U.S. patent application number 09/969423 was filed with the patent office on 2002-10-31 for compositions inducing cleavage of rna motifs.
This patent application is currently assigned to Ribozyme Pharmaceuticals, Inc.. Invention is credited to Ludwig, Janos, Sproat, Brian S..
Application Number | 20020161209 09/969423 |
Document ID | / |
Family ID | 25373400 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020161209 |
Kind Code |
A1 |
Ludwig, Janos ; et
al. |
October 31, 2002 |
Compositions inducing cleavage of RNA motifs
Abstract
Disclosed are compositions inducing cleavage of an RNA
substrate, as well as their use for inducing cleavage of RNA
substrates in vitro and in vivo. The compositions contain part of
an active center, with the other part of the active center provided
by the RNA substrate. The subunits of the active center region of
the compositions are nucleotides and/or nucleotide analogues. The
disclosed compositions also have flanking regions contributing to
the formation of a specific hybridization with an RNA substrate.
Preferred compositions form, in combination with an RNA substrate,
a structure resembling a hammerhead structure. The active center of
the disclosed compositions is characterized by the presence of
I.sup.15.1 which allows cleavage of RNA substrates having
C.sup.16.1.
Inventors: |
Ludwig, Janos; (Gottingen,
DE) ; Sproat, Brian S.; (Adelebsen, DE) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Ribozyme Pharmaceuticals,
Inc.
|
Family ID: |
25373400 |
Appl. No.: |
09/969423 |
Filed: |
October 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09969423 |
Oct 2, 2001 |
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08879078 |
Jun 19, 1997 |
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6300483 |
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Current U.S.
Class: |
536/23.1 ;
435/6.13; 544/245; 544/269; 544/276; 544/277; 544/280; 544/309 |
Current CPC
Class: |
C12N 2310/111 20130101;
C12N 15/1133 20130101; C12N 2310/322 20130101; C12N 15/113
20130101; C12N 2310/321 20130101; A61K 38/00 20130101; C12N 2310/33
20130101; C12N 2310/317 20130101; C12N 2310/321 20130101; C12N
15/1138 20130101; C12N 15/1131 20130101; C12N 15/1135 20130101;
C12N 2310/121 20130101; C12N 2310/3523 20130101 |
Class at
Publication: |
536/23.1 ;
544/245; 544/269; 544/276; 544/277; 544/280; 544/309; 435/6 |
International
Class: |
C12Q 001/68; C07H
021/04; C07D 473/18; C07D 473/16 |
Claims
We claim:
1. A composition that induces cleavage of an RNA substrate, the
composition comprising:5'-Z.sub.1-Z.sub.2-Z.sub.3-3'wherein Z.sub.1
and Z.sub.3 are oligomeric sequences which (1) are comprised of
nucleotides, nucleotide analogues, or both, or (2) are
oligonuleotide analogues, wherein the oligomeric sequences
specifically interact with the RNA substrate by hybridization.
wherein Z.sub.2 consists
of5'-X.sup.12X.sup.13X.sup.14X.sup.15.1-3',
or5'-X.sup.12X.sup.13X.sup.14- X.sup.15.1-3',wherein Z.sub.2 is
comprised of nucleotides, nucleotide analogues, or both, wherein
the nucleotides and nucleotide analogues each have the structure
3wherein each B is independently adenin-9-yl, cytosin-1-yl,
guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl,
thymin-1-yl, 5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl,
purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl,
5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl,
2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl,
2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl,
N.sup.2-dimethylguanin-9yl or a functional equivalent thereof,
wherein each V is independently an O, S, NH, or CH.sub.2 group,
wherein each W is independently selected from the group consisting
of --H, --OH, --COOH, --CONH.sub.2, --CONHR.sup.1,
--CONR.sup.1R.sup.2, --NH.sub.2, --NHR.sup.1, --NR.sup.1R.sup.2,
--NHCOR.sup.1, --SH, SR.sup.1, --F, --ONH.sub.2, --ONHR.sup.1,
--ONR.sup.1R.sup.2, --NHOH, --NHOR.sup.1, --NR.sup.2OH,
--NR.sup.2OR.sup.1, substituted or unsubstituted C.sub.1-C.sub.10
straight chain or branched alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 straight chain or branched alkenyl, substituted or
unsubstituted C.sub.2-C.sub.10 straight chain or branched alkynyl,
substituted or unsubstituted C.sub.1-C.sub.10 straight chain or
branched alkoxy, substituted or unsubstituted C.sub.2-C.sub.10
straight chain or branched alkenyloxy, and substituted or
unsubstituted C.sub.2-C.sub.10 straight chain or branched
alkynyloxy, wherein the substituents are independently halogen,
cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or
mercapto, wherein R.sup.1 and R.sup.2 are, independently,
substituted or unsubstituted alkyl, alkenyl, or alkynyl groups,
where the substituents are independently halogen, cyano, amino,
carboxy, ester, ether, carboxamide, hydroxy, or mercapto, wherein D
and E are residues which together form a phosphodiester or
phosphorothioate diester bond between adjacent nucleosides or
nucleoside analogues or together form an analogue of an
internucleosidic bond, wherein in X.sup.15.1, B is hypoxanthin-9-yl
or a functional equivalent thereof, wherein in X.sup.12, B is
independently guanin-9-yl, hypoxanthin-9-yl or 7-deazaguanin-9-yl;
wherein in X.sup.13 and X.sup.14, B is independently adenin-9-yl,
2,6-diaminopurin-9-yl, purin-9-yl or 7-deazaadenin-9-yl; wherein in
X .sup.12, B is independently adenin-9-yl, cytosin-1-yl,
guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl,
thymin-1-yl, 5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl,
purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl,
5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl,
2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl,
2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl,
N.sup.2-dimethylguanin-9-yl or a functional equivalent thereof.
2. The composition of claim 1 wherein the RNA substrate
comprises5'-Z.sub.3-C.sup.16.1-X.sup.17-S-Z.sub.4-Z.sub.1'-3',wherein
Z.sub.1' and Z.sub.3' interact with Z.sub.1 and Z.sub.3, wherein
C.sup.16.1 is cytidine, wherein X.sup.17 is adenosine, guanosine,
cytidine, or uridine, wherein S comprises a sequence capable of
forming a hairpin structure, wherein cleavage occurs 3' of
X.sup.17, wherein Z.sub.4 consists
of5'-X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9-3- ',
or5'-X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9 -3'wherein
X.sup.5 and X.sup.8 are guanosine, wherein X.sup.6 and X.sup.9 are
adenosine, wherein X.sup.4 is uridine, wherein X.sup.3 is cytidine,
and wherein X.sup.7 and X.sup.9 are independently adenosine,
guanosine, cytidine, or uridine.
3. The composition of claim 2 wherein X.sup.17 is adenosine,
cytidine, or uridine.
4. The composition of claim 1, wherein Z.sub.1 and Z.sub.3 do not
contain any pyrimidines that are ribonucleotides.
5. The composition of claim 1, wherein Z.sub.1 and Z.sub.3 do not
contain any ribonucleotides.
6. The composition of claim 1, wherein Z.sub.1 and Z3 are comprised
of nucleotides, nucleotide analogues, or both, wherein the
nucleotides and nucleotide analogues each have the structure
4wherein each B is independently adenin-9-yl, cytosin-1-yl,
guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl,
thymin-1-yl, 5-methylcytosin-1-yl; 2,6-diaminopurin-9-yl,
purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl,
5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl,
2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl,
2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl,
N.sup.2-dimethylguanin-9-yl or a functional equivalent thereof,
wherein each V is independently an 0, S, NH, or CH.sub.2 group,
wherein each W is independently selected from the group consisting
of substituted or unsubstituted C.sub.1-C.sub.10 straight chain or
branched alkyl, C.sub.2-C.sub.10 straight chain or branched
alkenyl, C.sub.2-C.sub.10 straight chain or branched alkynyl,
C.sub.1-C.sub.10 straight chain or branched alkoxy,
C.sub.2-C.sub.10 straight chain or branched alkenyloxy, and
C.sub.2-C.sub.10 straight chain or branched alkynyloxy, wherein D
and E are residues which together form a phosphodiester or
phosphorothioate diester bond between adjacent nucleosides or
nucleoside analogues or together form an analogue of an
internucleosidic bond.
7. The composition of claim 1, wherein Z.sub.1 and Z.sub.3 each
independently contain from 3 to 40 nucleotides, nucleotide
analogues, or a combination.
8. The composition of claim 1, wherein Z.sub.2 contains one or
several nucleotide analogues wherein each W is independently
selected from the group consisting of C.sub.1-C.sub.5 straight
chain or branched alkyl, C.sub.2-C.sub.5 straight chain or branched
alkenyl, C.sub.2-C.sub.5 straight chain or branched alkynyl,
C.sub.1-C.sub.5 straight chain or branched alkoxy, C.sub.2-C.sub.5
straight chain or branched alkenyloxy, and C.sub.2-C.sub.5 straight
chain or branched C.sub.2-C.sub.5 alkynyloxy.
9. The composition of claim 1, wherein the free 3' end is protected
against exonuclease degradation.
10. The composition of claim 1, wherein in X .sup.12 W is
independently NH.sub.2, OH-substituted C.sub.2-C.sub.4 alkyl,
OH-substituted C.sub.2-C.sub.4 alkenyl, OH-substituted
C.sub.1-C.sub.4 alkoxy or OH-substituted C.sub.2-C.sub.4
alkenyloxy.
11. The composition of claim 10, wherein in X .sup.12 W is
independently NH.sub.2, methoxy, 2-hydroxyethoxy, allyloxy or
allyl.
12. The composition of claim 1, wherein X.sup.12 is a
ribonucleotide.
13. The composition of claim 1, wherein X.sup.13 and X.sup.14, or a
combination is a nucleotide analogue in which each W is
independently C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl,
C.sub.1-C.sub.4 alkoxy, C.sub.2-C.sub.4 alkenyloxy, OH-substituted
C.sub.1-C.sub.4 alkyl, OH-substituted C.sub.2-C.sub.4 alkenyl,
OH-substituted C.sub.1-C.sub.4 alkoxy, or OH-substituted
C.sub.2-C.sub.4 alkenyloxy.
