U.S. patent application number 10/664835 was filed with the patent office on 2005-02-24 for rna catalyst for cleaving specific rna sequences.
This patent application is currently assigned to The Board of Regents for Northern Illinois University of DeKalb. Invention is credited to Hampel, Arnold E., Hicks, Margaret F., Tritz, Richard H..
Application Number | 20050042620 10/664835 |
Document ID | / |
Family ID | 27500247 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050042620 |
Kind Code |
A1 |
Hampel, Arnold E. ; et
al. |
February 24, 2005 |
RNA catalyst for cleaving specific RNA sequences
Abstract
A synthetic RNA catalyst capable of cleaving an RNA substrate,
the catalyst comprising a substrate binding portion and a "hairpin"
portion. The invention also provides an engineered DNA molecule and
a vector, each comprising a DNA sequence coding for an RNA catalyst
according to the invention. The invention further comprises host
cells transformed with the vectors of the invention which are
capable of expressing the RNA catalyst. Finally, the invention
provides a method of cleaving an RNA substrate which comprises
contacting the substrate with a synthetic RNA catalyst according to
the invention.
Inventors: |
Hampel, Arnold E.; (DeKalb,
IL) ; Tritz, Richard H.; (DeKalb, IL) ; Hicks,
Margaret F.; (Antioch, TN) |
Correspondence
Address: |
John P. White
Coopers & Dunham
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
The Board of Regents for Northern
Illinois University of DeKalb
Biotechnology Research and Develompent Corporation
|
Family ID: |
27500247 |
Appl. No.: |
10/664835 |
Filed: |
September 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10664835 |
Sep 15, 2003 |
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08475466 |
Jun 7, 1995 |
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08475466 |
Jun 7, 1995 |
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08078774 |
Jun 17, 1993 |
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5866701 |
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08078774 |
Jun 17, 1993 |
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07703427 |
May 14, 1991 |
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07703427 |
May 14, 1991 |
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07577658 |
Sep 4, 1990 |
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07577658 |
Sep 4, 1990 |
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07409666 |
Sep 20, 1989 |
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07409666 |
Sep 20, 1989 |
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07247100 |
Sep 20, 1988 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2; 536/23.1 |
Current CPC
Class: |
C12N 15/1132 20130101;
C12N 15/8216 20130101; C12N 15/8218 20130101; C12N 2310/111
20130101; C12N 2310/122 20130101; C12N 15/113 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 536/023.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C12P 019/34 |
Goverment Interests
[0002] This invention was made in part with Government support
under Grant No. DMB 8817576 awarded by the National Science
Foundation and Grant No. RO1 AI 29870 awarded by the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
We claim:
1. A synthetic RNA catalyst capable of cleaving an RNA substrate
which contains the sequence: 5'-F.sub.1-CS-F.sub.2-3', wherein, CS
is a cleavage sequence; and F.sub.1 and F.sub.2 each is a sequence
of bases flanking the cleavage sequence; the catalyst comprising a
substrate binding portion and a "hairpin" portion, the substrate
binding portion of the catalyst having the sequence:
3'-F.sub.4-L.sub.1-F.sub.3-5'wherein, F.sub.3 is a sequence of
bases selected so that F.sub.3 is substantially base paired with
F.sub.2 when the catalyst is bound to the substrate; F.sub.4 is a
sequence of bases selected so that F.sub.4 is substantially base
paired with F.sub.1 when the catalyst is bound to the substrate;
the sequences of F.sub.3 and F.sub.4 being selected so that each
contains an adequate number of bases to achieve sufficient binding
of the RNA substrate to the RNA catalyst so that cleavage of the
substrate can take place; and L.sub.1 is a sequence of bases
selected so that L.sub.1 does not base pair with CS when the
catalyst is bound to the substrate.
2. An RNA catalyst according to claim 1, the "hair-pin" portion of
the catalyst having the sequence: 6wherein, P.sub.1 and P.sub.4
each is a sequence of bases, the sequences of P.sub.1 and P.sub.4
being selected so that P.sub.1 and P.sub.4 are substantially base
paired; P.sub.1 is covalently linked to F.sub.4; S.sub.1 and
S.sub.2 each is a sequence of bases, the sequences of S.sub.1 and
S.sub.2 being selected so that S.sub.1 and S.sub.2 are
substantially unpaired; P.sub.2 and P.sub.3 each is a sequence of
bases, the sequences of P.sub.2 and P.sub.3 being selected so that
P.sub.2 and P.sub.3 are substantially base paired; and L.sub.2 is a
sequence of unpaired bases.
3. An RNA catalyst according to claim 1 or 2 which is capable of
cleaving an RNA substrate in which CS has the sequence 5'-NGUC-3',
wherein N is any base and the substrate is cleaved by the catalyst
between N and G.
4. An RNA catalyst according to claim 3 wherein L.sub.1 has the
sequence 3'-AAGA-5'.
5. An RNA catalyst according to claim 1 or 2 wherein F.sub.3 is at
least 3 bases in length and F.sub.4 is from 3 to 5 bases in length,
and the catalyst cleaves a substrate wherein F.sub.1 and F.sub.2
each is at least 3 bases in length.
6. An RNA catalyst according to claim 5 wherein F.sub.3 is from 6
to 12 bases in length and F.sub.4 is 4 bases in length, and the
catalyst cleaves a substrate wherein F.sub.1 is 4 bases in length
and F.sub.2 is from 6 to 12 bases in length.
7. An RNA catalyst according to claim 2 wherein P.sub.1 and P.sub.4
each is from 3 to 6 bases in length.
8. An RNA catalyst according to claim 7 wherein P.sub.1 has the
sequence 5'-ACCAG-31 and P.sub.4 has the sequence 5'-CUGGUA-3'.
9. An RNA catalyst according to claim 2 wherein S.sub.1 and S.sub.2
each is from 4 to 9 bases in length.
10. An RNA catalyst according to claim 9 wherein S.sub.1 has the
sequence 5'-AGAAACA-3' and S.sub.2 has the sequence
5'-GUAUAUUAC-3'.
11. An RNA catalyst according to claim 2 wherein P.sub.2 and
P.sub.3 each is from 3 to 9 bases in length.
12. An RNA catalyst according to claim 11 wherein P.sub.2 has the
sequence 5'-CAC-3' and P.sub.3 has the sequence 5'-GUG-3'.
13. An RNA catalyst according to claim 2 wherein L.sub.2 is at
least 3 bases in length.
14. An RNA catalyst according to claim 13 wherein L.sub.2 has the
sequence 5'-GUU-3'.
15. An RNA catalyst according to claim 2 wherein
5'-S.sub.1-P.sub.2-L.sub.- 2 has the sequence
5'AGAAACACACGUU-3'.
16. An RNA catalyst according to claim 2 wherein
5'-P.sub.2-L.sub.2-P.sub.- 3 has the sequence
5'-CACGGACUUCGGUCCGUG-3' [SEQ ID 46].
17. An RNA catalyst according to claim 1 or 2 which is capable of
cleaving an RNA substrate selected from the group consisting of
messenger RNA, transfer RNA, ribosomal RNA, viral RNA, nuclear RNA,
organellar RNA and other cellular RNA.
18. The catalyst of claim 17 which is capable of cleaving an RNA
substrate selected from the group consisting of HIV-1 virus RNA and
tobacco mosaic virus RNA.
19. An RNA catalyst according to claim 18 which is capable of
cleaving HIV-1 RNAs containing the sequence UGCCCGUCUGUUGUGU.
20. An RNA catalyst according to claim 2 containing the sequence:
7wherein, F.sub.1, F.sub.2, F.sub.3, F.sub.4, L.sub.1, L.sub.2,
S.sub.1, S.sub.2, P.sub.1, P.sub.2, P.sub.3 and P.sub.4 are as
defined in claims 1 and 2; and L.sub.3 is a sequence of unpaired
bases that covalently links the catalyst portion of the molecule
with the substrate portion to produce a synthetic autocatalytic RNA
catalyst.
