non-capped 5' terminal RNA fragments is calculated. In this
context, reference is also made to example 3.
[0282] For example, the degrees of capped and non-capped RNA,
respectively, can be calculated according to:
capped RNA ( % ) = measure capped RNA measures ( non - capped RNA +
capped RNA ) .times. 100 ##EQU00002## non - capped RNA ( % ) =
measure non-capped RNA measures ( non - capped RNA + capped RNA )
.times. 100 ##EQU00002.2##
[0283] In a preferred embodiment, the population of RNA molecules
comprises or consists of mRNA molecules.
[0284] In a preferred embodiment of the method for determining the
capping degree of a population of RNA molecules having a cleavage
site for a catalytic nucleic acid molecule, the orientation of the
cap on a capped 5' terminal RNA fragment is further determined as
described herein.
[0285] In a specific embodiment the method according to the present
invention is used as a quality control in the production process of
RNA molecules.
[0286] In a further aspect, the present invention also relates to
the use of a catalytic 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.
[0287] In the context of the present invention, it has been found
that, as explained above, catalytic nucleic acid molecules and
especially ribozymes are useful for determining the capping degree
of a population of RNA molecules. Consequently, the present
invention also relates to the use of said catalytic nucleic acid
molecules for said purpose.
[0288] In this context in a specific embodiment the catalytic
nucleic acid molecules used for determining the capping degree of a
population of RNA molecules may be used in the quality control of
the production process of RNA molecules.
[0289] All embodiments defined above for the method of the
invention also apply in the context of said use of the
invention.
[0290] In another aspect, the invention provides a novel use of a
catalytic nucleic acid molecule, preferably as defined herein, in a
method for analyzing an RNA molecule, preferably an mRNA molecule,
more preferably a modified RNA or mRNA molecule, having a cleavage
site for the catalytic nucleic acid molecule. In a preferred
embodiment, a catalytic nucleic acid molecule is used for analyzing
an RNA molecule as part of a quality control process during or
after RNA production, wherein the RNA, preferably an mRNA molecule,
more preferably a modified RNA or mRNA molecule, is preferably
produced by in vitro transcription. Preferably, the catalytic
nucleic acid molecule as defined herein, preferably a ribozyme, is
used in a method, which comprises determining the presence or
absence of a cap structure in an RNA molecule having a cleavage
site for the catalytic nucleic acid molecule. Alternatively or in
addition, the catalytic nucleic acid molecule as defined herein,
preferably a ribozyme, is used in a method, which comprises
determining the orientation of the cap structure at the 5' terminus
of a capped RNA molecule, preferably an mRNA molecule.
[0291] Furthermore, the invention provides a novel use of a
catalytic nucleic acid molecule, preferably as defined herein, in a
method for analyzing a population of RNA molecules, wherein the
population comprises at least one RNA molecule, preferably a
modified RNA molecule, having a cleavage site for a catalytic
nucleic acid molecule. In a preferred embodiment, a catalytic
nucleic acid molecule is used according to the invention for
analyzing a population of RNA molecules as part of a quality
control process during or after RNA production, wherein the RNA
molecules, preferably mRNA molecules, are preferably produced by in
vitro transcription and wherein at least one RNA molecule has a
cleavage site for the catalytic nucleic acid molecule.
[0292] In a preferred embodiment, the catalytic nucleic acid
molecule is used in a method for analyzing an RNA population,
wherein the population comprises at least one capped RNA molecule,
preferably at least one capped modified 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. Alternatively or in
addition, the catalytic nucleic acid molecule is used in a method
for analyzing an RNA population, wherein the population comprises
at least one correctly capped RNA molecule, preferably at least one
correctly capped modified 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.
[0293] In a particularly preferred embodiment, the invention
concerns the use of a catalytic nucleic acid molecule, preferably
as defined herein, more preferably a ribozyme, for analyzing an RNA
population, preferably an mRNA population, comprising at least one
RNA molecule, preferably a modified RNA molecule, having a cleavage
site for the catalytic nucleic acid molecule. More preferably, the
RNA population comprises at least one RNA molecule, preferably an
mRNA molecule, which was produced by an in vitro transcription
process. According to one embodiment, the catalytic nucleic acid
molecule is used for analyzing a population of RNA molecules as
part of a quality control process during or after RNA production,
wherein the production preferably involves an in vitro
transcription process. Therein, the population preferably comprises
at least one capped RNA molecule, preferably a capped mRNA
molecule, and the method is used for determining the relative
amount of capped RNA molecules having a cleavage site for the
catalytic nucleic acid molecule. In a particularly preferred
embodiment, the method further comprises determining the relative
amounts of correctly capped and reverse-capped RNA molecules,
respectively, in the RNA population.