14. The composition of claim 13, wherein X.sup.13 and X.sup.14, or
a combination is a nucleotide analogue in which each W is
independently methoxy, 2-hydroxyethoxy or allyloxy.
15. The composition of claim 1, wherein the RNA substrate is
selected from the group consisting of human dopamine D2 receptor
mRNA, human brain cholecystokinin receptor mRNA, human serotonin
5-HT3 receptor mRNA, human alpha-2-macroglobulin receptor RNA,
human tyrosine kinase-type receptor (HER2) mRNA, human interleukin
2 receptor beta chain mRNA, human MAD-3 mRNA, human bcl-1 mRNA,
human bcl-2 mRNA, human cyclin F mRNA, human cyclin G1 mRNA, human
bleomycin hydrolase mRNA, human acute myeloid leukemia 1 oncogene
mRNA, human polycystic kidney disease 1 protein (PKD1) mRNA,
transcripts of the bovine viral diarrhea virus, transcripts of the
foot and mouth disease virus 3D gene and transcripts of the
Epstein-Barr virus.
16. The composition of claim 1, wherein X.sup.15.1 is a
ribonucleotide.
17. A method for the specific cleavage of an RNA substrate, the
method comprising bringing into contact the composition of claim 1
and the RNA substrate.
18. The method of claim 17, wherein the RNA substrate is selected
from the group consisting of human dopamine D2 receptor mRNA, human
brain cholecystokinin receptor mRNA, human serotonin 5-HT3 receptor
mRNA, human alpha-2-macroglobulin receptor RNA, human tyrosine
kinase-type receptor (HER2) mRNA, human interleukin 2 receptor beta
chain mRNA, -human MAD-3 mRNA, human bcl-1 mRNA, human bcl-2 mRNA,
human cyclin F mRNA, human cyclin G1 mRNA, human bleomycin
hydrolase mRNA, human acute myeloid leukemia 1 oncogene mRNA, human
polycystic kidney disease 1 protein (PKD1) mRNA, transcripts of the
bovine viral diarrhea virus, transcripts of the foot and mouth
disease virus 3D gene and transcripts of the Epstein-Barr
virus.
19. A method of identifying the function of a gene, the method
comprising bringing into contact the composition of claim 1 and a
cell containing the gene, wherein the composition reduces
expression of the gene, and observing any change in the cell.
20. A method of treating a disease that is associated with an RNA
molecule, the method comprising administering to a subject having
the disease the composition of claim 1, wherein the RNA substrate
is the RNA molecule associated with the disease.
21. The method of claim 20 wherein the RNA molecule is an RNA
molecule that is overexpressed.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is in the field of compositions having
RNA-cleavage activity.
[0002] Hammerhead ribozymes are an example of catalytic RNA
molecules which are able to recognize and cleave a given specific
RNA substrate (Hotchins et al., Nucleic Acids Res. 14:3627 (1986);
Keese and Symons, in Viroids and viroid--like pathogens (J. J.
Semanchik, publ., CRC-Press, Boca Raton, Fla., 1987), pages 147).
The catalytic center of hammerhead ribozymes is flanked by three
stems and can be formed by adjacent sequence regions of the RNA or
also by regions which are separated from one another by many
nucleotides. FIG. 1 shows a diagram of such a catalytically active
hammerhead structure. The stems have been denoted I, II and III.
The nucleotides are numbered according to the standard nomenclature
for hammerhead ribozymes (Hertel et al., Nucleic Acids Res. 20:3252
(1992)). In this nomenclature, bases are denoted by a number which
relates their position relative to the 5' side of the cleavage
site. Furthermore, each base that is involved in a stem or loop
region has an additional designation (which is denoted by a decimal
point and then another number) that defines the position of that
base within the stem or loop. A designation of N.sup.11.3 would
indicate that this base is involved in a paired region and that it
is the third base in that stem going away for the core region. This
accepted convention for describing hammerhead derived ribozymes
allows for the nucleotides involved in the core of the enzyme to
always have the same number relative to all of the other
nucleotides. The size of the stems involved in substrate binding or
core formation can be any size and of any sequence, and the
position of A.sup.9, for example, will remain the same relative to
all of the other core nucleotides. Nucleotides designated, for
example, N .sup.12 or N.sup.9 represent an inserted nucleotide
where the position of the carrot ( ) relative to the number denotes
whether the insertion is before or after the indicated nucleotide.
Thus, N .sup.12 represents a nucleotide inserted before nucleotide
position 12, and N .sup.9 represent a nucleotide inserted after
nucleotide position 9.
[0003] The consensus sequence of the catalytic core structure is
described by Ruffner and Uhlenbeck (Nucleic Acids Res. 18:6025-6029
(1990)). Perriman et al. (Gene 113:157-163 (1992)) have meanwhile
shown that this structure can also contain variations, for example,
naturally occurring nucleotide insertions such as N.sup.9 and N
.sup.12. Thus, the positive strand of the satellite RNA of the
tobacco ring-spot virus does not contain any of the two nucleotide
insertions while the +RNA strand of the virusoid of the lucerne
transient streak virus (vLTSV) contains a N.sup.9 =U insertion
which can be mutated to C or G without loss of activity (Sheldon
and Symons, Nucleic Acids Res. 17:5679-5685 (1989)). Furthermore,
in this special case, N.sup.7=A and R.sup.15.1=A. On the other
hand, the minus strand of the carnation stunt associated viroid
(-CarSV) is quite unusual since it contains both nucleotide
insertions, that is N .sup.12=A and N.sup.9 =C (Hernandez et al.,
Nucleic Acids Res. 20:6323-6329 (1992)). In this viroid N.sup.7 =A
and R.sup.15.1=A. In addition, this special hammerhead structure
exhibits a very effective self-catalytic cleavage despite the more
open central stem.
[0004] Possible uses of hammerhead ribozymes include, for example,
generation of RNA restriction enzymes and the specific inactivation
of the expression of genes in, for example, animal, human or plant
cells and prokaryotes, yeasts and plasmodia. A particular
biomedical interest is based on the fact that many diseases,
including many forms of tumors, are related to the overexpression
of specific genes. Inactivating such genes by cleaving the
associated mRNA represents a possible way to control and eventually
treat such diseases. Moreover there is a great need to develop
antiviral, antibacterial and antifungal pharmaceutical agents.
Ribozymes have potential as such anti-infective agents since viral
expression can be blocked selectively by cleaving viral or
microbial RNA molecules vital to the survival of the organism can
be selectively destroyed.
[0005] In addition to needing the correct hybridizing sequences for
substrate binding, substrates for hammerhead ribozymes have been
shown to strongly prefer the triplet N.sup.16.2U.sup.16.1H.sup.17
where N can be any nucleotide, U is uridine, and H is either
adenosine, cytidine, or uridine (Koizumi et al., FEBS Lett. 228,
228-230 (1988); Ruffner et al., Biochemistry 29, 10695-10702
(1990); Perriman et al., Gene 113, 157-163 (1992)). The fact that
changes to this general rule for substrate specificity result in
non-functional substrates implies that there are "non core
compatible" structures which are formed when substrates are
provided which deviate from the stated requirements. Evidence along
these lines was recently reported by Uhlenbeck and co-workers
(Biochemistry 36:1108-1114 (1997)) when they demonstrated that the
substitution of a G at position 17 caused a functionally
catastrophic base pair between G.sup.17 and C.sup.3 to form, both
preventing the correct orientation of the scissile bond for
cleavage and the needed tertiary interactions of C.sup.3 (Murray et
al., Biochem. J. 311:487494 (1995)). The strong preference for a U
at position 16.1 may exist for similar reasons. Many experiments
have been done in an attempt to isolate ribozymes which are able to
efficiently relieve the requirement of a U at position 16.1,
however, attempts to find hammerhead type ribozymes which can
cleave substrates having a base other than a U at position 16.1
have proven impossible (Perriman et al., Gene 113,
[0006] Efficient catalytic molecules with reduced or altered
requirements in the cleavage region are highly desirable because
their isolation would greatly increase the number of available
target sequences that molecules of this type could cleave. For
example, it would be desirable to have a ribozyme variant that
could efficiently cleave substrates containing triplets other than
N.sup.16.2U.sup.161H.sup.17 since this would increase the number of
potential target cleavage sites.
[0007] Chemically modified oligonucleotides which contain a block
of deoxyribonucleotides in the middle region of the molecule have
potential as pharmaceutical agents for the specific inactivation of
the expression of genes (Giles et al., Nucleic Acids Res.
20:763-770 (1992)). These oligonucleotides can form a hybrid
DNA-RNA duplex in which the DNA bound RNA strand is degraded by
RNase H. Such oligonucleotides are considered to promote cleavage
of the RNA and so cannot be characterized as having an RNA-cleaving
activity nor as cleaving an RNA molecule (the RNase H is cleaving).
A significant disadvantage of these oligonucleotides for in vivo
applications is their low specificity, since hybrid formation, and
thus cleavage, can also take place at undesired positions on the
RNA molecules.
[0008] Previous attempts to recombinantly express catalytically
active RNA molecules in the cell by transfecting the cell with an
appropriate gene have not proven to be very effective since a very
high expression was necessary to inactivate specific RNA
substrates. In addition the vector systems which are available now
cannot generally be applied. Furthermore, unmodified ribozymes
cannot be administered directly due to the sensitivity of RNA to
degradation by RNases and their interactions with proteins. Thus,
chemically modified active substances have to be used in order to
administer hammerhead ribozymes exogenously (discussed, for
example, by Heidenreich et al., J. Biol. Chem. 269:2131-2138
(1994); Kiehntopf et al., EMBO J. 13:4645-4652 (1994); Paolella et
al., EMBO J. 11: 1913-1919 (1992); and Usman et al., Nucleic Acids
Symp. Ser. 31:163-164 (1994)).
[0009] U.S. Pat. No. 5,334,711 describes such chemically modified
active substances based on synthetic catalytic oligonucleotide
structures with a length of 35 to 40 nucleotides which are suitable
for cleaving a nucleic acid target sequence and contain modified
nucleotides that contain an optionally substituted alkyl, alkenyl
or alkynyl group with 1-10 carbon atoms at the 2'-O atom of the
ribose. These oligonucleotides contain modified nucleotide building
blocks and form a structure resembling a hammerhead structure.