21. An RNA catalyst according to claim 20 wherein CS has the
sequence 5'-NGUC-3', wherein N is any base, and the substrate is
cleaved by the catalyst between N and G.
22. An RNA catalyst according to claim 21 wherein L.sub.1 has the
sequence 3'-AAGA-5'.
23. An RNA catalyst according to claim 22 wherein
5'-P.sub.1-S.sub.1-P.sub- .2-L.sub.2-P.sub.3-S.sub.2-P.sub.4-3' has
the sequence 5'-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA-3'.
24. An RNA catalyst according to claim 23 wherein L.sub.3 has the
sequence 3'-CCUCC-5'.
25. A synthetic RNA catalyst which is capable of cleaving an RNA
substrate containing the sequence: 5'-F.sub.1-CS-F.sub.2-3', the
catalyst containing the sequence:
9 5'-F.sub.3-L.sub.1-F.sub.4-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGU
A-3',
and active variants thereof, wherein, CS is a cleavage sequence;
F.sub.1 and F.sub.2 each is a sequence of bases flanking the
cleavage sequence; F.sub.3 is a sequence of bases selected so that
F.sub.3 is substantially base paired with F.sub.2 when the catalyst
is bound to the substrate; F.sub.4 is a sequence of bases selected
so that F.sub.4 is substantially base paired with F.sub.1 when the
catalyst is bound to the substrate; the sequences of F.sub.3 and
F.sub.4 being selected so that each contains an adequate number of
bases to achieve sufficient binding of the RNA substrate to the RNA
catalyst so that cleavage of the substrate can take place; and
L.sub.1 is a sequence of bases selected so that L.sub.1 does not
base pair with CS when the catalyst is bound to the substrate.
26. A synthetic RNA catalyst which is capable of cleaving an RNA
substrate containing the sequence: 5'-F.sub.1-CS-F.sub.2-3', the
catalyst containing the sequence:
10 5'-F.sub.3-L.sub.1-F.sub.4-ACCAGAGAAACACACGGACUUCGGUCC [SEQ ID
47] GUG-GUAUAUUACCUGGUA-3'
wherein, CS is a cleavage sequence; F.sub.1 and F.sub.2 each is a
sequence of bases flanking the cleavage sequence; F.sub.3 is a
sequence of bases selected so that F.sub.3 is substantially base
paired with F.sub.2 when the catalyst is bound to the substrate;
F.sub.4 is a sequence of bases selected so that F.sub.4 is
substantially base paired with F.sub.1 when the catalyst is bound
to the substrate; the sequences of F.sub.3 and F.sub.4 being
selected so that each contains an adequate number of bases to
achieve sufficient binding of the RNA substrate to the RNA catalyst
so that cleavage of the substrate can take place; and L.sub.1 is a
sequence of bases selected so that L.sub.1 does not base pair with
CS when the catalyst is bound to the substrate.
27. An RNA catalyst according to claim 25 or 26 wherein F.sub.3 is
at least 3 bases in length and F.sub.4 is from 3 to 5 bases in
length, and the catalyst cleaves a substrate wherein F.sub.1 and
F.sub.2 each is at least 3 bases in length.
28. An RNA catalyst according to claim 27 wherein F.sub.3 is from 6
to 12 bases in length and F.sub.4 is 4 bases in length, and the
catalyst cleaves a substrate wherein F.sub.1 is 4 bases in length
and F.sub.2 is from 6 to 12 bases in length.
29. An RNA catalyst according to claim 25 or 26 which is capable of
cleaving an RNA substrate in which CS has the sequence 5'-NGUC-3',
wherein N is any base and the substrate is cleaved by the catalyst
between N and G.
30. AN RNA catalyst according to claim 29 wherein L.sub.1 has the
sequence 3'-AAGA-5'.
31. An RNA catalyst according to claim 25 or 26 which is capable of
cleaving an RNA substrate selected from the group consisting of
messenger RNA, transfer RNA, ribosomal RNA, viral RNA, nuclear RNA,
organellar RNA and other cellular RNA.
32. An RNA catalyst according to claim 31 which is capable of
cleaving an RNA substrate selected from the group consisting of
HIV-1 virus RNA and tobacco mosaic virus RNA.
33. An RNA catalyst according to claim 32 which is capable of
cleaving HIV-1 RNAs containing the sequence UGCCCGUCUGUUGUGU.
34. An engineered DNA molecule coding for an RNA catalyst according
to claim 1, 2, 20, 25 or 26.
35. A vector comprising a DNA sequence coding for an RNA catalyst
according to claim 1, 2, 20, 25 or 26, the DNA sequence being
operatively linked to expression control sequences.
36. The vector of claim 35 which is capable of self-replication in
a host.
37. The vector of claim 35 wherein the RNA catalyst encoded by the
vector is capable of cleaving an RNA substrate selected from the
group consisting of messenger RNA, transfer RNA, ribosomal RNA,
viral RNA, nuclear RNA, organellar RNA and other cellular RNA.
38. The vector of claim 37 wherein the RNA catalyst encoded by the
vector is capable of cleaving an RNA substrate selected from the
group consisting of HIV-1 virus RNA and tobacco mosaic virus
RNA.
39. The vector of claim 38 wherein the RNA catalyst encoded by the
vector is capable of cleaving HIV-1 RNAs containing the sequence
UGCCCGUCUGUUGUGU.
40. A host cell transformed with a vector according to claim 35 and
which is capable of expressing the RNA catalyst.
41. A method of cleaving an RNA substrate which contains the
sequence: 5'F.sub.1-CS-F.sub.2-3', wherein, CS is a cleavage
sequence; and F.sub.1 and F.sub.2 each is a sequence of bases
flanking the cleavage sequence; the method comprising contacting
the substrate with a synthetic RNA catalyst comprising a substrate
binding portion and a "hairpin" portion, the substrate binding
portion of the catalyst having the sequence:
3'F.sub.4-L.sub.1-F.sub.3-5'wherein, F.sub.3 is a sequence of bases
selected so that F.sub.3 is substantially base paired with F.sub.2
when the catalyst is bound to the substrate; F.sub.4 is a sequence
of bases selected so that F.sub.4 is substantially base paired with
F.sub.1 when the catalyst is bound to the substrate; the sequences
of F.sub.3 and F.sub.4 being selected so that each contains an
adequate number of bases to achieve sufficient binding of the RNA
substrate to the RNA catalyst so that cleavage of the substrate can
take place; and L.sub.1 is a sequence of bases selected so that
L.sub.1 does not base pair with CS when the catalyst is bound to
the substrate.
42. The method of claim 41 wherein the "hairpin" portion of the
catalyst has the sequence: 8wherein, P.sub.1 and P.sub.4 each is a
sequence of bases, the sequences of P.sub.1 and P.sub.4 being
selected so that P.sub.1 and P.sub.4 are substantially base paired;
P.sub.1 is covalently linked to F.sub.4; S.sub.1 and S.sub.2 each
is a sequence of bases, the sequences of S.sub.1 and S.sub.2 being
selected so that S.sub.1 and S.sub.2 are substantially unpaired;
P.sub.2 and P.sub.3 each is a sequence of bases, the sequences of
P.sub.2 and P.sub.3 being selected so that P.sub.2 and P.sub.3 are
substantially base paired; and L.sub.2 is a sequence of unpaired
bases.
43. The method of claim 42 wherein the catalyst has the sequence:
9wherein, F.sub.1, F.sub.2, F.sub.3, F.sub.4, L.sub.1, L.sub.2,
S.sub.1, S2, P.sub.1, P.sub.2, P.sub.3 and P.sub.4 are as defined
in claims 41 and 42; and L.sub.3 is a sequence of unpaired bases
that covalently links the catalyst portion of the molecule with the
substrate portion to produce a synthetic autocatalytic RNA
catalyst.