[0294] The invention further provides the use of a catalytic
nucleic acid molecule in a method for analyzing an RNA molecule,
wherein the method comprises at least one of the features described
herein with respect to the inventive methods.
[0295] Furthermore, the invention provides a 5' terminal RNA
fragment obtainable by the methods according to invention. In a
preferred embodiment, the invention concerns the isolated 5'
terminal RNA fragment obtainable by the methods according to
invention.
[0296] In addition, the invention provides an 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.
[0297] In one embodiment, the 5' terminal RNA fragment according to
the invention or the RNA molecule according to the invention
comprises or consists of SEQ ID NO: 6, wherein the RNA fragment or
the RNA molecule optionally comprises a cap structure at the 5'
terminus.
TABLE-US-00004 SEQ ID NO: 6: GGGAGAAAGC
[0298] In a preferred embodiment, 5' terminal RNA fragment
according to the invention or the RNA molecule according to the
invention have the general structure according to the following
formula:
5'-cap-N.sub.10-NUH-3',
wherein NUH is preferably as defined above, and N is selected from
A, G, U and C.
[0299] In a preferred embodiment, N.sub.10 in the formula above is
the nucleic acid sequence defined by SEQ ID NO: 6.
[0300] Preferably, the 5' terminal RNA fragment according to the
invention or the RNA molecule according to the invention comprise
at least one modification as defined herein.
[0301] The invention further concerns the use of the 5' terminal
RNA fragment according to the invention or the RNA molecule
according to the invention in a method for analyzing an RNA
molecule. In a preferred embodiment of the invention, the 5'
terminal RNA fragment according to the invention or the RNA
molecule according to the invention are used in a method for
analyzing RNA or in a method for analyzing an RNA population,
wherein the method further comprises at least one of the features
as described above with respect to the inventive methods.
[0302] The invention is further explained with the help of the
following figures and examples, which are intended to explain, but
not to limit the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0303] The figures shown in the following are merely illustrative
and shall describe the present invention in a further way. These
figures shall not be construed to limit the present invention
thereto.
[0304] FIG. 1: G/C optimized mRNA sequence coding for Homo sapiens
New York Esophageal Squamous Cell Carcinoma 1 antigen (HsNY-ESO-1;
SEQ ID NO: 3).
[0305] FIG. 2: G/C optimized mRNA sequence coding for Photinus
pyralis Luciferase (PpLuc; SEQ ID NO: 4).
[0306] FIG. 3: G/C optimized mRNA sequence coding for Homo sapiens
prostate stem cell antigen (HsPSCA; SEQ ID NO: 5).
[0307] FIGS. 4A-B: Schematic diagram of non-capped (A) and capped
mRNA (B).
[0308] FIG. 5: Diagram of hammerhead ribozyme annealed to target
RNA sequence (highlighted in bold).
[0309] FIG. 6: Hammerhead ribozyme HHNUH2d annealed to 5' UTR of
target mRNA sequence (highlighted in bold). Recognition site marked
by circle and AUG start codon underlined. Cleavage at the indicated
site yields a 13mer 5'fragment of non-capped RNA (or a 14mer
fragment if the RNA is capped).
[0310] FIGS. 7A-B: Separation of capped and non-capped RNA
fragments by denaturing polyacrylamide gel electrophoresis (dPAGE).
RNAs were synthesized in the absence (-) or presence (+) of a cap
analog as described in Example 2 and subsequently incubated without
(-) or with (+) hammerhead (HH) ribozyme HHNU2d as described in
Example 3. (A) Full gel and (B) enlarged part of gel with capped
and non-capped RNA fragments.
[0311] FIGS. 8A-B: Separation of capped 5' mRNA fragments,
non-capped 5' mRNA fragments, 3' mRNA fragments and hammerhead
ribozyme by HPLC. The mRNA sample was prepared by mixing 60%
enzymatically capped mRNA coding for Photinus pyralis Luciferase
(PpLuc) and 40% non-capped mRNA coding for Photinus pyralis
Luciferase (PpLuc). Subsequently, this sample was incubated with
the hammerhead (HH) ribozyme HHNU2d as described in Example 3 and
analysed by HPLC. [0312] (A) Full chromatogram showing the
separation of the hammerhead (HH) ribozyme from the 3' mRNA
fragment. [0313] (B) Enlarged area of the chromatogram showing the
separation of capped and non-capped 5' mRNA fragments.