These oligonucleotides are able to cleave specific RNA substrates.
Examples of oligonucleotides are described having an active center
which has a length of 14 nucleotides and which contains several
ribonucleotides. These ribonucleotides increase the sensitivity of
the oligonucleotide to enzymes which cleave RNA. A further
disadvantage is the length of the active center which can often
lead to unspecific hybridization.
[0010] WO 95/11304 describes RNA-cleaving nucleic acids with an
active center that is free of ribonucleotide building blocks but
instead contains deoxyribonucleotides. However, the
deoxyribonucleotides used in the active center result in a very low
RNA cleavage activity. Thus, it was reported that a 13-mer
deoxyribozyme of the "GAAA" type based on LTSV was not able to
cleave a 41-mer oligoribonucleotide substrate while the
corresponding 13-mer ribozyme exhibited catalytic activity
(Jeffries and Symons, Nucleic Acids Res. 17:1371-1377 (1989)).
[0011] WO 97/18312 describes oligomers which contain only part of a
catalytic core resembling a hammerhead catalytic core. These
oligomers, when associated with an RNA substrate having a motif
resembling the complementary part of a catalytic core, induce
cleavage of the RNA substrate. The RNA substrates for use with
these oligomers all have a U at position 16.1.
[0012] The use of a large number of deoxyribonucleotides in the
hybridization arms or in the active center can lead to a loss of
specificity due to an activation of RNase H since sequences which
are related to the desired target sequence can also be cleaved.
Moreover, catalytic DNA oligomers are not particularly well suited
for in vivo applications due to interactions with proteins, and
lack of resistance to degradation by nucleases.
[0013] The shortest ribozymes that have been previously used have a
minimum length of 15+N+M nucleotides, the active center being 15
nucleotides long and N and M being the length of the recognition
sequences (Benseler et al., J. Am. Chem. Soc. 115:8483-8484
(1993)). Such ribozymes also contain ribonucleotides in at least
five positions of the catalytic center (Paolella et al., EMBO J.
11:1913-1919 (1992), and Yang et al., Bio-chemistry 31:5005-5009
(1992)).
[0014] It is therefore an object of the present invention to
provide compositions that induce cleavage of RNA, and in particular
to provide oligomers that induce cleavage of RNA and which at the
same time have a high stability, activity, and specificity.
[0015] It is another object of the present invention to provide
compositions that induce cleavage of RNA substrates having a
cleavage site triplet other than N.sup.16.2U.sup.16.1H.sup.17.
SUMMARY OF THE INVENTION
[0016] Disclosed are compositions inducing cleavage of an RNA
substrate, as well as their use for inducing cleavage of RNA
substrates in vitro and in vivo. The compositions contain part of
an active center, with the other part of the active center provided
by the RNA substrate. The subunits of the active center region of
the compositions are nucleotides and/or nucleotide analogues. The
disclosed compositions also have flanking regions contributing to
the formation of a specific hybridization with an RNA substrate.
Preferred compositions form, in combination with an RNA substrate,
a structure resembling a hammerhead structure. The active center of
the disclosed compositions is characterized by the presence of
I.sup.15.1 which allows cleavage of RNA substrates having
C.sup.16.1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a diagram of a hammerhead structure and the,
corresponding nomenclature (SEQ ID NO:1). Cleavage occurs between
H.sup.17 and N.sup.1.1 to generate the 2'-3'-cyclic phosphate at H
.sup.17.
[0018] FIG. 2 is a diagram of an RNA substrate (SEQ ID NO:3) in
association with an example of an oligomer (SEQ ID NO:2) that
induces cleavage of the RNA substrate. The structure formed by the
oligomer and the substrate resembles the structure of a hammerhead
ribozyme, with each providing a part of the elements corresponding
to the catalytic core. In this case, the substrate makes up half of
stems II and III and all of stem I, and loops II and III are not
present. Cleavage occurs 3' of HI.sup.17.
[0019] FIG. 3 is a diagram showing the interaction of the
A.sup.15.1-U.sup.16.1 base pair in hammerhead ribozymes (top), and
the predicted isostructural interaction of a I.sup.15.1-C.sup.16.1
base pair (bottom) that replaces the A.sup.15.1 -U.sup.16.1 base
pair.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Disclosed are compositions inducing cleavage of an RNA
substrate, as well as their use for inducing cleavage of RNA
substrates in vitro and in vivo. The compositions contain part of
an active center, with the other part of the active center provided
by the RNA substrate. The subunits of the active center region of
the compositions are nucleotides and/or nucleotide analogues. The
disclosed compositions also have flanking regions contributing to
the formation of a specific hybridization with an RNA substrate.
Preferred compositions form, in combination with an RNA substrate,
a structure resembling a hammerhead structure. The active center of
the disclosed compositions is characterized by the presence of
I15.1 which allows cleavage of RNA substrates having
C.sup.16.1.
[0021] All naturally occurring hammerhead ribozymes have an
A.sup.15.1-U.sup.16.1 base pair. In addition, it is known that
substrates for ribozymes based on the consensus hammerhead sequence
strongly prefer a substrate that contains an
N.sup.16.2U.sup.16.1H.sup.17 triplet in which H.sup.17 is not a
guanosine (Koizumi et al., FEBS Lett. 228, 228-230 (1988); Ruffner
et al., Biochemistry 29, 10695-10702 (1990); Perriman et al., Gene
113, 157-163 (1992)). Many experiments have been done in an attempt
to isolate ribozymes which are able to efficiently relieve the
requirement of a U at position 16.1, however, attempts to find
ribozymes which can cleave substrates having a base other than a U
at position 16.1 have proven impossible (Perriman et al., Gene 113,
157-163 1992, Singh et al., Antisense and Nucleic Acid Drug
Development 6:165-168 (1996)).
[0022] However, examination of the recently published X-ray crystal
structures (Pley et al., Nature 372:68-74 (1994), Scott et al.,
Cell 81:991-1002 (1995), and Scott et al., Science 274:2065-2069
(1996)) led to the realization that the A.sup.15.1-U.sup.16.1
interaction is a non-standard base pair with a single hydrogen bond
between the exocyclic amine (N6) of the adenosine and the 4-oxo
group of the uridine. Modeling studies (based on the crystal
structure) then led to the discovery that the interaction of the
wild-type A.sup.15.1-U.sup.16.1 base pair can be spatially mimicked
by replacement with an I.sup.15.1-C.sup.16.1 base pair that adopts
an isostructural orientation and which preserves the required
contact of the 2-keto group of C.sup.16.1 with A.sup.6 of the
uridine turn. In the model, the polarity of the stabilizing
hydrogen bond between positions 15.1 and 16.1 is reversed in the
I.sup.15.1-C.sup.16.1 interaction, but the correct orientation of
the bases around this bond is maintained.
[0023] It has been discovered that Gerlach type ribozyme analogues
containing an inosine at position 15.1 readily cleave RNA
substrates containing an N.sup.16.2C.sup.16.1H.sup.17 triplet.
Based on this, disclosed are compositions, preferably synthetic
oligomers, which induce cleavage of a nucleic acid target sequence
containing the structure
5'-Z.sub.3'-C.sup.16.1-X.sup.17-S-Z.sub.4-Z.sub.1'-3' where S is
capable of forming a stem and loop and Z.sub.4 corresponds to part
of an active center. It is preferred that X.sup.17 is not
guanosine. The ability to induce cleavage of substrates having
N.sup.16.1C.sup.16.1X.sup.17 triplets effectively doubles the
number of targets available for cleavage using compositions of the
type disclosed.
Compositions Inducing RNA Cleavage in a Substrate
[0024] Specifically disclosed is a composition that induces
cleavage of an RNA substrate, where the composition includes a
structure 5'-Z.sub.1-Z.sub.2-Z.sub.3-3'. Elements Z.sub.1 and
Z.sub.3 are each oligomeric sequences which are made up of
nucleotides, nucleotide analogues, or a combination of both, or are
oligonucleotide analogues. The oligomeric sequences of elements
Z.sub.1 and Z.sub.3 specifically interact with the RNA substrate,
preferably by hybridization.
[0025] In these preferred compositions, element Z.sub.2 has a
structure of
5'-X.sup.12X.sup.13X.sup.14X.sup.15.1-3', or
5-X .sup.12X.sup.12X.sup.13X.sup.14X.sup.15.1-3'.
[0026] Element Z.sub.2 in these preferred compositions is made up
of nucleotides, nucleotide analogues, or a combination of both. The
nucleotides and nucleotide analogues in element Z.sub.2 each have
the structure 1
[0027] In structure (I) each B can be adenin-9-yl, cytosin-1-yl,
guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl,
thymin-1-yl, 5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl,
purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl,
5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl,
2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl,
2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl,
N.sup.2-dimethylguanin-9-yl or a functional equivalent thereof;
[0028] Each V can be an O, S, NH, or CH.sub.2 group.
[0029] Each W can be --H, --OH, --COOH, --CONH.sub.2,
--CONHR.sup.1, --CONR.sup.1R.sup.2, --NH.sub.2, --NHR.sup.1,
--NR.sup.1R.sup.2, --NHCOR.sup.1, --SH, SR.sup.1, --F, --ONH.sub.2,
--NHR.sup.1, --ONR.sup.1R.sup.2, --NHOH, --NHOR.sup.1,
--NR.sup.2OH, --NR.sup.2OR.sup.1, substituted or unsubstituted
C.sub.1-C.sub.10 straight chain or branched alkyl, substituted or
unsubstituted C.sub.2-C.sub.10 straight chain or branched alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 straight chain or
branched alkynyl, substituted or unsubstituted C.sub.1-C.sub.10
straight chain or branched alkoxy, substituted or unsubstituted
C.sub.2-C.sub.10 straight chain or branched alkenyloxy, and
substituted or unsubstituted C.sub.2-C.sub.10 straight chain or
branched alkynyloxy. The substituents for W groups are
independently halogen, cyano, amino, carboxy, ester, ether,
carboxamide, hydroxy, or mercapto. R.sup.1 and R.sup.2 can be
substituted or unsubstituted alkyl, alkenyl, or alkynyl groups,
where the substituents are independently halogen, cyano, amino,
carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
[0030] D and E are residues which together form a phosphodiester or
phosphorothioate diester bond between adjacent nucleosides or
nucleoside analogues or together form an analogue of an
internucleosidic bond.