44. A method of cleaving an RNA substrate containing the sequence:
5'-F.sub.1-CS-F.sub.2-3', comprising contacting the substrate with
a synthetic RNA catalyst containing the sequence:
11 5'-F.sub.3-L.sub.1-F.sub.4-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGU
A-3',
and active variants thereof, wherein, CS is a cleavage sequence;
F.sub.1 and F.sub.2 each is a sequence of bases flanking the
cleavage sequence; F.sub.3 is a sequence of bases selected so that
F.sub.3 is substantially base paired with F.sub.2 when the catalyst
is bound to the substrate; F.sub.4 is a sequence of bases selected
so that F.sub.4 is substantially base paired with F.sub.1 when the
catalyst is bound to the substrate; the sequences of F.sub.3 and
F.sub.4 being selected so that each contains an adequate number of
bases to achieve sufficient binding of the RNA substrate to the RNA
catalyst so that cleavage of the substrate can take place; and
L.sub.1 is a sequence of bases selected so that L.sub.1 does not
base pair with CS when the catalyst is bound to the substrate.
45. A method of cleaving an RNA substrate containing the sequence:
5'-F.sub.1-CS-F.sub.2-3', comprising contacting the substrate with
a synthetic RNA catalyst containing the sequence:
12 5'-F.sub.3-L.sub.1-F.sub.4-ACCAGAGAAACACACGGACUUCGGUCC [SEQ ID
47] GUGG-UAUAUUACCUGGUA-3'
wherein, CS is a cleavage sequence; F.sub.1 and F.sub.2 each is a
sequence of bases flanking the cleavage sequence; F.sub.3 is a
sequence of bases selected so that F.sub.3 is substantially base
paired with F.sub.2 when the catalyst is bound to the substrate;
F.sub.4 is a sequence of bases selected so that F.sub.4 is
substantially base paired with F.sub.1 when the catalyst is bound
to the substrate; the sequences of F.sub.3 and F.sub.4 being
selected so that each contains an adequate number of bases to
achieve sufficient binding of the RNA substrate to the RNA catalyst
so that cleavage of the substrate can take place; and L.sub.1 is a
sequence of bases selected so that L.sub.1 does not base pair with
CS when the catalyst is bound to the substrate.
46. The method of claim 41, 42, 43, 44 or 45 wherein the cleavage
occurs under physiological conditions.
47. The method of claim 46 wherein the cleavage occurs in vivo in a
host cell which has been transformed with a vector comprising a DNA
sequence coding for the RNA catalyst, the DNA sequence being
operatively linked to expression control sequences.
48. A synthetic RNA transcript comprising an autocatalytic portion
which has the formula: 10wherein, CS is a cleavage sequence;
F.sub.1 and F.sub.2 each is a sequence of bases flanking the
cleavage sequence; F.sub.3 is a sequence of bases selected so that
F.sub.3 is substantially base paired with F.sub.2; F.sub.4 is a
sequence of bases selected so that F.sub.4 is substantially base
paired with F.sub.1; the sequences of F.sub.3 and F.sub.4 being
selected so that each contains an adequate number of bases to
achieve sufficient binding with F.sub.1 and F.sub.2 so that
cleavage can take place; L.sub.1 is a sequence of bases selected so
that L.sub.1 does not base pair with CS; P.sub.1 and P.sub.4 each
is a sequence of bases, the sequences of P.sub.1 and P.sub.4 being
selected so that P.sub.1 and P.sub.4 are substantially base paired;
S.sub.1 and S.sub.2 each is a sequence of bases, the sequences of
S.sub.1 and S.sub.2 being selected so that S.sub.1 and S.sub.2 are
substantially unpaired; P.sub.2 and P.sub.3 each is a sequence of
bases, the sequences of P.sub.2 and P.sub.3 being selected so that
P.sub.2 and P.sub.3 are substantially base paired; L.sub.2 is a
sequence of unpaired bases; and L.sub.3 is a sequence of unpaired
bases.
49. A method of terminating an RNA transcript comprising:
transforming a host cell with a vector comprising DNA coding for an
RNA transcript according to claim 48; culturing the host cell so
that RNA is transcribed and the autocatalytic portion cleaves the
RNA transcript to terminate the transcript.
Description
[0001] This application is a continuation-in-part of co-pending
application Ser. No. 07/577,658 filed Sep. 4, 1990, which was a
continuation-in-part of application Ser. No. 07/409,666 filed Sep.
20, 1989, which was a continuation-in-part of application Ser. No.
07/247,100 filed Sep. 20, 1988, now abandoned.
FIELD OF THE INVENTION
[0003] The present invention relates to an RNA catalyst which
cleaves specific RNA sequences into a fragment having a 5' hydroxyl
and a fragment having a 2',3' cyclic phosphate. The products of the
reaction described herein resemble those resulting from the natural
hydrolysis of RNA.
BACKGROUND OF THE INVENTION
[0004] Certain naturally occurring satellite, virusoid and viroid
RNAs possess the property of self-catalyzed cleavage. Self-cleavage
has been demonstrated in vitro for avocado sunblotch viroid (ASBV)
(Hutchins, C. J., Rathjen, P. D., Forster, A. C. and Symons, R. H.
(1986) Nucleic Acids Res., 14: 3627-3640), satellite RNA from
tobacco ringspot virus (sTRSV) (Prody, G. A., Bakos, J. T.,
Buzayan, J. M., Schneider, I. R. and Bruening, G. (1986) Science,
231: 1577-1580; Buzayan, J. M., Gerlach, W. L. and Bruening, G. B.
(1986) Proc. Natl. Acad. Sci. U.S.A. 83: 8859-8862) and lucerne
transient streak virus (vLTSV) (Forster, A. C. and Symons, R. H.
(1987) Cell, 49: 211-220). These self-catalyzed RNA cleavage
reactions share a requirement for divalent metal ions and neutral
or higher pH and cleave target RNA sequences to give 5' hydroxyl
and 2',3'-cyclic phosphate termini (Prody, G. A., Bakos, J. T.,
Buzayan, J. M., Schneider, I. R. and Bruening, G. (1986) Science,
213: 1577-1580; Forster, A. C. and Symons, R. H. (1987) Cell, 49:
211-220; Epstein, L. M. and Gall, J. G. (1987) Cell, 48: 535-543;
Buzayan, J. M. Gerlach, W. L., Bruening, G. B., Keese, P. and
Gould, A. R. (1986) Virology, 151: 186-199).
[0005] A "hammerhead" model has been proposed and accurately
describes the catalytic center of (+)sTRSV RNA, the. (+) and (-)
strands of ASBV and the (+) and (-) strands of vLTSV (Forster, A.
C. and Symons, R. H. (1987) Cell, 49: 211-220). The single
exception is (-)sTRSV RNA which does not fit the "hammerhead" model
(Forster, A. C. and Symons, R. H. (1987) Cell, 49: 211-220;
Buzayan, J. M., Gerlach, W. L. and Bruening, G. (1986) Nature, 323:
349-352; Buzayan, J. M., Hampel, A. and Bruening, G. B. (1986)
Nucleic Acids Res., 14: 9729-9743), and the structure of whose
catalytic center was unknown prior to the present invention. It is
therefore understandable that the primary scientific focus has been
on studying the "hammerhead" consensus structure and, as regards
sTRSV, on studying the (+) strand.
[0006] Intermolecular cleavage of an RNA substrate by an RNA
catalyst that fits the "hammerhead" model was first shown in 1987
(Uhlenbeck, O. C. (1987) Nature, 328: 596-600). The RNA catalyst
was recovered and reacted with multiple RNA molecules,
demonstrating that it was truly catalytic.
[0007] Catalytic RNAs designed based on the "hammerhead" motif have
been used to cleave specific target sequences by making appropriate
base changes in the catalytic RNA to maintain necessary base
pairing with the target sequences (Haseloff and Gerlach, Nature,
334, 585 (1988); Walbot and Bruening, Nature, 334, 196 (1988);
Uhlenbeck, O. C. (1987) Nature, 328: 596-600; Koizumi, M., Iwai, S.
and Ohtsuka, E. (1988) FEBS Lett., 228: 228-230). This has allowed
use of the catalytic RNA to cleave specific target sequences and
indicates that catalytic RNAs designed according to the
"hammerhead" model may possibly cleave specific substrate RNAs in
vivo. (see Haseloff and Gerlach, Nature, 334, 585 (1988); Walbot
and Bruening, Nature, 334, 196 (1988); Uhlenbeck, O. C. (1987)
Nature, 328: 596-600).