[0314] FIGS. 9A-D: Resolution of co-transcriptionally capped,
non-capped, and enzymatically capped 5' mRNA fragments separated by
dPAGE and HPLC. Photinus pyralis luciferase (PpLuc) RNAs were
synthesized in the absence (no cap) or presence (cotx) of a cap
analog, non-capped RNAs were subsequently enzymatically capped
(Ecap) as described in Example 2. The RNAs were incubated with
hammerhead (HH) ribozyme HHNU2d as described in Example 3 and
analysed by dPAGE and HPLC. [0315] (A) Enlarged part of a dPAGE gel
with capped (cotx, Ecap) and non-capped (no cap) RNA fragments. Two
bands were detected for co-transcriptionally capped RNA. [0316]
(B-D) Enlarged area of the HPLC chromatogram showing the separation
of capped and non-capped 5' mRNA fragments. (B) Non-capped PpLuc
RNA, (C) enzymatically capped PpLuc RNA originating from (B), (D)
co-transcriptionally capped PpLuc RNA. Five peaks were detected for
co-transcriptionally capped mRNA.
[0317] FIG. 10: Fractionation of co-transcriptionally capped 5'
mRNA fragments via HPLC. Photinus pyralis luciferase (PpLuc) RNAs
were synthesized in the presence of a cap analog as described in
Example 2, incubated with hammerhead (HH) ribozyme HHNU2d as
described in Example 3, and analyzed via HPLC. Fractions were
collected over the course of time as indicated on the x-axis of the
diagram. Double peaks at 13.30-13.80 minutes and 14.30-14.80
minutes could not be separated via HPLC and were thus pooled prior
to MALDI analysis (sample S1=fractions 12-14, S2=21-24,
S3=28-30).
[0318] FIGS. 11A-B: MALDI-TOF spectrum for samples S1 and S2
obtained after HPLC separation of ribozyme-cleaved 5' terminal RNA
fragments of co-transcriptionally capped Photinus pyralis
luciferase (PpLuc) RNAs. Analyses were performed as described in
Example 3.
[0319] FIGS. 12A-D: Overlay of HPLC analyses of
co-transcriptionally capped, non-capped, and enzymatically capped
5' terminal RNA fragments. Photinus pyralis luciferase (PpLuc) RNAs
were synthesized in the absence (no cap) or presence of a cap
analog, non-capped RNAs were subsequently enzymatically capped
(Ecap) as described in Example 2. In addition, enzymatically capped
Photinus pyralis Luciferase (PpLuc) RNAs lacking the initial 5'
guanosine (Ecap-G1) were synthesised analogously. The RNAs were
incubated with hammerhead (HH) ribozyme HHNU2d as described in
Example 3 and analysed by HPLC.
[0320] FIGS. 13A-D: Quantitation of different RNA populations. Peak
areas (mAU*min) for non-capped, correctly capped and reverse-capped
RNA populations (FIG. 12 D, full-length and minus1G (`n-1`) RNA)
were determined using Chromeleon software. Relative proportions
were plotted: (A) distribution of single populations; (B) combined
capped versus non-capped populations; (C) combined minusG1 RNA
(correctly capped and reverse-capped) and combined full-length RNA
(correctly capped and reverse-capped) versus non-capped; (D)
combined correctly capped RNA (full-length and minus1G), combined
reverse-capped RNA (full-length and minusG1) versus non-capped
RNA.
EXAMPLES
[0321] The examples shown in the following are merely illustrative
and shall describe the present invention in a further way. These
examples shall not be construed to limit the present invention
thereto.
Example 1: Preparation of Hammerhead Ribozymes
[0322] 1. A hammerhead ribozyme can be directed to cleave 3' of any
NUH sequence as shown in FIG. 5 (N=G,A,C,U; H=A,C,U) (Haseloff and
Gerlach, 1988. Nature 334: 585-591; McCall et al., 2000. Molecular
Biotechnology 14: 5-17). The schematic diagram of FIG. 5 shows how
helix I and helix III anneal to the target RNA sequence.
[0323] 2. The trans-acting hammerhead ribozyme HHNUH2d was designed
to target the 5' region of the RNA sequences shown in FIGS. 1 to 3,
forming helix III with mRNA positions 1-12, and helix I with mRNA
positions 14-18 (FIG. 6). The 5' region of the target RNA sequence
contains two possible recognition sites, NUH1 (positions 10-12) and
NUH2 (positions 11-13), of which NUH2 is the preferred target
site.