[0031] B is hypoxanthin-9-yl, or a functional equivalent thereof,
in X.sup.15.1; B can be guanin-9-yl, hypoxanthin-9-yl or
7-deazaguanin-9-yl in X.sup.12; B can be adenin-9-yl,
2,6-diaminopurin-9-yl, purin-9-yl or 7-deazaadenin-9-yl in X.sup.13
and X.sup.14; and B can be adenin-9-yl, cytosin-1-yl, guanin-9-yl,
uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl,
5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl, purin-9-yl,
7-deazaadenin-9-yl, 7-deazaguanin-9-yl, 5-propynylcytosin-1-yl,
5-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-yl,
6-methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-yl,
quinazoline-2,4-dione-1-yl, xanthin-9-yl,
N.sup.2-dimethylguanin-9-yl or a functional equivalent thereof in X
.sup.12. B of X.sup.15.1 is preferably an analog of
hypoxanthin-9-yl, preferably where no hydrogen bond can form
between any group at the 2 position of the base and the 2-oxo group
of C.sup.16.1. Preferably, B is not guanin-9-yl in X.sup.15.1.
[0032] B in X.sup.12, X.sup.13, and X.sup.14 can also be a
functionally equivalent nucleobase within the context of the
catalytic core of a hammerhead ribozyme.
[0033] The disclosed compositions have significant advantages. For
example, the disclosed compositions require only 4+N+M or 5+N+M
monomeric units (for example, nucleotides) in which N and M are
preferably numbers in the range of 5 to 10. Furthermore, the
disclosed compositions can contain a significantly smaller number
of natural ribonucleotides without loss of activity. Due to the
reduced length and reduced number of ribonucleotides, the disclosed
compositions are more conveniently and easily synthesized, and can
be more stable in vivo, than Gerlach type ribozymes. The in vivo
stability can be increased by a further reduction in the number of
ribonucleotides.
Definitions
[0034] As used herein, oligomer refers to oligomeric molecules
composed of subunits where the subunits can be of the same class
(such as nucleotides) or a mixture of classes. It is preferred that
the disclosed oligomers be oligomeric sequences. It is more
preferred that the disclosed oligomers be oligomeric sequences.
Oligomeric sequences are oligomeric molecules where each of the
subunits includes a nucleobase (that is, the base portion of a
nucleotide or nucleotide analogue) which can interact with other
oligomeric sequences in a base-specific manner. The hybridization
of nucleic acid strands is a preferred example of such
base-specific interactions. Oligomeric sequences preferably are
comprised of nucleotides, nucleotide analogues, or both, or are
oligonucleotide analogues.
[0035] As used herein, nucleoside refers to adenosine, guanosine,
cytidine, uridine, 2'-deoxyadenosine, 2'-deoxyguanosine,
2'-deoxycytidine, or thymidine. A nucleoside analogue is a
chemically modified form of nucleoside containing a chemical
modification at any position on the base or sugar portion of the
nucleoside. As used herein, the term nucleoside analogue
encompasses, for example, both nucleoside analogues based on
naturally occurring modified nucleosides, such as inosine and
pseudouridine, and nucleoside analogues having other modifications,
such as modifications at the 2' position of the sugar. As used
herein, nucleotide refers to a phosphate derivative of nucleosides
as described above, and a nucleotide analogue is a phosphate
derivative of nucleoside analogues as described above. The subunits
of oligonucleotide analogues, such as peptide nucleic acids, are
also considered to be nucleotide analogues.
[0036] As used herein, a ribonucleotide is a nucleotide having a 2'
hydroxyl function. Analogously, a 2'-deoxyribonucleotide is a
nucleotide having only 2' hydrogens. Thus, ribonucleotides and
deoxyribonucleotides as used herein refer to naturally occurring
nucleotides having nucleoside components adenosine, guanosine,
cytidine, and uridine, or 2'-deoxyadenosine, 2'-deoxyguanosine,
2'-deoxycytidine, and thymidine, respectively, without any chemical
modification. Ribonucleosides, deoxyribonucleosides, ribonucleoside
analogues and deoxyribonucleoside analogues are similarly defined
except that they lack the phosphate group, or an analogue of the
phosphate group, found in nucleotides and nucleotide analogues.
[0037] As used herein, oligonucleotide analogues are polymers of
nucleic acid-like material with nucleic acid-like properties, such
as sequence dependent hybridization, that contain at one or more
positions, a modification away from a standard RNA or DNA
nucleotide. A preferred example of an oligonucleotide analogue is
peptide nucleic acid.
[0038] As used herein, base pair refers to a pair of nucleotides or
nucleotide analogues which interact through one or more hydrogen
bonds. The term base pair is not limited to interactions generally
characterized as Watson-Crick base pairs, but includes
non-canonical or sheared base pair interactions (Topal and Fresco,
Nature 263:285 (1976); Lomant and Fresco, Prog. Nucl. Acid Res.
Mol. Biol. 15:185 (1975)). Thus, nucleotides A.sup.15.1 and
U.sup.16.1 form a base pair in hammerhead ribozymes (see FIG. 1)
but the base pair is non-canonical (see FIG. 3).
[0039] The internucleosidic linkage between two nucleosides can be
achieved by phosphodiester bonds or by modified phospho bonds such
as by phosphorothioate groups or other bonds such as, for example,
those described in U.S. Pat. No. 5,334,711.
Flanking Elements Z.sub.1 and Z.sub.3
[0040] The monomeric subunits of elements Z.sub.1 and Z.sub.3 which
flank the active center (formed by element Z.sub.2) are preferably
nucleotides and/or nucleotide analogues. Elements Z.sub.1 and
Z.sub.3 are designed so that they specifically interact, preferably
by hybridization, with a given RNA substrate and, together with the
element Z.sub.2, form a structure (preferably a structure
resembling part of a hammerhead ribozyme) which induces specific
cleavage of the RNA substrate.
[0041] The subunits of elements Z.sub.1 and Z.sub.3 can, on the one
hand, be ribonucleotides. However, it is preferred that the number
of ribonucleotides be as small as possible since the presence of
ribonucleotides reduces the in vivo stability of the oligomers.
Elements Z.sub.1 and Z.sub.3 (and also the active center Z.sub.2)
preferably do not contain any ribonucleotides at the positions
containing pyrimidine nucleobases. Such positions preferably
contain nucleotide analogues.
[0042] The use of a large number of deoxyribonucleotides in
elements Z.sub.1 and Z.sub.3 is also less preferred since undesired
interactions with proteins can occur or an unintended RNase
H-sensitive DNA-RNA hybrid could form. Thus, elements Z.sub.1 and
Z.sub.3 each preferably contain (1) no ribonucleotides, and (2) no
sequences of more than 3 consecutive deoxyribonucleotides.
[0043] The subunits of elements Z.sub.1 and Z.sub.3 are preferably
nucleotides, nucleotide analogues, or a combination. Preferably,
the nucleotides and nucleotide analogues in elements Z.sub.1 and
Z.sub.3 each have the structure 2
[0044] In structure (I) each B can be adenin-9-yl, cytosin-1-yl,
guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl,
thymin-1-yl, 5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl,
purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl,
5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl,
2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl,
2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl,
N2-dimethylguanin-9-yl or a functional equivalent thereof;
[0045] Each V can be an O, S, NH, or CH.sub.2 group.
[0046] Each W can be --H, --OH, --COOH, --CONH.sub.2,
--CONHR.sup.1, --CONR.sup.1R.sup.2, --NH.sub.2, --NHR.sup.1,
--NR.sup.1R.sup.2, --NHCOR.sup.1, --SH, SR.sup.1, --F, --ONH.sub.2,
--ONHR.sup.1, --ONR.sup.1R.sup.2, --NHOH, --NHOR.sup.1,
--NR.sup.2OH, --NR.sup.2OR.sup.1, substituted or unsubstituted
C.sub.1-C.sub.10 straight chain or branched alkyl, substituted or
unsubstituted C.sub.2-C.sub.10 straight chain or branched alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 straight chain or
branched alkynyl, substituted or unsubstituted C.sub.1-C.sub.10
straight chain or branched alkoxy, substituted or unsubstituted
C.sub.2-C.sub.10 straight chain or branched alkenyloxy, and
substituted or unsubstituted C.sub.2-C.sub.10 straight chain or
branched alkynyloxy. The substituents for W groups are
independently halogen, cyano, amino, carboxy, ester, ether,
carboxamide, hydroxy, or mercapto. R.sup.1 and R.sup.2 can be
substituted or unsubstituted alkyl, alkenyl, or alkynyl groups,
where the substituents are independently halogen, cyano, amino,
carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
[0047] D and E are residues which together form a phosphodiester or
phosphorothioate diester bond between adjacent nucleosides or
nucleotide analogues or together form an analogue of an
internucleosidic bond.
[0048] For elements Z.sub.1 and Z.sub.3 having nucleotide and/or
nucleotide analogues of structure (I), it is preferred that each W
is substituted or unsubstituted C.sub.1-C.sub.10 straight chain or
branched alkoxy, C.sub.2-C.sub.10 straight chain or branched
alkenyloxy, or C.sub.2-C.sub.10 straight chain or branched
alkynyloxy.
[0049] In addition, the flanking elements Z.sub.1 and Z3 can also
contain nucleotide analogues such as peptide nucleic acids (also
referred to as peptidic nucleic acids; see for example Nielsen et
al., Science 254:1497-1500 (1991), and Dueholm et al., J. Org.
Chem. 59:5767-5773 (1994)). In this case the coupling of individual
subunits can, for example, be achieved by acid amide bonds.
Elements Z.sub.1 and Z.sub.3, when based on peptide nucleic acids,
can be coupled to element Z.sub.2, based on nucleotides or
nucleotide analogues, using either suitable linkers (see, for
example, Petersen et al., BioMed. Chem. Lett. 5:1119-1121 (1995))
or direct coupling (Bergmann et al., Tetrahedron Lett. 36:6823-6826
(1995)). Where elements Z.sub.1 and Z3 contain a combination of
nucleotides (and/or nucleotide analogues) and peptide nucleic acid,
similar linkages can be used to couple the different parts.