[0008] However, catalytic RNAs such as those that were designed
based on the "hammerhead" model have several limitations which
restrict their use in vitro and may forestall their use in vivo.
For example, the temperature optimum for the reaction is
50-55.degree. C., which is well above physiological, and the kcat
(turnover number) is only 0.5/mm even at 55.degree. C. (Uhlenbeck,
O. C. (1987) Nature, 328:596-600; Haseloff and Gerlach, Nature,
334, 585 (1988)). In addition, the Km is 0.6 uM (Uhlenbeck, O. C.
(1987) Nature, 328:596-600), meaning that the reaction requires
high concentrations of substrate which makes it difficult, if not
impossible, for the catalytic RNA to cleave low levels of target
RNA substrate such as would be encountered in vivo.
[0009] Cech et al. published application WO 88/04300 and U.S. Pat.
No. 4,987,071 also report the preparation and use of certain
synthetic ribozymes that have several activities, including
endoribonuclease activity. The design of these ribozymes is based
on the properties of the Tetrahymena ribosomal RNA self-splicing
reaction. A temperature optimum of 50.degree. C. is reported (page
39 of WO 88/04300; col. 20, lines 4-5, of U.S. Pat. No. 4,987,071)
for the endoribonuclease activity, and the Km and kcat reported for
this activity are 0.8 uM and 0.13/minute, respectively (Example VI,
last paragraph).
[0010] In view of the above, there is a need for an RNA catalyst
having a lower temperature optimum, preferably near physiological
temperatures, a higher turnover number and a smaller Km and which
can be engineered to cut specific target RNA substrates.
Accordingly, based on the discovery of a totally different
structure disclosed hereinafter, it is an object of the present
invention to provide such an RNA catalyst. Other objects and
features of the invention will be in part apparent and in part
pointed out. The invention, accordingly, comprises the products and
methods hereinafter described and their equivalents, the scope of
the invention being indicated in the appended claims.
SUMMARY OF THE INVENTION
[0011] The invention comprises a synthetic RNA catalyst capable of
cleaving an RNA substrate which contains the sequence:
5'-F.sub.1-CS-F.sub.2-3',
[0012] wherein,
[0013] CS is a cleavage sequence; and
[0014] F.sub.1 and F.sub.2 each is a sequence of bases flanking the
cleavage sequence.
[0015] The catalyst comprises a substrate binding portion and a
"hairpin" portion. The substrate binding portion of the catalyst
has the sequence:
3'-F.sub.4-L.sub.1-F.sub.3-5'
[0016] wherein,
[0017] F.sub.3 is a sequence of bases selected so that F.sub.3 is
substantially base paired with F.sub.2 when the catalyst is bound
to the substrate;
[0018] F.sub.4 is a sequence of bases selected so that F.sub.4 is
substantially base paired with F.sub.1 when the catalyst is bound
to the substrate;
[0019] the sequences of F.sub.3 and F.sub.4 being selected so that
each contains an adequate number of bases to achieve sufficient
binding of the RNA substrate to the RNA catalyst so that cleavage
of the substrate can take place; and
[0020] L.sub.1 is a sequence of bases selected so that L.sub.1 does
not base pair with CS when the catalyst is bound to the
substrate.
[0021] The "hairpin" portion is a portion of the catalyst that
assumes a hairpin-like configuration when the substrate-catalyst
complex is modeled in two dimensions for minimum energy folding.
The "hairpin" portion of the catalyst preferably has the sequence:
1
[0022] wherein,
[0023] P.sub.1 and P.sub.4 each is a sequence of bases, the
sequences of P.sub.1 and P.sub.4 being selected so that P.sub.1 and
P.sub.4 are substantially base paired;
[0024] P.sub.1 is covalently attached to F.sub.4;
[0025] S.sub.1 and S.sub.2 each is a sequence of bases, the
sequences of S.sub.1 and S.sub.2 being selected so that S.sub.1 and
S.sub.2 are substantially unpaired;
[0026] P.sub.2 and P.sub.3 each is a sequence of bases, the
sequences of P.sub.2 and P.sub.3 being selected so that P.sub.2 and
P.sub.3 are substantially base paired; and
[0027] L.sub.2 is a sequence of unpaired bases.
[0028] RNA catalysts according to the invention can cleave
substrates of any length or type as long as they contain an
appropriate cleavage sequence. In particular, the catalysts can be
used to cleave a specific sequence in naturally-occurring RNA
having a cleavage sequence, as well as RNAs which have been
engineered to contain a cleavage sequence.
[0029] The invention further comprises an engineered DNA molecule
and a vector, each of which comprises a DNA sequence that codes for
an RNA catalyst according to the invention. The invention also
comprises a host transformed with the vector, the host being
capable of expressing the RNA catalyst. In particular, hosts can be
transformed with vectors that, when transcribed, will produce RNA
catalysts which can cleave any RNA, native or foreign, found in the
host. For example, hosts can be transformed with vectors that, when
transcribed, produce RNA catalysts which can regulate the
expression of genes by cleaving messenger RNA or which act as
anti-viral agents by cleaving viral RNA. Thus, the invention has
application in vitro and in vivo in prokaryotes and eukaryotes of
plant or animal origin in regulating gene expression and for
controlling viral infections.
[0030] Finally, the invention includes a method of cleaving an RNA
substrate comprising contacting the substrate with an RNA catalyst
according to the invention. The reaction is unique because it
occurs under physiological conditions, having a temperature optimum
near 37.degree. C., with very favorable reaction parameters. The
method can be practiced in vitro or in vivo. For instance, the
method may be practiced in vivo in host cells that have been
transformed with a vector that codes for an RNA catalyst according
to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows the (-)sTRSV RNA substrate-catalyst complex
that fits the "hairpin" model of catalytic RNA in accordance with
the present invention.
[0032] FIG. 2 shows minimum energy folding of (-)sTRSV RNA.
[0033] FIG. 3 shows the time course of catalysis of a substrate RNA
by the catalytic RNA.
[0034] FIG. 4 shows the Michaelis-Menten kinetics of the RNA
catalytic reaction.
[0035] FIG. 5 shows the temperature dependence of the RNA catalytic
reaction.
[0036] FIG. 6 shows the dependence of the rate of reaction on
concentration of catalytic RNA.
[0037] FIG. 7 shows the reaction properties of a smaller RNA
substrate.
[0038] FIG. 8 shows the loss of catalytic activity when the
terminal A at position 175 or the terminal bases AU at positions
175 and 176 are removed from the catalytic RNA.
[0039] FIG. 9 shows loss of catalytic activity when bases 195-203
in the catalytic RNA sequence are removed.
[0040] FIG. 10 shows loss of catalytic activity when bases AAA at
positions 203, 202 and 201 are changed to CGU respectively.
[0041] FIGS. 11A-C show that there is no effect on catalytic
activity when base A at position 49 in the substrate is changed to
a G, U or C.
[0042] FIG. 12 shows that different target RNA sequences can be
used as long as the base pairing with the catalytic RNA in the
regions flanking the cleavage sequence is maintained.
[0043] FIG. 13 shows that an RNA sequence found in tobacco mosaic
virus can be cleaved at a specific site with the catalytic RNA of
the present invention.
[0044] FIGS. 14A-C show three substrates having sequences found in
the sequence of the messenger. RNA coding for chloramphenicol
acetyl transferase. FIGS. 14A-C also show the separation patterns
on acrylamide gels of the reaction products obtained by reacting
these substrates with catalytic RNAs designed to base pair with the
substrates in the regions flanking the AGUC cleavage sequence.
[0045] FIG. 15 shows the sequence of a substrate having a sequence
found in the sequence coding for the gag protein of the HIV-1 virus
which causes AIDS. FIG. 15 also shows the separation patterns on
acrylamide gels of the reaction products obtained by reacting this
substrate with a catalytic RNA designed to base pair with the
substrate in the regions flanking the CGUC cleavage sequence of the
substrate.