[0324] Sequence of the trans-acting hammerhead ribozyme HHNUH2d
(SEQ ID NO: 2):
TABLE-US-00005 5'-GCAUGGCUGAUGAGGCCUCGACCGAUAGGUCGAGGCCGAAAAGCU
UUCUCCC-3'
[0325] 3. The ribozyme HHNUH2d was synthesized and HPLC purified by
Biomers.net GmbH (Ulm, Germany), and 200 .mu.g were resolved on a
preparative denaturing 10 cm.times.8 cm.times.1.0 mm acrylamide gel
for purification (8 M urea (Applichem), 20% acrylamid:bisacrylamid
19:1 (Applichem), 1.times.TBE, 1% APS (AppliChem), 0.1% TEMED
(AppliChem); 180 V, 2 hours, Mini-PROTEAN.RTM. Tetra Cell
(BioRad)). The ribozyme band was identified by UV shadowing (E-BOX
VX2 gel documentation system with 312 nm-UV Transilluminator
(Peqlab)) over a TLC plate (Kieselgel 60 F254, Merck), excised and
eluted from the gel slice in 10 mM Tris/HCl, pH 7.5 (room
temperature, 16 hours). The supernatant was filtered through
Corning.RTM. Costar.RTM. Spin-X columns (Sigma) (1 minute, 16.000
g, room temperature), and RNAs were precipitated (300 mM NaOAc, pH
5, 75% ethanol, 16 hours, -20.degree. C.). Following centrifugation
(30 minutes, 16.000 g, 4.degree. C.), pellets were washed in 75%
ethanol (invert, centrifuge 5 minutes, 16.000 g, 4.degree. C.),
dried and re-dissolved in H.sub.2O.
Example 2: Preparation of the mRNA
[0326] 1. Preparation of DNA and mRNA Constructs
[0327] For the present example DNA sequences encoding HsNY-ESO-1
mRNA according to SEQ ID NO: 3 (FIG. 1), PpLuc mRNA according to
SEQ ID NO: 4 (FIG. 2) and HsPSCA RNA according to SEQ ID NO: 5
(FIG. 3) were prepared and used for subsequent in vitro
transcription reactions.
[0328] According to a first preparation, the DNA sequences coding
for the above mentioned mRNAs were prepared. The constructs were
prepared by modifying the wild type coding sequence by introducing
a GC-optimized sequence for stabilization, followed by a
stabilizing sequence derived from the alpha-globin-3'-UTR (muag
(mutated alpha-globin-3'-UTR)), a stretch of 64 adenosines
(poly-A-sequence), a stretch of 30 cytosines (poly-C-sequence), and
a histone stem loop. In FIGS. 1 to 3 the sequences of the
corresponding mRNAs are shown.
[0329] The 5' region of the target RNA sequence contains two
possible recognition sites, NUH1 (positions 10-12) and NUH2
(positions 11-13), of which NUH2 is the preferred target site.
Cleavage occurs 3' the H of the NUH recognition site.
[0330] 2. In Vitro Transcription
[0331] The respective DNA plasmids prepared according to paragraph
1 were transcribed in vitro using T7 RNA polymerase.
[0332] 3. In Vitro Transcription in the Presence of Cap Analog
[0333] For the production of 5'-capped RNAs using cap analog,
transcription was carried out in 5.8 mM m7G(5.sup.-)ppp(5.sup.-)G
Cap analog, 4 mM ATP, 4 mM CTP, 4 mM UTP, and 1.45 mM GTP (all
Thermo Fisher Scientific).
[0334] 4. In Vitro Transcription of Non-Capped RNAs
[0335] For the production of non-capped, 5' triphosphate RNAs,
transcription was carried out in the presence of 4 mM of each ATP,
GTP, CTP and UTP (all Thermo Fisher Scientific).
[0336] 5. Enzymatic Capping of mRNA
[0337] Enyzmatic capping was performed using the ScriptCap.TM.
m.sup.7G Capping System (Cell Script) according to the
manufacturer's instructions. In brief, per reaction, 60 .mu.g of
non-capped RNAs were heat-denatured (10 minutes, 65.degree. C.) in
a volume of 68.5 .mu.l and immediately cooled on ice (5 minutes).
Following addition of reaction components (1.times. ScriptCap
Capping buffer, 1 mM GTP, 0.1 mM SAM, 1000 U/ml ScripGuard RNase
Inhibitor, 400 U/ml ScriptCap Capping Enzyme) to a final volume of
100 .mu.l, reactions were incubated for 1 hour at 37.degree. C.
RNAs were precipitated in 2.86 M LiCl for 16 hours at -20.degree.