[0050] The subunits of the flanking elements Z.sub.1 and Z3 contain
nucleobases or nucleobase analogues which can hybridize or interact
with nucleobases that occur naturally in RNA molecules. The
nucleobases are preferably selected from naturally occurring bases
(that is, adenine, guanine, cytosine, thymine and uracil) as well
as nucleobase analogues, such as 2,6-diaminopurine, hypoxanthine,
5-methylcytosine, pseudouracil, 5-propynyluracil, and
5-propynylcytosine, which enable a specific binding to the target
RNA.
[0051] A strong and sequence-specific interaction (that is, a more
stable hybrid between the RNA substrate and the oligomer) between
the RNA substrate and elements Z.sub.1 and Z.sub.3 is preferred.
For this purpose, it is preferred that the following nucleobase
analogues be used in oligomeric sequences of elements Z.sub.1 and
Z3 in place of the standard nucleobases: 2,6-diaminopurine instead
of adenine; thymine or 5-propynyluracil instead of uracil; and
5-methylcytosine or 5-propynylcytosine instead of cytosine.
2-Amino-2'-O-alkyladenosines are also preferred (Lamm et al.,
Nucleic Acids Res. 19:3193-3198 (1991)). Furthermore, aromatic
systems can be linked to positions 4 and 5 of uracil to produce
nucleobase analogues such as phenoxazine, which can improve the
stability of the double-strand (Lin et al., J. Am. Chem. Soc.
117:3873-3874 (1995)).
[0052] Preferred RNA substrates for cleavage using the disclosed
compositions have the structure
5'-Z.sub.3'-C.sup.16.1-X.sup.17-S-Z.sub.4-Z.sub.1'-3',
[0053] where Z.sub.1' and Z.sub.3' interact with Z.sub.1 and
Z.sub.3, respectively, where C.sup.16.1 is cytidine, and where
X.sup.17 is adenosine, guanosine, cytidine, or uridine. S is an RNA
sequence capable of forming a hairpin structure with a length of
preferably from 6 to 60 and more preferably of from 6 to 20 bases.
Cleavage occurs 3' of X.sup.17. Preferably, X.sup.17 is adenosine,
cytidine, or uridine, more preferably X.sup.17 is adenosine or
cytidine, and most preferably X.sup.17 is adenosine. Preferably,
X.sup.16.2 (that is, the 3' nucleoside in element Z.sub.3') is
adenosine or guanosine. The target sites in substrates which can be
cleaved using the disclosed compositions are distinct from target
sites for hammerhead ribozymes since hammerhead ribozymes require a
uridine in position 16.1 of the substrate.
[0054] Element Z.sub.4 of the substrate has the structure
5'-X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9-3', or
5'-X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.9 -3'
[0055] where X.sup.5 and X.sup.8 are both guanosine, X.sup.6 and
X.sup.9 are both adenosine, X.sup.4 is uridine, X.sup.3 is
cytidine, and X.sup.7 and X.sup.9 are adenosine, guanosine,
cytidine, or uridine. The disclosed composition, in combination
with an RNA substrate containing a structure of element Z.sub.4,
can form a structure resembling a hammerhead as shown in FIG.
2.
[0056] It is preferred that Z.sub.1 interact with Z.sub.1' in such
a way as to stabilize the interactions between Z.sub.2 and Z.sub.4.
Although preferred, it is not required that element Z.sub.1 be
present in the disclosed compositions. In this case, it is
preferred that element Z.sub.1' (in the substrate) include a G at
the 5' end (that is, at the junction of elements Z.sub.4 and
Z.sub.1'). Taira and co-workers (Amontov and Taira, J. Am. Chem.
Soc. 118:1624-1628 (1996)) have shown that the stacking energy
gained from a guanosine juxtaposed to R.sup.9 of a hammerhead-like
ribozyme stabilizes the formation of a catalytic structure. Thus,
it is preferred that the 5' nucleotide of Z.sub.1' is G.
[0057] The cleavage motif C.sup.16.1-X.sup.17-S-Z.sub.4 occurs only
rarely (approximately one motif for every 5000 to 10,000
nucleotides). This, taken together with the individually selected
recognition sequences, means that, statistically, a composition as
disclosed should induce cleavage of only the selected target RNA
within the entire human RNA pool. Only an unproductive binding but
no cleavage occurs at other potential binding sites, since
C.sup.16.1, X.sup.17, and elements S and Z.sub.4 are required for
cleavage. In addition, the disclosed compositions need not activate
RNase H since they can be made with a low content of
deoxyribonucleotides. This prevents induction of any unwanted
non-specific cleavage.
[0058] Computer algorithms can be used to identify RNA substrates
in sequence databases suitable for use with the disclosed
compositions. An example of such an algorithm is (using the
numbering according to FIG. 2):
[0059] i: find all C.sup.3 UGANGA(N)R sequences in a given
mRNA;
[0060] ii: identify N.sup.2.1 and find potential
N.sup.1.1-N.sup.2.1 base pairs (in which N.sup.1.1 must be part of
an N.sup.16.2-C.sup.16.1-N.sup.- 17-N.sup.1.1 sequence) in a region
positioned approximately 30 nucleotides from C.sup.3 in the 3'
direction;
[0061] iii: calculate stem stabilities for stems which terminate at
the above-mentioned N.sup.1.1-N.sup.2.1 base pairs;
[0062] iv: sort according to stem stability.
[0063] A program based on these algorithms enables a very efficient
search in databases or individual sequences. As a result, in
addition to a suitable RNA target sequence, one obtains the
sequence of the oligomer which is necessary to induce cleavage of
this target sequence. In this connection it is important to also
take into consideration potential target sites containing
incomplete base pairs in the region of the stem structure (that is,
element S) since several incomplete base pairs (mismatches) can be
tolerated in this section.
[0064] Preferred RNA substrates for cleavage using the disclosed
compositions are human cellular transcripts and transcripts of
human or animal viruses as well as transcripts of bacteria and
fungi that infect humans. Preferred RNA substrates are human
dopamine D2 receptor mRNA, human brain cholecystokinin receptor
mRNA, human serotonin 5-HT3 receptor mRNA, human
alpha-2-macroglobulin receptor RNA, human tyrosine kinase-type
receptor (HER2) mRNA, human interleukin 2 receptor beta chain mRNA,
human MAD-3 mRNA, human bcl-1 mRNA, human bcl-2 mRNA, human cyclin
F mRNA, human cyclin G1 mRNA, human bleomycin hydrolase mRNA, human
acute myeloid leukemia 1 oncogene mRNA, human polycystic kidney
disease 1 protein (PKD1) mRNA, transcripts of the bovine viral
diarrhea virus, transcripts of the foot and mouth disease virus 3D
gene and transcripts of the Epstein-Barr virus.
[0065] Particularly preferred cleavage motifs are located at the
following positions of the RNA substrates (the name of the
respective sequence in the EMBL Nucleotide Sequence Database 49th
or 50th Edition is given in parentheses):
[0066] human dopamine D2 receptor mRNA (HSDRD2A) with N.sup.16.2 at
position 2355 and a cleavage after the triplet UCU;
[0067] human brain cholecystokinin receptor mRNA (HSBRACHE) with
N.sup.16.2 at position 1519 and a cleavage after the triplet
ACA;
[0068] human serotonin 5-HT3 receptor mRNA (HSSSHT3RA) with
N.sup.16.2 at position 467 and a cleavage after the triplet
ACA;
[0069] human alpha-2-macroglobulin receptor RNA (HS2MRLR08) with
N.sup.16.2 at position 776 and a cleavage after the triplet
GCC;
[0070] human tyrosine kinase-type receptor (HER2) mRNA (HSHER2A)
with N.sup.16.2 at position 3330 and a cleavage after the triplet
ACU;
[0071] human interleukin 2 receptor beta chain mRNA (HSIL2RBC) with
N.sup.16.2 at position 937 and a cleavage after the triplet
ACA;
[0072] human MAD-3 mRNA (HSMAD3A) with N.sup.16.2 at position 138
and a cleavage after the triplet GCC;
[0073] human bcl-1 mRNA (HSBCL1G) with N.sup.16.2 at position 777
and a cleavage after the triplet GCA;
[0074] human bcl-2 mRNA (HSBCL2A) with N.sup.16.2 at position 4152
and a cleavage after the triplet ACC;
[0075] human cyclin F mRNA (HSCYCLF) with N.sup.16.1 at position
378 and a cleavage after the triplet ACA;
[0076] human cyclin G1 mRNA (HSCYCGIR) with N.sup.16.2 at position
166 and a cleavage after the triplet GCC;
[0077] human bleomycin hydrolase mRNA (HSBLEO) with N.sup.16.2 at
position 1352 and a cleavage after the triplet ACA;
[0078] human acute myeloid leukemia 1 oncogene mRNA (HSAML1) with
N.sup.16.2 at position 883 and a cleavage after the triplet
GCC;
[0079] human polycystic kidney disease 1 protein mRNA (HSPKD1A)
with N.sup.16.2 at position 11354 and a cleavage after the triplet
GCC;
[0080] transcripts of the bovine viral diarrhea virus (BV25053)
with N.sup.16.2 at position 616 and cleavage after the triplet
GCC;
[0081] transcripts of the foot and mouth disease virus 3D gene
(FMDV3D) with N.sup.16.2 at position 1291 and a cleavage after the
triplet GCA; and
[0082] transcripts of the Epstein-Barr virus (HEEBVT2R) with
N.sup.16.2 at position 1647 and a cleavage after the triplet
GCA.
[0083] Flanking elements Z.sub.1 and Z3 preferably contain,
independently of each other, from 3 to 40, and more preferably from
5 to 10, nucleotides or nucleotide analogues. It is preferred that
Z.sub.1 and Z.sub.1' interact to form a stem of at least three base
pairs, and that Z.sub.3' and Z.sub.3' interact to form a stem of at
least three base pairs. It is more preferred that these stems are
adjacent to Z.sub.2. It is most preferable that Z.sub.1 and
Z.sub.1' interact to form a stem of more than three base pairs, and
that Z.sub.3 and Z.sub.3'interact to form a stem of more than three
base pairs.