[0046] FIG. 16 shows the sequence of a substrate having a sequence
found in the sequence coding for the regulatory tat protein of the
HIV-1 virus. FIG. 16 also shows the separation patterns on an
acrylamide gel of the reaction products obtained by reacting this
substrate with a catalytic RNA designed to base pair with the
substrate in the regions flanking the UGUC cleavage sequence of the
substrate.
[0047] FIG. 17 shows the sequence of a substrate having four
non-native U's added to the 3' end of the sequence of the native
(-)sTRSV substrate shown in FIG. 1. FIG. 17 also shows the
separation patterns on an acrylamide gel of the reaction products
obtained by reacting this substrate with different concentrations
of a catalytic RNA designed to base pair with the substrate in the
regions flanking the cleavage sequence of the substrate, including
with the four non-native U's.
[0048] FIG. 18 summarizes the sequence requirements for the target
region of the substrate RNA. Only GUC is required for cleavage.
[0049] FIG. 19 shows the positions of base changes (open boxes)
made in the sequence of the catalytic RNA shown in FIG. 1 in order
to prove the existence of 3 and 4 predicted by the "hairpin" model
for (-)sTRSV RNA. FIG. 19 also shows the separation patterns on
acrylamide gels of the reaction products obtained by reacting the
various catalytic RNAs with substrate RNA S17.
[0050] FIG. 20 shows the RNA sequence of an autocatalytic cassette
that has utility in terminating transcription at a very specific
site. FIG. 20 also shows the separation pattern on an acrylamide
gel of the reaction products obtained when this catalyst was
transcribed and cleaved autocatalytically.
[0051] FIG. 21 shows the positions of two base changes that were
made in the native (-)sTRSV catalytic RNA sequence shown in FIG. 1.
FIG. 21 also shows the separation patterns on acrylamide gels of
the reaction products obtained by reacting these catalysts or
catalyst R51 (control gel) with substrate S17.
[0052] FIG. 22A shows a plasmid map illustrating pertinent features
of vector pHC-CAT which contains the CAT gene linked to the
"hairpin" autocatalytic cassette of the invention. Also shown is
the expected RNA transcript of the illustrated region of pHC-CAT
and the expected 5' fragment of the autocatalytic cleavage.
[0053] FIG. 22B shows the results of Northern blot analysis of RNA
produced by host cells transformed with pHC-CAT.
[0054] FIG. 23: Map of the pMHC-CAT mammalian expression
vector.
[0055] FIG. 24: Map of plasmid pHC19R.
[0056] FIG. 25: Shows the results of S1 nuclease protection assay
of RNA from transfected CHO cells and RNA controls.
[0057] FIG. 26: Map of viral vector constructions (CMV) with the
hairpin autocatalytic cassette in the sense orientation (pCS101HC7)
and the antisense orientation (pCS101HC9).
[0058] FIG. 27A: Shows the results of S1 nuclease protection assay
of RNA from infected turnip plants and RNA controls.
[0059] FIG. 27B: Results of Northern blot analysis of the RNA
isolated from control and infected plants.
[0060] FIG. 27C: Results of PCR amplification of viral sequences
from control and infected turnip plants.
[0061] FIGS. 27D and 27E: Results of Western immunoblot analysis of
protein extracts of turnips leaves from control and infected
plants.
[0062] FIG. 28: HIV-1 target sequence.
[0063] FIG. 29: Conservation of the HIV-1 target sequence in
various HIV isolates.
[0064] FIG. 30: Sequences of HIV-1 substrate ("SHIV") containing
the conserved target sequence and of an engineered "hairpin"
catalytic RNA ("RHIV") designed to cleave the substrate.
[0065] FIGS. 31 and 32: Results of the cleavage of SHIV substrate
RNA by RHIV catalytic RNA.
[0066] FIG. 33: Sequence of longer HIV-1 transcript and results of
its cleavage by RHIV.
[0067] FIG. 34: Map of pHR and partial sequence.
[0068] FIG. 35: Results of cleavage of substrate SHIV by RHIV and
by catalytic RNA produced by T7 RNA polymerase transcription of
pHR.
[0069] FIG. 36: Map of pMSGRHIV and partial sequence.
[0070] FIG. 37: Map of plasmid pMRHPT and partial sequence.
[0071] FIG. 38: Selection and testing scheme for RHGPT.
[0072] FIG. 39: Results of S1 nuclease assay for reduction of HGPRT
mRNA in CHO cells transfected with pMRHPT and pMSG-dhfr.
[0073] FIG. 40: Map of the plasmid pMCATRCAT and partial
sequence.
[0074] FIG. 41: Results of S1 nuclease assay for reduction of CAT
mRNA in CHO cells transfected with pMCATRCAT and pMSG-dhfr.
[0075] FIGS. 42A-C: Summary of mutagenesis experiments with the
(-)sTRSV RNA substrate-catalyst complex.
[0076] FIG. 42D: A more refined secondary structure model for the
(-)sTRSV RNA substrate-catalyst complex.
[0077] FIG. 43: Separation patterns on an acrylamide gel of the
reaction products obtained by reacting substrate with "hairpin"
catalytic RNA having the loop that closes the "hairpin" replaced by
the sequence GGAC(UUCG)GUCC.
DETAILED DESCRIPTION OF THE DRAWINGS
[0078] FIG. 1. The "hairpin" model for RNA catalysis was developed
by determining the minimum energy folding predicted by computer
modeling of the catalytic complex containing the minimum size
catalytic RNA and substrate RNA of (-)sTRSV RNA. It is this minimum
energy folding which is shown in FIG. 1. Two molecules are shown
folded: (1) catalytic RNA which contains 50 bases of satellite RNA
(224-175) and (2) substrate RNA which contains 14 bases of
satellite RNA (53-40) The arrow represents the site of
cleavage.
[0079] The 50-base catalytic RNA and the 14-base substrate RNA are
the "minimum size" in the sense that reductions in their length
result in a substantial or total loss of catalytic activity as is
shown in the Examples below. Thus, this length of (-)sTRSV catalyst
sequence is preferred to shorter lengths. Also, substrate RNA
having at least the degree of base-pairing with the catalyst
exhibited by the 14-base substrate is preferred.
[0080] FIG. 2. Minimum energy folding of (-)sTRSV RNA. The molecule
was folded using the Wisconsin RNA folding program (Zucker, M. and
Stiegler, P. (1981) Nucleic Acids Res., 9: 133-148; Devereux, J.,
Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res., 12:
387-395) with base numbers corresponding to (+)sTRSV (Buzayan, J.
M., Gerlach, W. L., Bruening, G. B., Keese, P. and Gould, A. R.
(1986) Virology, 151: 186-199). With this numbering scheme the
5'-3' direction of the molecule is with decreasing base number. The
minimum catalytic complex is identified. The substrate RNA sequence
is between bases 53-40 and the catalytic RNA sequence is between
bases 224-175. The arrow is the site of cleavage.
[0081] The folding identifies regions of expected base pairing and
expected non-base pairing, loops. This model does not preclude
higher order interactions occurring between the loops.
[0082] FIG. 3. Time course of substrate S17 cleavage by catalytic
RNA R51. The reaction was carried out under standard conditions,
which were 37.degree. C. in 12 mM MgCl.sub.2, 40 mM Tris pH 7.5 and
2 mM spermidine, for the following times: lane 1, 30 sec; lane 2, 5
min; lane 3, 15 min; lane 4, 30 min; lane 5, 60 min; lane 6, 90
min; lane 7, 150 min. Concentrations were as follows: [R51]=0.0032
uM and [S17]=0.09 uM. RNA was separated on 7M urea, 20% acrylamide
gels, bands cut out and counted in the liquid scintillation counter
for FIGS. 3-17 and 19-21. Throughout the figures, the designations
5'F and 3'F are the products of cleavage of the substrate and
represent the resulting 5' fragments and 3' fragments,
respectively.
[0083] FIG. 4. Eadie Hofstee plot of catalytic RNA R51 cleavage of
substrate RNA S17. The reaction was carried out at 37.degree. C. in
12mM MgCl.sub.2, 40 mM Tris pH 7.5 and 2 mM spermidine.