C., followed by centrifugation (30 minutes, 16.000 g, 4.degree.
C.). Pellets were washed in 0.5 reaction volumes 75% ethanol
(invert, centrifuge 5 minutes, 16.000 g, 4.degree. C.), dried and
re-dissolved in H.sub.2O.
[0338] Subsequently the mRNA was purified using PureMessenger.RTM.
(CureVac, Tubingen, Germany; WO2008/077592A1).
TABLE-US-00006 TABLE 2 Target RNAs Length of Description Sequence
SEQ (Name) (nucleotides) ID NO Experiment HsNY-ESO-1 mRNA 760 3
Capped mRNA (used for PAGE, FIG. 7) PpLuc mRNA 1870 4
co-transcriptionally capped mRNA (used for HPLC/PAGE) PpLuc mRNA
1870 4 enzymatically capped mRNA (used for HPLC/PAGE) PpLuc mRNA
1870 4 non-capped RNA (used for HPLC/PAGE as no cap control) HsPSCA
mRNA 589 5 non-capped RNA (used for PAGE, FIG. 7 as no cap
control)
Example 3: Cap Analysis Assay
[0339] 1. Principle of the Assay
[0340] The hammerhead ribozyme HHNUH2d of example 1 was incubated
with the in vitro transcribed RNAs of example 2 (Table 2) and the
cleavage products were separated by denaturing polyacrylamide gel
electrophoresis (PAGE) or high performance liquid chromatography
(HPLC).
[0341] 2. Ribozyme Cleavage Reaction
[0342] Reaction scales for gel analysis were usually 1.times. (10
pmol RNA). For HPLC analysis, 15.times. reaction (150 pmol RNA)
were set up, allowing a more sensitive detection and thus a more
precise determination of the respective mRNA populations. Per
reaction, 10 pmol of HHNUH2d and 10 pmol of the respective
substrate RNA were annealed in 0.625 mM EDTA in a total volume of 6
.mu.l (2 min at 95.degree. C., 0.1.degree. C./sec to 25.degree. C.,
10 min at 25.degree. C.). After addition of 4 .mu.l of 100 mM
MgCl.sub.2, 125 mM Tris/HCl, pH 7.5 (final concentration 40 mM
MgCl.sub.2, 50 mM Tris/HCl), the reaction was incubated at
25.degree. C. for 1 hour. For analysis via polyacrylamide gel
electrophoresis (PAGE), the 1.times. reaction was stopped with 30
.mu.l 95% formamide, 20 mM EDTA. For HPLC analysis, the 15.times.
reaction was stopped with 24 .mu.l 250 mM EDTA (final concentration
40 mM).
[0343] 3. Gel Separation, Quantification of Cleavage Products and
Calculation of Capping Degree
[0344] Stopped reactions were heat-denatured (heated to 80.degree.
C. for 2 minutes, immediately put on ice for 5 minutes) and
separated on a 10 cm.times.8 cm.times.1.5 mm 20% denaturing PAGE (8
M urea (AppliChem), 20% acrylamid:bisacrylamid 19:1 (AppliChem),
1.times.TBE, 1% APS (AppliChem), 0.1% TEMED (AppliChem); 180 V, 2
hours, Mini-PROTEAN.RTM. Tetra Cell (BioRad)). Gels were stained
for 10 minutes in 1:10,000 SYBR Gold (Invitrogen) in TBE and
documented on a E-BOX VX2 gel documentation system with 312 nm-UV
Transilluminator (Peqlab) (excitation maximum for SYBR Gold:
.about.300 nm, emission: .about.537 nm).
[0345] To determine the capped proportion in the mRNA preparations,
bands of the respective 13-mer (derived from the non-capped
fraction) or 14-mer (derived from the capped fraction) cleavage
products can be quantified using Quantity One 1-D Analysis Software
(BioRad).
[0346] The degrees of capped and non-capped RNA, respectively, can
be calculated according to:
capped RNA ( % ) = signal intensity 14 mer signal intensities ( 13
mer + 14 mer ) .times. 100 ##EQU00003## non - capped RNA ( % ) =
signal intensity 14 mer signal intensities ( 13 mer + 14 mer )
.times. 100 ##EQU00003.2##
[0347] As can be seen in FIG. 7, the capped and uncapped RNA
fragments produced by ribozyme cleavage of long mRNA molecules can
be resolved by denaturing PAGE.