Catalytic Core
[0084] Elements Z.sub.2 and Z.sub.4 are considered to form the
catalytic core of the combination of a disclosed composition and an
RNA substrate (see FIG. 2). Z.sub.2 is preferably made up of
nucleotide analogues. In element Z.sub.2 it is preferred that each
W (in structure (I)) is C.sub.1-C.sub.5 straight chain or branched
alkyl, C.sub.2-C.sub.5 straight chain or branched alkenyl,
C.sub.2-C.sub.5 straight chain or branched alkynyl, C.sub.1-C.sub.5
straight chain or branched alkoxy, C.sub.2-C.sub.5 straight chain
or branched alkenyloxy, and C.sub.2-C.sub.5 straight chain or
branched C.sub.2-C.sub.5 alkynyloxy. It is also preferred that in X
.sup.12, W is NH.sub.2, OH-substituted C.sub.1-C.sub.4 alkyl,
OH-substituted C.sub.2-C.sub.4 alkenyl, OH-substituted
C.sub.1-C.sub.4 alkoxy or OH-substituted C2-C.sub.4 alkenyloxy. It
is more preferred that in X .sup.12, W is NH.sub.2, methoxy,
2-hydroxyethoxy, allyloxy or allyl. It is also preferred that in
X.sup.12, W is --H or --OH. It is also preferred that in each
X.sup.13 and X.sup.14, W is C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4
alkenyl, C.sub.1-C.sub.4 alkoxy, C.sub.2-C.sub.4 alkenyloxy,
OH-substituted C.sub.1-C.sub.4 alkyl, OH-substituted
C.sub.2-C.sub.4 alkenyl, OH-substituted C.sub.1-C.sub.4 alkoxy, or
OH-substituted C.sub.2-C.sub.4 alkenyloxy. It is more preferred
that in each X.sup.13 and X.sup.14, W is methoxy, 2-hydroxyethoxy
or allyloxy.
[0085] The subunits in element Z.sub.2 are preferably nucleotide
analogues which can only hybridize weakly with ribonucleotides.
Examples of such subunits are nucleotide analogues that contain a
substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy,
alkenyloxy or alkynyloxy group, with preferably 1 to 5 carbon
atoms, at the 2' position of ribose. Preferred nucleobases which
can be used in element Z.sub.2 for this purpose are adenin-9-yl,
purin-9-yl, guanin-9-yl and hypoxanthin-9-yl.
[0086] The following nucleotides and nucleotide analogues are
preferred for element Z.sub.2 (referring to components of structure
(I)):
[0087] Position X.sup.12: B=guanin-9-yl, V=O, W=H;
B=7deazaguanin-9-yl, V=O, W=OH; or B=guanin-9-yl, V=O, W=OH;
[0088] Position X.sup.13: B=adenin-9-yl, V=O, W=allyloxy; or
B=adenin-9-yl, V=O, W=2-hydroxyethoxy; B=purin-9-yl, V=O,
W=allyloxy;
[0089] Position X.sup.14: B=adenin-9-yl, V=O, W=allyloxy;
B=purin-9-yl, V=O, W=OH; or B=adenin-9-yl, V=O, W=2-hydroxyethoxy;
B=purin-9-yl, V=O, W=allyloxy;
[0090] Position X.sup.15.1: B=hypoxanthin-9-yl or a functional
equivalent thereof, V=O, W=OH.
[0091] Elements Z.sub.2 and Z.sub.4 interact in a way that allows
for the formation of a catalytic structure. In preferred
compositions Z.sub.2 and Z.sub.4 interact in a way that allows for
the formation of a catalytic structure resembling a hammerhead
catalytic structure. One way Z.sub.2 and Z.sub.4 can interact to
form a catalytic structure is through the interaction of the
nucleotides and/or nucleotide analogues making up Z.sub.2 and the
nucleotides making up Z.sub.4. The disclosed compositions are able
to induce cleavage of an RNA substrate independent of RNase H. That
is, the disclosed compositions are able to induce cleavage of an
RNA substrate without involving RNase H. Although the disclosed
compositions may also be capable of promoting cleavage of RNA by
RNase H, it is preferred that they do not.
[0092] The 3' end of the disclosed compositions can be protected
against degradation by exonucleases by, for example, using a
nucleotide analogue that is modified at the 3' position of the
ribose sugar (for example, by including a substituted or
unsubstituted alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl or
alkynyloxy group as defined above). The disclosed compositions can
also be stabilized against degradation at the 3' end by
exonucleases by including a 3'-3'-linked dinucleotide structure
(Ortigao et al., Antisense Research and Development 2:129-146
(1992)) and/or two modified phospho bonds, such as two
phosphorothioate bonds.
[0093] The disclosed compositions can also be linked to a
prosthetic group in order to improve their cellular uptake and/or
to enable a specific cellular localization. Examples of such
prosthetic groups are polyamino acids (for example, polylysine),
lipids, hormones or peptides. These prosthetic groups are usually
linked via the 3' or 5' end of the oligomer either directly or by
means of suitable linkers (for example, linkers based on
6-aminohexanol or 6-mercaptohexanol). These linkers are
commercially available and techniques suitable for linking
prosthetic groups to the oligomer are known to a person skilled in
the art.
[0094] Increasing the rate of hybridization can be important for
the biological activity of the disclosed compositions since in this
way it is possible to achieve a higher activity at low
concentrations of the composition. This is important for
short-lived RNA substrates or RNA substrates that occur less often.
A substantial acceleration of the hybridization can be achieved by,
for example, coupling positively charged peptides (containing, for
example, several lysine residues) to the end of an oligonucleotide
(Corey J. Am. Chem. Soc. 117:9373-9374 (1995)). The disclosed
compositions can be simply modified in this manner using the
linkers described above. Alternatively, the rate of hybridization
can also be increased by incorporation of subunits which contain
sperminyl residues (Schmid and Behr, Tetrahedron Lett. 36:1447-1450
(1995)). Such modifications of the disclosed compositions also
improve the ability to bind to RNA substrates having secondary
structures.
Synthesis of Oligomers
[0095] The disclosed compositions can be synthesized using any
suitable method. Many synthesis methods are known. The following
techniques are preferred for synthesis of the disclosed
compositions. 2'-O-Allyl modified oligomers that contain residual
purine ribonucleotides, and bearing a suitable 3'-terminus such as
an inverted thymidine residue (Ortigao et al., Antisense Research
and Development 2:129-146 (1992)) or two phosphorothioate linkages
at the 3'-terminus to prevent eventual degradation by
3'-exonucleases, can be synthesized by solid phase
.beta.-cyanoethyl phosphoramidite chemistry (Sinha et al., Nucleic
Acids Res. 12:45394557 (1984)) on any commercially available
DNA/RNA synthesizer. A preferred method is the
2'-O-tert-butyldimethylsilyl (TBDMS) protection strategy for the
ribonucleotides (Usman et al., J. Am. Chem. Soc. 109:7845-7854
(1987)), and all the required 3'-O-phosphoramidites are
commercially available. In addition, the use of
aminomethylpolystyrene is preferred as the support material due to
its advantageous properties (McCollum and Andrus Tetrahedron
Letters 32:40694072 (1991)). Fluorescein can be added to the 5'-end
of a substrate RNA during the synthesis by using commercially
available fluorescein phosphoramidites. In general, a desired
oligomer can be synthesized using a standard RNA cycle. Upon
completion of the assembly, all base labile protecting groups are
removed by an 8 hour treatment at 55.degree. C. with concentrated
aqueous ammonia/ethanol (3:1 v/v) in a sealed vial. The ethanol
suppresses premature removal of the 2'-O-TBDMS groups which would
otherwise lead to appreciable strand cleavage at the resulting
ribonucleotide positions under the basic conditions of the
deprotection (Usman et al., J. Am. Chem. Soc. 109:7845-7854
(1987)). After lyophilization the TBDMS protected oligomer is
treated with a mixture of triethylamine
trihydrofluoride/triethylamine/N-methylpyrrolidi- none for 2 hours
at 60.degree. C. to afford fast and efficient removal of the silyl
protecting groups under neutral conditions (Wincott et al., Nucleic
Acids Res. 23:2677-2684 (1995)). The fully deprotected oligomer can
then be precipitated with butanol according to the procedure of
Cathala and Brunel (Nucleic Acids Res. 18:201 (1990)). Purification
can be performed either by denaturing polyacrylamide gel
electrophoresis or by a combination of ion-exchange HPLC (Sproat et
al., Nucleosides and Nucleotides 14:255-273 (1995)) and reversed
phase HPLC. For use in cells, it is preferred that synthesized
oligomers be converted to their sodium salts by precipitation with
sodium perchlorate in acetone. Traces of residual salts are then
preferably removed using small disposable gel filtration columns
that are commercially available. As a final step it is preferred
that the authenticity of the isolated oligomers is checked by
matrix assisted laser desorption mass spectrometry (Pieles et al.,
Nucleic Acids Res. 21:3191-3196 (1993)) and by nucleoside base
composition analysis. In addition, a functional cleavage test with
the oligomer and the corresponding chemically synthesized short
oligoribonucleotide substrate is also preferred.
Cleavage of RNA Substrates
[0096] The disclosed compositions can have a very high in vivo
activity since the RNA cleavage will be promoted by protein factors
that are present in the nucleus or cytoplasm of the cell. Examples
of such protein factors (which can increase the activity of
hammerhead ribozymes) are, for example, the nucleocapsid protein
NCp7 of HIVI (Muller et al., J. Mol. Biol. 242:422-429 (1994)) and
the heterogeneous nuclear ribonucleoprotein Al (Heidenreich et al.,
Nucleic Acids Res. 23:2223-2228 (1995)). Thus, cleavage of long RNA
transcripts can be efficiently induced within the cell by the
disclosed compositions.