Concentrations were as follows: [R51]=0.0004 uM and [S17]=0.125 uM
(lane 1), 0.0625 uM (lane 2), 0.0417 uM (lane 3), 0.031 uM (lane
4), 0.021 uM (lane 5), 0.0156 uM (lane 6), 0.0078 uM (lane 7) and
0.0039 uM (lane 8).
[0084] FIG. 5. Temperature dependence of the rate of cleavage of
substrate RNA S17 by catalytic RNA R51. The reaction was carried
out in 12 mM MgCl.sub.2, 40 mM Tris pH 7.5 and 2 mM spermidine at
45.degree. C. (lane 6), 41.degree. C. (lane 5), 37.degree. C. (lane
4), 33.degree. C. (lane 3), 27.degree. C. (lane 2) and 20.degree.
C. (lane 1). The concentrations used were: [R51]=0.003 uM and
[S17]=0.09 uM. R51 was unlabeled. The velocities shown in the graph
in FIG. 5 were calculated by the use of time points of 8 and 16
minutes. The separation patterns of the reaction products on the
acrylamide gel shown in the figure are for the 16-minute time
point.
[0085] FIG. 6. Rate of cleavage of substrate RNA S17 at varying
concentrations of catalytic RNA R51. The reaction was carried out
at 37.degree. C. in 12 mM MgCl.sub.2, 40 mM Tris pH 7.5 and 2 mM
spermidine for 40 min (lane 1 and 2), 20 min (lane 3), 10 min (lane
4) and 5 min (lane 5). The concentration of substrate used was
0.175 uM.
[0086] FIG. 7. Eadie Hofstee plot of catalytic RNA R51 cleavage of
substrate RNA S10. The substrate S10 containing the RNA sequence:
GACAGUCCUG was prepared from a DNA template containing the T-7
promoter as described in Example 2. This substrate was mixed with
the catalytic RNA, R51, from Example 2 under standard conditions:
37.degree. C. in 12 mM MgCl.sub.2, 40 mM Tris pH 7.5 and 2 mM
spermidine for 10 min. Concentrations of substrate used were as
follows: 0.115 uM, 0.77 uM, 0.038 uM, 0.029 uM, 0.014 uM. The
concentration of catalytic RNA, R51, used was 1 nM. The line was
fit by linear regression analysis and intercept, kcat, and
Michaelis constant, Km, calculated.
[0087] FIG. 8. Removal of the terminal "A" at position 175 of the
catalytic RNA. The "A" at base position 175 (circled) was removed
and the resulting catalytic RNA, R50, reacted with substrate S17.
In addition, two bases were removed, to give R49 which had both
A175 and U176 removed. Concentration of substrate S17 was 0.3 uM
and all catalytic RNA concentrations were 4 nM. The reaction times
were 20 min under standard conditions. Lane 1 R51/S17; Lane 2
R50/S17; Lane 3 R49/S17; Lane 4 S17 alone. A 75% loss of activity
was seen with R50 and R49.
[0088] FIG. 9. Loss of activity when bases 195-203 in the catalytic
RNA sequence are removed. When the underlined bases were removed
and the adjacent bases ligated together, no catalytic activity was
seen.
[0089] FIG. 10. Loss of activity when bases AAA at positions 203,
202 and 201 are changed to CGU respectively. When the circled AAA
bases were replaced by the underlying 5'-CGU-3' bases, no catalytic
activity was seen.
[0090] FIG. 11A. No effect on activity when base A49 in the
substrate is changed to a "G". The circled "A" base 49 in the
substrate was changed to a "G" and no effect on activity was seen.
The concentration of R51 was 0.016 uM, [S17]=0.4 uM, and
[S17(A->G)]=0.2 uM. Reaction under standard conditions was for
40 min. Lane 1 S17; Lane 2 S17/R51; Lane 3 S17(A->G); Lane 4 S17
(A->G)/R51.
[0091] FIG. 11B. No effect on activity was seen when base A49 in
the substrate was changed to a "U" (S17(A-->U)). The
concentration of substrate RNA S17 (A-->U) used was 0.12 uM and
the concentration of R51 was 0.0065 uM. Reaction was for 60 minutes
under standard conditions. The catalytic RNA was unlabeled.
[0092] FIG. 11C. No effect on activity was seen when base A49 in
the substrate was changed to a "C" (S17(A-->C)). The
concentration of substrate RNA S17 (A-->C) used was 0.08 uM and
the concentration of R51 was 0.0065 uM. Reaction was for 60 minutes
under standard conditions. The catalytic RNA was unlabeled.
[0093] FIG. 12. Different target RNA sequences can be used as long
as the base pairing is maintained with the catalytic RNA. The C:G
base pair predicted by the "hairpin" model of the catalytic complex
of (-)sTRSV, FIG. 1, was changed to a G:C base pair (circled) and
activity was maintained. In this experiment the usual substrate S17
was not used; instead a new substrate was used with the exact same
sequence except the first two vector bases GC were eliminated. The
resulting sequence of this new substrate S15 was gUGACAGUCCUGUUU.
The substrate containing the C50->G50 base change was
S15(C->G) and the catalytic RNA with the G214->C214 base
change was R51(G->C). The reactions were run under standard
conditions for 60 min at [R51]=0.07 uM, [S15]=0.12 uM,
[S15(C->G)]=0.15 uM, [R51(G->C)]=0.05 uM. Lane 1 R51/S1 S;
Lane 2 R51(G->C)/S15; Lane 3 S15; Lane 4
R5(G->C)/S15(C->G); Lane 5 S15(C->G).
[0094] FIG. 13. An RNA sequence found in tobacco mosaic virus (TMV)
can be cleaved at a specific site. The substrate RNA shown is that
beginning with nucleotide #538 in the tobacco mosaic virus
sequence. The catalytic RNA was synthesized to base pair with the
TMV substrate RNA in the stem regions of the "hairpin" as shown by
the circled base pairs. Reaction was for 20 min under standard
conditions with a catalytic RNA concentration of 3 nM and a
substrate concentration of 0.06 uM. Lane 1 TMV substrate RNA only;
Lane 2 TMV catalytic RNA/TMV substrate RNA.
[0095] FIGS. 14A-C. The sequences of three substrate RNAs having
sequences found in the messenger RNA for chloramphenicol acetyl
transferase (CAT) are shown. They have 14-, 16- and 18-base long
target sites, and the length of the 3' regions flanking the AGUC
cleavage sequence is extended in substrates (B) and (C) as compared
to substrate (A). Catalytic RNAs designed to base pair with the
substrate RNAs in both the 3' and 5' regions flanking the cleavage
sequence AGUC were synthesized. The open boxed bases are those
which are different from those in the native (-)sTRSV substrate RNA
sequence shown in FIG. 1.
[0096] FIG. 15. This figure shows the sequence of a substrate RNA
having a sequence found in the region of the HIV-1 viral RNA which
specifies the gag protein. A catalytic RNA was made whose sequence
was designed so that the catalyst would base pair with the
substrate RNA in both the 3' and 5' regions flanking the cleavage
sequence. The open boxed bases are those which are different than
those of the native (-)sTRSV sequence shown in FIG. 1. The
catalytic RNA cleaved the substrate RNA at the arrow.
[0097] FIG. 16. Shown is the sequence of a substrate RNA having the
sequence found at the beginning of the coding region for the
regulatory protein tat of the HIV-1 virus. A catalytic RNA was made
which was designed so that it would base pair with the substrate
RNA in both the 3' and 5' regions flanking the UGUC catalytic
cleavage sequence. The open boxes are bases which are different
from those of the native (-)sTRSV substrate sequence shown in FIG.
1. Cleavage occurred at the arrow as shown.
[0098] FIG. 17. Shown is the sequence of a substrate RNA having
four non-native bases (UUUU) added to the 3' end of the sequence of
the native (-)sTRSV substrate RNA shown in FIG. 1. A corresponding
catalytic RNA was made whose sequence was designed so it would base
pair with the substrate in both the 3' and 5' regions flanking the
AGUC cleavage sequence. Cleavage rates with a constant catalytic
RNA concentration and various concentrations of substrate RNA were
determined by cutting out the bands of the acrylamide gels,
counting the radioactivity and plotting the data using
Michaelis-Menton procedures to calculate Km and kcat.