[0348] 4. HPLC Separation, Quantification of Cleavage Products and
Calculation of Capping Degree
[0349] For the experiment shown in FIG. 8 an mRNA sample was
prepared by mixing 60% enzymatically capped mRNA coding for
Photinus pyralis Luciferase (PpLuc) and 40% non-capped mRNA coding
for Photinus pyralis Luciferase (PpLuc). Subsequently this sample
was incubated with the hammerhead (HH) ribozyme HHNU2d as described
above and analysed by HPLC.
[0350] Analysis was performed via ion-pair, reversed-phase
chromatography on a Dionex Parallel-HPLC U3000 CV-P-1247, equipped
with analytical pump (DPG-3600SD), column oven (TCC-3000SD) and
UV/Vis-4-channel-detectors (2.times.VWD-3400R5) with analytical SST
measuring cell (11 .mu.L, 10 mm, for VWD-3x00 detector). An AQUITY
UPLC OST C18 column (2.1.times.50 mm, 1.7 .mu.m particle size;
Waters Corporation, Milford, Mass., USA) was used. Column
temperature was set to 60.degree. C. Buffer A contained 0.1 M
triethylammonium acetate (TEAA), pH 6.8, buffer B 0.1 M TEAA, pH
7.3, 25% acetonitrile. The column was equilibrated with 14% buffer
B.
[0351] For sample preparation, HPLC equilibration buffer (86%
buffer A, 14% buffer B) was added to the stopped hammerhead
ribozyme reactions to obtain a final volume of 1700 .mu.l.
[0352] 1650 .mu.l of the RNA solution were loaded using a
SEMIPREP-Autosampler (WPS-3000SL, Dionex) and run with a stepped
gradient beginning with 14% buffer B for 3 minutes, increasing to
19% buffer B over 2 minutes, to 21% buffer B over 9 minutes. 21%
buffer B was held for 1 minute, then increased to 100% B over 5
minutes, held for 3.5 minutes, then decreased to 14% buffer B over
1.5 minutes.
[0353] Signal integration was done using Chromeleon software 6.80
SR11 Build 3161 (Dionex). The relative peak areas of capped 5' RNA
fragment (Peak 1, FIG. 8B) and non-capped 5' RNA fragments (Peak 2,
FIG. 8B) were determined. The degree of capped RNA was calculated
by dividing the relative peak area of Peak 1 by the sum of peak
areas 1 and 2. Deviation from the expected capping degree was
determined by dividing the calculated capping degree by the
expected capping degree.
TABLE-US-00007 TABLE 3 Determination of capping degree after HPLC
separation of cleavage products Relative Relative % peak area peak
area % % Deviation capped Peak 1 Peak 2 capped calculated/ expected
capped non-capped calculated expected 60 67.6 32.4 67.6 12.7
[0354] As can be seen from FIG. 8, the capped 5' mRNA fragment,
non-capped 5' mRNA fragment, 3' mRNA fragment and hammerhead
ribozyme can be separated by HPLC and the capping degree was
calculated as explained above.
Example 4: Determination of Cap Orientation
[0355] For the experiment shown in FIG. 9, non-capped,
enzymatically capped and co-transcriptionally capped RNA samples
encoding Photinus pyralis luciferase (PpLuc) were prepared as
described in Example 2. Subsequently, these samples were incubated
with the hammerhead (HH) ribozyme HHNU2d as described in Example 3
and analysed in parallel by denaturing polyacrylamide gel
electrophoresis (dPAGE) and HPLC. On the dPAGE gel, two bands are
detected for co-transcriptionally capped RNA (FIG. 9A), whereas
five peaks are detected in the HPLC chromatogram for the same
sample (FIG. 9D).
[0356] In order to characterize the peaks in the HPLC chromatogram
shown in FIG. 9, they were separated by HPLC according to the
protocol described above and collected (fraction collection by
time: 12-17 min, 20 sec/fraction) (FIG. 10). Double peaks at
13.30-13.80 min and 14.30-14.80 min could not be separated and were
thus pooled prior to Matrix-assisted laser desorption/ionization
(MALDI-TOF) analysis (sample S1=fractions 12-14, S2=21-24,
S3=28-30). MALDI-TOF mass spectrometry was performed by using an
AnchorChip target at the service provider PANAteqs (Heilbronn,
Germany).
[0357] Whereas two double peaks were detected by HPLC (FIG. 10),
mass spectrometry only revealed a single mass (in addition to minor
salt adducts) for each double peak (FIG. 11), corresponding to the
expected capped 5' fragment (FIG. 10 Peak S2, FIG. 11B) and a
further double peak (FIG. 10 Peak S1, FIG. 11A), respectively.