[0097] The disclosed compositions can be used in pharmaceutical
compositions that contain one or several oligomers as the active
substance, and, optionally, pharmaceutically acceptable auxiliary
substances, additives and carriers. Such pharmaceutical
compositions are suitable for the production of an agent to
specifically inactivate the expression of genes in eukaryotes,
prokaryotes and viruses, especially of human genes such as tumor
genes or viral genes or RNA molecules in a cell. Further areas of
application are the inactivation of the expression of plant genes
or insect genes. Thus, the disclosed compositions can be used as
drugs for humans and animals as well as a pesticide for plants.
[0098] A variety of methods are available for delivering the
disclosed compositions to cells. For example, in general, the
disclosed compositions can be incorporated within or on
microparticles. As used herein, microparticles include liposomes,
virosomes, microspheres and microcapsules formed of synthetic
and/or natural polymers. Methods for making microcapsules and
microspheres are known to those skilled in the art and include
solvent evaporation, solvent casting, spray drying and solvent
extension. Examples of useful polymers which can be incorporated
into various microparticles include polysaccharides,
polyanhydrides, polyorthoesters, polyhydroxides and proteins and
peptides.
[0099] Liposomes can be produced by standard methods such as those
reported by Kim et al., Biochim. Biophys. Acta, 728:339-348 (1983);
Liu et al., Biochim. Biophys. Acta, 1104:95-101 (1992); and Lee et
al., Biochim. Biophys. Acta., 1103:185-197 (1992); Wang et al.,
Biochem., 28:9508-9514 (1989)). Such methods have been used to
deliver nucleic acid molecules to the nucleus and cytoplasm of
cells of the MOLT-3 leukemia cell line (Thierry and Dritschilo,
Nucl. Acids Res., 20:5691-5698 (1992)). Alternatively, the
disclosed compositions can be incorporated within microparticles,
or bound to the outside of the microparticles, either ionically or
covalently.
[0100] Cationic liposomes or microcapsules are microparticles that
are particularly useful for delivering negatively charged compounds
such as the disclosed compounds, which can bind ionically to the
positively charged outer surface of these liposomes. Various
cationic liposomes have previously been shown to be very effective
at delivering nucleic acids or nucleic acid-protein complexes to
cells both in vitro and in vivo, as reported by Felgner et al.,
Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987); Felgner, Advanced
Drug Delivery Reviews, 5:163-187 (1990); Clarenc et al.,
Anti-Cancer Drug Design, 8:81-94 (1993). Cationic liposomes or
microcapsules can be prepared using mixtures including one or more
lipids containing a cationic side group in a sufficient quantity
such that the liposomes or microcapsules formed from the mixture
possess a net positive charge which will ionically bind negatively
charged compounds. Examples of positively charged lipids that may
be used to produce cationic liposomes include the aminolipid
dioleoyl phosphatidyl ethanolamine (PE), which possesses a
positively charged primary amino head group; phosphatidylcholine
(PC), which possess positively charged head groups that are not
primary amines; and N[1-(2,3-dioleyloxy)propyl]--
N,N,N-triethylammonium ("DOTMA," see Felgner et al., Proc. Natl.
Acad. Sci USA, 84:7413-7417 (1987); Felgner et al., Nature,
337:387-388 (1989); Felgner, Advanced Drug Delivery Reviews,
5:163-187 (1990)).
[0101] A preferred form of microparticle for delivery of the
disclosed compositions are heme-bearing microparticles. In these
microparticles, heme is intercalated into or covalently conjugated
to the outer surface of the microparticles. Heme-bearing
microparticles offer an advantage in that since they are
preferentially bound and taken up by cells that express the heme
receptor, such as hepatocytes, the amount of drug required for an
effective dose is significantly reduced. Such targeted delivery may
also reduce systemic side effects that can arise from using
relatively high drug concentrations in non-targeted delivery
methods. Preferred lipids for forming heme-bearing microparticles
are 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP) and
dioleoyl phosphatidyl ethanolamine (DOPE). The production and use
of heme-bearing microparticles are described in PCT application WO
95/27480 by Innovir.
[0102] The disclosed compositions can also be encapsulated by or
coated on cationic liposomes which can be injected intravenously
into a mammal. This system has been used to introduce DNA into the
cells of multiple tissues of adult mice, including endothelium and
bone marrow, where hematopoietic cells reside (see, for example,
Zhu et al., Science, 261:209-211 (1993)).
[0103] Liposomes containing the disclosed compositions can be
administered systemically, for example, by intravenous or
intraperitoneal administration, in an amount effective for delivery
of the disclosed compositions to targeted cells. Other possible
routes include trans-dermal or oral, when used in conjunction with
appropriate microparticles. Generally, the total amount of the
liposome-associated oligomer administered to an individual will be
less than the amount of the unassociated oligomer that must be
administered for the same desired or intended effect.
[0104] Compositions including various polymers such as the
polylactic acid and polyglycolic acid copolymers, polyethylene, and
polyorthoesters and the disclosed compositions can be delivered
locally to the appropriate cells by using a catheter or syringe.
Other means of delivering such compositions locally to cells
include using infusion pumps (for example, from Alza Corporation,
Palo Alto, Calif. or incorporating the compositions into polymeric
implants (see, for example, Johnson and Lloyd-Jones, eds., Drug
Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987),
which can effect a sustained release of the therapeutic
compositions to the immediate area of the implant.
[0105] For therapeutic applications the active substance is
preferably administered at a concentration of 0.01 to 10,000
.mu.g/kg body weight, more preferably of 0.1 to 1000 .mu.g/kg body
weight. The administration can, for example, be carried out by
injection, inhalation (for example as an aerosol), as a spray,
orally (for example as tablets, capsules, coated tablets etc.),
topically or rectally (for example as suppositories).
[0106] The disclosed compositions can be used in a method for the
specific inactivation of the expression of genes in which an active
concentration of the composition is taken up into a cell so that
the composition induces specific cleavage of a predetermined RNA
molecule which is present in the cell, the cleavage preferably
occurring catalytically. Similar compositions, which are described
in U.S. Pat. No. 5,334,711, have been used successfully in mice to
inactivate a gene (Lyngstadaas et al., EMBO J. 14:5224-5229
(1995)). This process can be carried out in vitro on cell cultures
as well as in vivo on living organisms (prokaryotes or eukaryotes
such as humans, animals or plants).
[0107] The disclosed compositions can also be used as RNA
restriction enzymes to induce cleavage of RNA molecules (in, for
example, cell free in vitro reactions). The disclosed compositions
can also be used in a reagent kit for the restriction cleavage of
RNA molecules which contains, for example, an oligomer and suitable
buffer substances. In this case the oligomer and the buffer
substances can be present in the form of solutions, suspensions or
solids such as powders or lyophilisates. The reagents can be
present together, separated from one another or optionally also on
a suitable carrier. The disclosed compositions can also be used as
a diagnostic agent or to identify the function of unknown
genes.
[0108] The present invention will be further understood by
reference to the following non-limiting examples.
Examples
[0109] The following examples demonstrate that compositions having
motifs forming structures resembling hammerheads but which include
I.sup.15.1 and C.sup.16.1 can result in specific cleavage of an RNA
molecule. Although the examples involve the use of oligomers having
RNA cleaving activity and including motifs corresponding to both
elements Z.sub.2 and Z.sub.4 (the oligomers in the examples are
thus analogous to a Gerlach type ribozyme), the activities are
indicative of the cleavage inducing activity of the disclosed
compositions.
Example 1
Cleavage Reactions which Indicate that an Inosine Substitution at
Position 15.1 can Effectively Cleave
N.sup.16.2C.sup.16.1H.sup.17
[0110] A set of 12 substrates was synthesized which covered each
permutation of the N.sup.16.2C.sup.16.1H.sup.17 motif where
H.sup.17 is not guanosine. The oligomers and the corresponding
substrates used in the cleavage assays are shown in Table 1. Each
of the substrates was labeled with fluorescein at the 5' end and an
inverted thymidine cap was used on the 3'-end. A set of four
catalytic oligomers was synthesized, providing an appropriately
matched catalytic oligomer for each of the substrates. Each of
these catalytic oligomers had an inosine at position 15.1. The
catalytic oligomers were similar to those described in U.S. Pat.
No. 5,334,711 except for the substitution of I for A at position
15.1. The catalytic oligomers includes, in a single molecule, the
equivalent of elements Z.sub.4, Z.sub.1', Z.sub.1, Z.sub.2 and
Z.sub.3 in the compositions and RNA substrates as described above.
A control substrate and catalytic oligomer were also synthesized in
which there was a U at position 16.1 of the substrate and an A at
position 15.1 of the catalytic oligomer.
1TABLE 1 N.sup.16.2N.sup.16.1H.sub.17 Triplet Substrate sequence
ACC Fl-GAAUACCGGUCGC*T (SEQ ID NO: 4) ACA Fl-GAAUACAGGUCGC*T (SEQ
ID NO: 5) ACU Fl-GAAUACUGGUCGC*T (SEQ ID NO: 6) GCC
Fl-GAAUACAGGUCGC*T (SEQ ID NO: 7) GCA Fl-GAAUGCAGGUCGC*T (SEQ ID
NO: 8) GCU Fl-GAAUGCUGGUCGC*T (SEQ ID NO: 9) CCC Fl-GAAUCCCGGUCGC*T
(SEQ ID NO: 1O) CCA Fl-GAAUCCAGGUCGC*T (SEQ ID NO: 11) CCU
Fl-GAAUCCUGGUCGC*T (SEQ ID NO: 12) UCC Fl-GAAUUCCGGUCGC*T (SEQ ID
NO: 13) UCA Fl-GAAUUCAGGUCGC*T (SEQ ID NO: 14) UCU
Fl-GAAUUCUGGUCGC*T (SEQ ID NO: 15) GUC Fl-GAAUGUCGGUCGC*T (SEQ ID
NO: 16) Targeted Catalytic triplet oligomer sequence ACH
gcgacccuGAuGaggccgug (SEQ ID NO: 17) aggccGaaIuauuc*T GCH
gcgacccuGAuGaggccgug (SEQ ID NO: 18) aggccGaaIcauuc*T CCH
gcgacccuGAuGaggccgug (SEQ ID NO: 19) aggccGaaIgauuc*T UCH
gcgacccuGAuGaggccgug (SEQ ID NO: 20) aggccGaaIaauuc*T GUC
gcgacccuGAuGaggccgug (SEQ ID NO: 21) aggccGaaAcauuc*T Fl =
Fluorescein label *T = 3'--3' inverted thymidine A, C, G, I, U =
ribonucleotides (I is inosine) a, c, g, u =
2'-O-allyl-ribonucleotides
[0111] The above substrates and catalytic oligomers were used in
cleavage reactions to determine the ability of an inosine at
position 15.1 to overcome the requirement of a U at position 16.1
for cleavage. All of the reactions were performed using the
following protocol. The reactions were typically done in 100 .mu.l
and they contained distilled, autoclaved H.sub.2O, 10 mM
MgCl.sub.2, 10 mM Tris-HCl pH 7.4, 5 .mu.M ribozyme, and 0.25 .mu.M
substrate. The catalytic oligomer, substrate, and buffer were added
together and heated to 95.degree. C. for 5 minutes. After cooling
to room temperature over 5 minutes the reactions were brought to 10
mM MgCl.sub.2, mixed, and placed at 37.degree. C. 10 .mu.L aliquots
were removed at specific time intervals (10, 30, 60, and 120
minutes) and added to 3 .mu.l of loading buffer (95% formamide, 100
mM EDTA pH 8.0, 0.05% bromophenol blue) to quench the reaction.