[0099] FIG. 18. Summary of the sequence requirements for the target
region of substrate RNA. Only a GUC sequence is required for
cleavage of the substrate as long as the short sequences of bases
in the regions of the substrate flanking the cleavage sequence are
substantially base paired with corresponding regions of the RNA
catalyst. The regions of base pairing are labeled Helix 1 and Helix
2 in the figure. Also, the regions of base pairing predicted by the
"hairpin" model for (-)sTRSV to exist in the "hairpin" portion of
the catalyst are labeled Helices 3 and 4.
[0100] FIG. 19. Confirmation of the existence of Helices 3 and 4
predicted by the "hairpin" model for (-)sTRSV RNA. A G->C base
mutation in base 35 (count bases from the 5' end of the catalytic
RNA sequence shown) of the (-)sTRSV catalytic RNA sequence shown in
FIG. 1 resulted in an RNA with no catalytic activity (Lanes 3 and 4
("mismatch")). A double mutant, G35->C; C27->G had restored
catalytic activity (Lanes 5 and 6 ("substitute b.p.")). These two
base changes are in the Helix 4 region whose existence is predicted
by the "hairpin" model for (-)sTRSV. Also, a catalytic RNA having a
single base change at position 47 (G47->C) was inactive (Lanes 9
and 10), while a double mutant, with a second mutation C17->G,
had restored activity (Lanes 11 and 12). These two base changes are
in the Helix 3 region whose existence is predicted by the "hairpin"
model. The control (Lanes 1, 2, 7 and 8) is cleavage of the
substrate RNA S17 having the native (-)sTRSV sequence by catalytic
RNA sequence R51 having the native sequence.
[0101] FIG. 20. The RNA sequence of a synthetic "hairpin"
autocatalytic cassette is shown. The sequence shown in FIG. 20 is
the same as that of the catalyst shown in FIG. 1, but with
additional 5' bases added to form a loop at the 5' end of the
catalyst and to provide a substrate target sequence (i.e., a
cleavage sequence and upstream and downstream flanking bases) which
can bind to the substrate binding portion of the catalyst sequence.
Such an RNA was prepared. When transcription was performed, the
cassette autocatalytically cleaved at the expected site to give the
appropriate 3'F and 5'F products.
[0102] FIG. 21. Shown are two base changes that were made in the
native (-)sTRSV catalytic sequence shown in FIG. 1. The two bases
changes were an "A" to "U" mutation at position 217 and a "G" to
"C" mutation at position 216. FIG. 21 also shows the separation
patterns on acrylamide gels of the reaction products obtained by
reacting one of these catalysts or R51 (control) with substrate
S17. Both base changes produced catalysts that were inactive when
the catalysts were reacted with substrate S17 under standard
conditions for 15 minutes.
[0103] FIG. 22A shows a plasmid map illustrating pertinent portions
of vector pHC-CAT containing the CAT gene linked to the "hairpin"
autocatalytic cassette of the invention so that the "hairpin"
autocatalytic RNA would be expected to serve as a chain terminator
for the CAT gene. Also shown is the expected RNA transcript of the
illustrated region ("CAT-cassette RNA") and the expected 5'
fragment of the autocatalytic cleavage ("Cleaved CAT-cassette
RNA"). Finally, the figure illustrates the location of DNA probes
designed to hybridize with different regions of the CAT-cassette
RNA transcript and Cleaved CAT-cassette 5' fragment.
[0104] FIG. 22B shows the results of Northern blot analysis of RNA
isolated from Escherichia coli host cells transformed with pHC-CAT.
When the CAT probe was used, both the full length transcript and
the expected 5' cleavage fragment were detected, indicating that
cleavage took place in vivo. When the "hairpin" autocatalytic
cassette probe was used, only the full length CAT-cassette RNA
transcript was detected. The fact that the 5' fragment did not
hybridize with this probe was to be expected, since most of the
"hairpin" autocatalytic cassette transcript would be in the 3'
fragment after cleavage. Although, it would be expected that the
"hairpin" autocatalytic cassette probe would hybridize to the 3'
fragment, the fact that the 3' fragment was not detected by
Northern blot analysis is not surprising. The 5' terminus of the 3'
fragment would contain a 5'-OH and not the 5'-ppp which is
ordinarily seen in RNA transcripts. Thus, the 3' fragment would be
expected to be very labile in vivo and was likely degraded
immediately after the autocatalytic cleavage.
[0105] FIG. 23. Shown is the map of the vector pMHC-CAT which was
constructed by excising the "hairpin" autocatalytic cassette from
pHC (prepared as described in Example 23) with SmaI/SalI and
ligating it to vector pMSG at the SmaI/XhoI sites to give the
vector pMHC. Then, the CAT gene was excised from pMAM-NEO-CAT with
SmaI/XhoI and was ligated into pMHC at the SmaI/XhoI sites to give
pMHC-CAT as shown.
[0106] FIG. 24. Shown is the map of the vector pHC19R which was
prepared by excising the "hairpin" autocatalytic cassette from pHC
with BamHI/SalI and ligating it to the SalI/BamHI sites of pTZ19R
to give pHC19R as shown.
[0107] FIG. 25. Shown are the results of an S1 nuclease protection
assay performed on RNA isolated from CHO cells that had been
transfected with pMHC-CAT. In the gel shown in FIG. 25, the lanes
contain: Lane 1---probe (148 nt); Lane 2---S1 nuclease digested
probe; Lane 3---in vitro transcribed "hairpin" autocatalytic
cassette that had been hybridized to probe and S1 nuclease digested
(uncleaved 134 nt, 3'F 87 nt, 5'F 47 nt); Lane 4---RNA isolated
from uninduced, pMHC-CAT-transfected CHO cells, hybridized to probe
and S1 nuclease digested; and Lane 5---RNA isolated from
dexamethasone induced, pMHC-CAT-transfected CHO cells, hybridized
to probe and S1 nuclease digested (uncleaved 111 nt, 3'F 69 nt, 5'F
42 nt). All mobilities were as expected.
[0108] FIG. 26. Shown are catalytic RNA/viral vector constructions
used to infect plants. In these constructions, the "hairpin"
autocatalytic cassette from the vector pHC was ligated to the
cauliflower mosaic virus (CMV) in vector pCS101 to give the two
engineered CMV viral constructs shown. The "hairpin" autocatalytic
cassette is in the sense orientation in pCS101HC7 and in the
antisense orientation in pCS101HC9.
[0109] FIG. 27A: Shown are the results of an S1 nuclease protection
assay performed on RNA isolated from turnip plants that had been
infected with pCS101HC7 or pCS101HC9. Lane 1 is undigested
"hairpin" autocatalytic RNA probe (148 nt) and Lane 2 is RNA
transcribed from HindIII-digested plasmid pHC which gave three RNA
products--uncleaved "hairpin" autocatalytic cassette (158 nt), 3'F
(87 nt) and 5'F (71 nt). The remaining lanes are all S1 nuclease
digests of probe hybridized to the following RNA preparations: Lane
3--RNA from uninfected plants; Lane 413 RNA from plants infected by
the virus control (pCS101); Lane 5--RNA from plants infected with
pCS101HC7 (uncleaved 112 nt, 3'F 70 nt and 5'F 42 nt); Lane 6--RNA
from plants infected with pCS101HC9.
[0110] FIG. 27B: Results of Northern blot analysis of the RNA
isolated from plants that were mock infected, infected with pCS101
(wild-type CMV) or infected with pCS101HC7. The probe was labelled
"hairpin" autocatalytic RNA. Lane 1, RNA from mock-infected plants;
Lane 2, RNA from plants infected with pCS101 (wild-type CMV); and
Lane 3, RNA from plants infected with pCS101HC7.