Double peak S1 indicated a capped RNA population lacking one
nucleotide in the 5' terminal RNA fragment. It was speculated that
in vitro transcription from the template used in Example 2 (SEQ ID
NO: 2, see FIG. 2) does not only yield the desired full-length
transcript, but also an aberrant transcript lacking the 5' terminal
guanine nucleotide of SEQ ID NO: 2. It was further speculated that
double peak S1 in the chromatogram of FIG. 9 was derived from said
aberrant transcripts.
[0358] To verify the mass spectrometry results indicating a capped
5' terminal RNA fragment derived from an RNA population lacking one
guanosine phosphate, enzymatically capped Photinus pyralis
luciferase (PpLuc) RNAs lacking the 5' terminal guanosine (Ecap-G1)
were synthesized as described above. The RNAs were incubated with
hammerhead (HH) ribozyme HHNU2d as described in Example 3 and
analysed by HPLC.
[0359] Overlay of the chromatograms of co-transcriptionally capped
RNA and the control construct Ecap(-G1) confirmed the MALDI results
(FIG. 12), identifying double peak S1 in the chromatogram of FIG. 9
as RNA shortened by one guanosine at the 5' end, i.e. lacking the
first G nucleotide in SEQ ID NO: 2. Therein, the first peak of the
first double peak (S1) co-eluted with the enzymatically capped
shortened control molecule (Ecap(-G1)). Likewise, the first peak of
the second double peak (S2) eluted simultaneously with the
enzymatically capped control (Ecap), again confirming the MALDI
results.
[0360] In enzymatically capped RNA, the cap is present in the
correct orientation ("correctly capped"). As confirmed by the
simultaneous elution of the first peak of the double peaks with the
capped controls (Ecap and Ecap(-G1), respectively), the respective
first peaks thus correspond to correctly capped RNA. The respective
second peaks correspond to those RNA molecules, which have, in
contrast, incorporated the cap in the reverse orientation
("reverse-capped"). The reverse-capped fractions displayed delayed
elution due to higher hydrophobicity at the 5' end (Dickman, 2011.
Ion Pair Reverse-Phase Chromatography: A Versatile Platform for the
Analysis of RNA. Chromatography Today, March 2011, p. 22-26). The
capping degree for the different populations was calculated as
explained above (FIG. 13).
TABLE-US-00008 TABLE 4 Determination of the relative amounts of
correctly capped, reverse- capped and non-capped RNA populations
after HPLC separation of cleavage products of co-transcriptionally
produced mRNA RNA population % of total RNA Correct cap minusG1
13.0 Reverse cap minusG1 14.2 Correct cap full-length 28.8 Reverse
cap full-length 38.0 No cap 6.1
Sequence CWU 1
1
6153RNAArtificial Sequencehammerhead ribozyme
RNAmisc_feature(1)..(7)n is a, c, g, or umisc_feature(15)..(38)n is
a, c, g, or umisc_feature(43)..(53)n is a, c, g, or u 1nnnnnnncug
augannnnnn nnnnnnnnnn nnnnnnnnga aannnnnnnn nnn 53252RNAArtificial
Sequencehammerhead ribozyme HHNUH2d 2gcauggcuga ugaggccucg
accgauaggu cgaggccgaa aagcuuucuc cc 523760RNAHomo sapiens
3gggagaaagc uuaccaugca ggccgagggc cgcggcaccg gcggcucgac cggcgacgcc
60gacgggcccg gcggcccggg caucccggac ggcccgggcg ggaacgcggg cggcccgggc
120gaggccggcg ccaccggcgg gcggggcccg cggggcgccg gcgccgcccg
ggcgagcggc 180cccggcgggg gcgccccgcg gggcccgcac ggcggcgccg
ccagcggccu gaacgggugc 240ugccggugcg gcgcccgcgg cccggagagc
cggcuccugg aguucuaccu ggccaugccg 300uucgcgaccc cgauggaggc
cgagcuggcc cggcggagcc uggcccagga cgccccgccg 360cugcccgugc