Samples were analyzed by 20% polyacrylamide gel electrophoresis.
Gels were analyzed on a Molecular Dynamics Fluorescence Imager. The
results of cleavage reactions of this type, using the substrates
and catalytic oligomers shown in Table 1, are shown in Table 2.
2 TABLE 2 N.sup.16.2 N.sup.16.1 H.sup.17 After Triplet mixing 10 30
60 120 I.sup.15.1 U.sup.15.2 Catalytic oligomer ACC 4.4 28.2 58.1
91.5 91.5 ACA 7.7 71.8 84.7 93.1 94.8 ACU 1.8 58.7 70.5 I.sup.15.1
C.sup.15.2 Catalytic oligomer GCC 1.62 39.6 59.9 82.0 87.0 GCA 13.7
65.3 78.7 89.7 93.1 GCU -- 64.3 74.8 I.sup.15.1 G.sup.15.2
Catalytic oligomer CCC -- 34.33 45.38 CCA 1.1 18.8 45.5 70.8 80.63
CCU 2.0 28.4 36.7 I.sup.15.1 A.sup.15.2 Catalytic oligomer UCC 6.8
57.0 64.7 UCA 1.6 39.6 60.8 UCU 3.3 41.1 53.1 A.sup.15.1 C.sup.15.2
Catalytic oligomer GUC 1.6 38.5 66.5 93.5
[0112] The numbers represent the percentage of substrate cleaved at
the indicated time point (which were at 0, 10, 30, 60, and 120
minutes after starting the reaction). The results indicate that
substrates with a C at position 16.1 are able to be cleaved by
catalytic oligomers containing an I at position 15.1. While there
are differences between the various substrates at the 120 minute
time point, the data show that a substrate with a C at position
16.1 in conjunction with a catalytic oligomer with an I at position
15.1 is able to effectively cleave in all backgrounds, indicating
that the substitution of an I at position 15.1 does in fact allow
for the cleavage of any appropriate substrate containing a
N.sup.16.2C.sup.16.1H.sup.17 site.
[0113] Initial rates of cleavage of the twelve substrates having
C.sup.16.1, and the control substrate having U.sup.16.1, by the
corresponding catalytic oligomers (all shown in Table 1) were
determined using single turnover kinetics. Single turnover kinetics
were assessed by mixing 2.5 .mu.l of a 100 .mu.M ribozyme solution,
2.5 .mu.l of a 10 .mu.M solution of 5' fluorescein labeled
substrate, and 10 .mu.l of a 100 mM Tris-HCl pH 7.4 solution. The
mixture was diluted to a final volume of 90 .mu.l, heated to
95.degree. C. for 5 minutes, and cooled to 37.degree. C. The
reaction was started by adding 10 .mu.l of a 100 mM MgCl.sub.2
solution. The final concentrations of the reaction components were
250 nM substrate, 2.5 .mu.mol ribozyme, and 10 mM MgCl.sub.2. Ten
microliter samples were removed at various times and mixed with 10
.mu.l of a 100 mM EDTA, bromphenol blue solution to stop the
reaction. Cleavage products were separated from unreacted substrate
by PAGE and were quantitated on a Molecular-Dynamics Fluorescence
Imager.
[0114] The data, measured in fraction of substrate cleaved versus
time, were fitted to the equation:
frac[P]=H.sub.0(1-e.sup.-k.sup..sub.2.sup.t)/S.sub.0
[0115] as described by Jankowsky and Schwenzer, Nucl. Acids Res.
24:433 (1996). The calculated values of k.sub.2 for the various
ribozymes are shown in Table 3.
3TABLE 3 N.sup.16.2N.sup.16.1H.sup.17 Triplet k.sub.2 (min.sup.-1)
Substrate sequence gcgacccuGAuGaggccgugaggccGaaIuauuc*T (SEQ ID NO:
17) ACC 0.07 Fl-GAAUACCGGUCGC*T (SEQ ID NO: 4) ACA 0.36
Fl-GAAUACAGGUCGC*T (SEQ ID NO: 5) ACU 0.026 Fl-GAAUACUGGUCGC*T (SEQ
ID NO: 6) gcgacccuGAuGaggccgugaggccGaaIcauuc*T (SEQ ID NO: 18) GCC
0.12 Fl-GAAUGCCGGUCGC*T (SEQ ID NO: 7) GCA 0.48 Fl-GAAUGCAGGUCGC*T
(SEQ ID NO: 8) GCU 0.05 Fl-GAAUGCUGGUCGC*T (SEQ ID NO: 9)
gcgacccuGAuGaggccgugaggccGaaIgauuc*T (SEQ ID NO: 19) CCC <0.01
Fl-GAAUCCCGGUCGC*T (SEQ ID NO: 10) CCA 0.04 Fl-GAAUCCAGGUCGC*T (SEQ
ID NO: 11) CCU <0.01 Fl-GAAUCCUGGUCGC*T (SEQ ID NO: 12)
gcgacccuGAuGaggccgugaggccGaaIaauuc*T (SEQ ID NO: 20) UCC <0.01
Fl-GAAUUCCGGUCGC*T (SEQ ID NO: 13) UCA <0.01 Fl-GAAUUCAGGUCGC*T
(SEQ ID NO: 14) UCU <0.01 Fl-GAAUUCUGGUCGC*T (SEQ ID NO: 15)
gcgacccuGAuGaggccgugaggccGaaAcauuc*T (SEQ ID NO: 21) GUC 0.13
Fl-GAAUGUCGGUCGC*T (SEQ ID NO: 16) Fl = Fluorescein label *T =
3'--3' inverted thymidine A, C, G, I, U = ribonucleotides (I is
inosine) a, c, g, u = 2'-O-allyl-ribonucleotides
[0116] The results show that substrates with
A.sup.16.2C.sup.16.1H.sup.17 and G.sup.16.2C.sup.16.1H.sup.17
triplets are cleaved at a high rate. Comparison to the control
catalytic oligomer having an A at position 15.1 (to cleave a
substrate with a G.sup.16.2U.sup.16.1C.sup.17 triplet) shows that
substrates with A.sup.16.2C.sup.16.1A.sup.17 and
G.sup.16.2C.sup.16.1A.sup.17 triplets (to be cleaved by a catalytic
oligomer with an I at position 15.1) have an initial rate of
cleavage that is higher than the corresponding control reactions
involving reactants with a standard A.sup.15.1-U.sup.16.1 base
pair.
[0117] Publications cited herein and the material for which they
are cited are specifically incorporated by reference.
[0118] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
21 1 57 RNA Artificial Sequence Description of Artificial Sequence
Hammerhead ribozyme 1 nnnnnnnnnn nnnnnncuga nganrnnnnn nnnnnnnyng
aarnnnnnnn nnnnnuh 57 2 14 RNA Artificial Sequence Description of
Artificial Sequence oligomer that induces cleavage of the RNA
substrate 2 nnnnyngaan nnnn 14 3 35 RNA Artificial Sequence
Description of Artificial Sequence RNA substrate 3 nnnnchnnnn
nnnnnnnnnn nncugangan rnnnn 35 4 14 RNA Artificial Sequence
Description of Artificial Sequence oligonucleotide substrate 4
gaauaccggu cgcn 14 5 14 RNA Artificial Sequence Description of
Artificial Sequence oligonucleotide substrate 5 gaauacaggu cgcn 14
6 14 RNA Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 6 gaauacuggu cgcn 14 7 14 RNA Artificial
Sequence Description of Artificial Sequence oligonucleotide
substrate 7 gaaugccggu cgcn 14 8 14 RNA Artificial Sequence
Description of Artificial Sequence oligonucleotide substrate 8
gaaugcaggu cgcn 14 9 14 RNA Artificial Sequence Description of
Artificial Sequence oligonucleotide substrate 9 gaaugcuggu cgcn 14
10 14 RNA Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 10 gaaucccggu cgcn 14 11 14 RNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 11 gaauccaggu cgcn 14 12 14 RNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 12 gaauccuggu cgcn 14 13 14 RNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 13 gaauuccggu cgcn 14 14 14 RNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 14 gaauucaggu cgcn 14 15 14 RNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 15 gaauucuggu cgcn 14 16 14 RNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide substrate 16 gaaugucggu cgcn 14 17 35 RNA
Artificial Sequence Description of Artificial Sequence Catalytic
Oligomer 17 gcgacccuga ugaggccgug aggccgaanu auucn 35 18 35 RNA
Artificial Sequence Description of Artificial Sequence Catalytic
Oligomer 18 gcgacccuga ugaggccgug aggccgaanc auucn 35 19 35 RNA
Artificial Sequence Description of Artificial Sequence Catalytic
Oligomer 19 gcgacccuga ugaggccgug aggccgaang auucn 35 20 35 RNA
Artificial Sequence Description of Artificial Sequence Catalytic
Oligomer 20 gcgacccuga ugaggccgug aggccgaana auucn 35 21 35 RNA
Artificial Sequence Description of Artificial Sequence Catalytic
Oligomer 21 gcgacccuga ugaggccgug aggccgaaac auucn 35
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