cgggcgugcu ccugaaggag uucacgguga gcggcaacau ccugaccauc
420cggcugaccg ccgcggacca ccggcagcug cagcugucga ucagcagcug
ccuccagcag 480cugagccugc ugauguggau cacccagugc uuccugccgg
uguuccuggc ccagccgccc 540agcggccagc gccggugacc acuaguuaua
agacugacua gcccgauggg ccucccaacg 600ggcccuccuc cccuccuugc
accgagauua auaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaugca uccccccccc cccccccccc
720cccccccccc ccaaaggcuc uuuucagagc caccagaauu
76041870RNAArtificial SequencePpLuc - Photinus pyralis Luciferase
4gggagaaagc uuaccaugga ggacgccaag aacaucaaga agggcccggc gcccuucuac
60ccgcuggagg acgggaccgc cggcgagcag cuccacaagg ccaugaagcg guacgcccug
120gugccgggca cgaucgccuu caccgacgcc cacaucgagg ucgacaucac
cuacgcggag 180uacuucgaga ugagcgugcg ccuggccgag gccaugaagc
gguacggccu gaacaccaac 240caccggaucg uggugugcuc ggagaacagc
cugcaguucu ucaugccggu gcugggcgcc 300cucuucaucg gcguggccgu
cgccccggcg aacgacaucu acaacgagcg ggagcugcug 360aacagcaugg
ggaucagcca gccgaccgug guguucguga gcaagaaggg ccugcagaag
420auccugaacg ugcagaagaa gcugcccauc auccagaaga ucaucaucau
ggacagcaag 480accgacuacc agggcuucca gucgauguac acguucguga
ccagccaccu cccgccgggc 540uucaacgagu acgacuucgu cccggagagc
uucgaccggg acaagaccau cgcccugauc 600augaacagca gcggcagcac
cggccugccg aagggggugg cccugccgca ccggaccgcc 660ugcgugcgcu
ucucgcacgc ccgggacccc aucuucggca accagaucau cccggacacc
720gccauccuga gcguggugcc guuccaccac ggcuucggca uguucacgac
ccugggcuac 780cucaucugcg gcuuccgggu gguccugaug uaccgguucg
aggaggagcu guuccugcgg 840agccugcagg acuacaagau ccagagcgcg
cugcucgugc cgacccuguu cagcuucuuc 900gccaagagca cccugaucga
caaguacgac cugucgaacc ugcacgagau cgccagcggg 960ggcgccccgc
ugagcaagga ggugggcgag gccguggcca agcgguucca ccucccgggc
1020auccgccagg gcuacggccu gaccgagacc acgagcgcga uccugaucac
ccccgagggg 1080gacgacaagc cgggcgccgu gggcaaggug gucccguucu
ucgaggccaa ggugguggac 1140cuggacaccg gcaagacccu gggcgugaac
cagcggggcg agcugugcgu gcgggggccg 1200augaucauga gcggcuacgu
gaacaacccg gaggccacca acgcccucau cgacaaggac 1260ggcuggcugc
acagcggcga caucgccuac ugggacgagg acgagcacuu cuucaucguc
1320gaccggcuga agucgcugau caaguacaag ggcuaccagg uggcgccggc
cgagcuggag 1380agcauccugc uccagcaccc caacaucuuc gacgccggcg
uggccgggcu gccggacgac 1440gacgccggcg agcugccggc cgcgguggug
gugcuggagc acggcaagac caugacggag 1500aaggagaucg ucgacuacgu
ggccagccag gugaccaccg ccaagaagcu gcggggcggc 1560gugguguucg
uggacgaggu cccgaagggc cugaccggga agcucgacgc ccggaagauc
1620cgcgagaucc ugaucaaggc caagaagggc ggcaagaucg ccgugugagg
acuaguuaua 1680agacugacua gcccgauggg ccucccaacg ggcccuccuc
cccuccuugc accgagauua 1740auaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800aaaaaaugca uccccccccc
cccccccccc cccccccccc ccaaaggcuc uuuucagagc 1860caccagaauu
18705589RNAHomo sapiens 5gggagaaagc uuaccaugaa ggccgugcug
cucgcgcugc ugauggccgg ccuggcccug 60cagccgggga ccgcccugcu gugcuacagc
ugcaaggccc aggucucgaa cgaggacugc 120cugcaggugg agaacugcac
gcagcugggc gagcagugcu ggaccgcccg gauccgcgcc 180gugggccugc
ucaccgugau cagcaagggc ugcagccuga acugcgugga cgacagccag
240gacuacuacg ugggcaagaa gaacaucacc ugcugcgaca ccgaccugug
caacgccagc 300ggcgcccacg cccugcagcc cgcggccgcc auccuggccc
ugcugcccgc ccugggccug 360cugcucuggg gccccggcca gcugugacca
cuaguuauaa gacugacuag cccgaugggc 420cucccaacgg gcccuccucc
ccuccuugca ccgagauuaa uaaaaaaaaa aaaaaaaaaa 480aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaugcau cccccccccc
540cccccccccc cccccccccc caaaggcucu uuucagagcc accagaauu
589610DNAArtificial Sequence5' terminal sequence 6gggagaaagc 10
* * * * *