[0111] FIG. 27C: Total DNA from plants that were mock infected,
infected with pCS101 (wild-type CMV) or infected with pCS101HC7 was
amplified by the polymerase chain reaction (PCR). Lane M, molecular
weight markers; Lane 1, PCR-amplified DNA from mock-infected
plants; Lane 2, PCR-amplified DNA from plants infected with pCS101
(wild-type CMV); Lane 3, PCR-amplified DNA from plants infected
with pCS101HC7; Lane 4, PCR-amplified DNA from the pCS101 plasmid;
and Lane 5 PCR-amplified DNA from pCS101HC7 plasmid.
[0112] FIGS. 27D and 27E: Results of Western immunoblot analysis of
protein extracts of turnips leaves from plants that were mock
infected, infected with pCS101 (wild-type CMV) or infected with
pCS101HC7. FIG. 27D shows the results for plants one month after
inoculation, and FIG. 27E shows the results for plants two months
after inoculation. The lanes are the same in both figures. Lane 1,
protein from mock-infected plants; Lane 2, protein from plants
infected with pCS101 (wild-type CMV); and Lane 3, protein from
plants infected with pCS101HC7. CP=coat protein, (M)=molecular
weight markers.
[0113] FIG. 28: HIV-1 target sequence. The 16-base target sequence
is found in the 5'-leader region of all 9 HIV-1 mRNAs. The
nucleotide numbering starts at the first base transcribed in the
HXB2 clone (HIV Sequence Data Base, prepared and distributed by
Gerald Myers et al., Los Alamos National Laboratory, Los Alamos,
N.Mex., telephone (505) 665-0480).
[0114] FIG. 29: Conservation of the HIV-1 target sequence in
various HIV isolates. All sequences are from the HIV Sequence Data
Base and are listed 5'-->3'. All homologies are +107 to capsite
(+1), except MAL which is +105. The capsite sequence searched for
was GGT CTC TCT. Only two isolates (MAL and MN) had variations in
the target sequence, and the variations were, in each case, a
single base change (G-->A). In the figure, cg indicates that the
complete genome was contained in the sequence file, and "----"
indicates proviral DNA without any homology which contained
sequence information that started too late or ended too soon. It is
likely that a homologous sequence does indeed occur in these
strains.
[0115] FIG. 30: Sequence of HIV-1 substrate ("SHIV") having the
target sequence of FIG. 28 plus additional GCG vector bases at its
5' end. Also shown is the sequence of engineered "hairpin"
catalytic RNA ("RHIV") designed to cleave this substrate. The
catalytic RNA also has additional 5' vector bases 3'-CUGAGGG-5' as
shown.
[0116] FIG. 31: Time course of cleavage of the substrate RNA SHIV
by RHIV (both depicted in FIG. 30). Shown are the catalytic RNA
RHIV (R), substrate RNA SHIV (S), 3' cleavage fragment (3'F), and
5' cleavage fragment (5'F). Since 35% of the substrate was
uncleavable, the remaining 65% was normalized to 100% on the
ordinate of the graph.
[0117] FIG. 32: Kinetics of the cleavage of the substrate RNA SHIV
by RHIV (both depicted in FIG. 30). Shown are the catalytic RNA
RHIV (R), substrate RNA SHIV (S), 3' cleavage fragment (3'F), and
5' cleavage fragment (5'F). The incubation time was 5 minutes, and
the concentration of RHIV was 0.005 uM. The concentration of SHIV
was: Lane 1--0.10 uM; Lane 2--0.05 uM; Lane 3--0.025 uM; Lane
4--0.012 uM; Lane 5--0.006 uM; and Lane 6--0.025 uM (this is
control lane at zero time). From the graph, the Km was found to be
100 nM, and the kcat to be 1.6/min. From the time course shown in
FIG. 31, it was determined that 35% of the substrate was
uncleavable. This was subtracted from these calculations.
[0118] FIG. 33: Sequence of longer HIV-1 transcript and results of
its cleavage by RHIV. Shown on the gel are the uncleaved transcript
of 183 nt and the two cleavage products (5'F of 111 nt and 3'F of
72 nt). The control lane at 0 minutes showed no cleavage. The gel
was calibrated with standards, and all mobilities were as
expected.
[0119] FIG. 34: Map of pHR and partial sequence.
[0120] FIG. 35: Results of cleavage of the substrate SHIV by the
101 nt catalytic RNA (designated as "PRHIV") produced by T7 RNA
polymerase transcription of pHR. Shown on the gel are RHIV (R),
PRHIV (PR), SHIV (S), 3' cleavage fragment (3'F), and 5' cleavage
fragment (5'F). Times of incubation were 0 and 15 minutes.
[0121] FIG. 36: Map of pMSGRHIV and partial sequence.
[0122] FIG. 37: Map of plasmid pMRHPT. This mammalian expression
vector contains DNA encoding an engineered "hairpin" catalytic RNA
("RHGPT") under control of the dexamethasone-inducible MMTV
promoter and terminated by the "hairpin" autocatalytic
cassette.
[0123] FIG. 38: Selection and testing scheme for RHGPT.
[0124] FIG. 39: Results of S1 nuclease assay for reduction of HGPRT
mRNA in CHO cells transfected with pMRHPT and pMSG-dhfr. Lane 1
contains the P.sup.32-labelled 148 nt probe which hybridizes to
HGPRT mRNA; Lane 2 contains P.sup.32-labelled "hairpin"
autocatalytic RNA used as a standard (seen only on longer
exposures); Lanes 3 and 4 contain S1-nuclease digested RNA isolated
from cells transformed with pMRHPT and pMSG-dhfr and induced (Lane
4) or not induced (Lane 3) with dexamethasone. The arrow shows the
location of the probe-protected RNA which corresponds to HGPRT
mRNA.
[0125] FIG. 40: Map of the plasmid pMCATRCAT. This plasmid contains
DNA encoding a "hairpin" RNA catalyst engineered to cleave CAT mRNA
operatively linked to the "hairpin" autocatalytic cassette, all
driven by the dexamethasone-inducible MMTV promoter. The CAT gene
is on the same plasmid and is driven by the SV40 promoter.
[0126] FIG. 41: Results of S1 nuclease assay for reduction of CAT
mRNA in CHO cells transfected with pMCATRCAT and pMSG-dhfr. Lane 1
contains the P.sup.32-labelled 119 nt probe which hybridizes to CAT
mRNA; Lane 2 contains RNA from untransfected CHO cells; Lane 3
contains mRNA transcribed from the CAT gene on vector pHC-CAT in
vitro; Lanes 4 and 5 contain S1-nuclease digested RNA isolated from
cells transfected with pMCATRCAT and pMSG-dhfr and induced (Lane 5)
or not induced (Lane 4) with dexamethasone.
[0127] FIGS. 42A-C: Summary of mutagenesis experiments with the
(-)sTRSV "hairpin" substrate-catalyst complex. Each base or
combination of bases enclosed in a circle represents a separate
mutational experiment.
[0128] FIG. 42D: A more refined secondary structure model for the
(-)sTRSV "hairpin" substrate-catalyst complex.
[0129] FIG. 43: Separation patterns on an acrylamide gel of the
reaction products obtained by reacting substrate with a "hairpin"
catalytic RNA having the loop that closes the "hairpin" replaced by
the hairpin sequence GGAC(UUCG)GUCC.
DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
[0130] An RNA catalyst has been identified comprising an RNA
sequence which can be engineered to cut, with great precision,
target RNAs having a cleavage sequence. In particular, the
invention comprises certain synthetic RNA catalysts capable of
cleaving an RNA substrate which contains the target sequence
5'-F.sub.1-CS-F.sub.2-3'.
[0131] "Synthetic RNA catalyst," as used herein, means a catalyst
which is not a naturally-occurring RNA catalyst, although
"synthetic catalysts" may be truncated or altered versions of
naturally-occurring catalysts. "Synthetic catalysts" are catalysts
designed according to the principles set forth herein based on the
"hairpin" model to bind to and cleave a selected target sequence in
a selected RNA substrate. "Synthetic catalysts" are also sometimes
referred to herein as "engineered catalysts." "Synthetic catalysts"
include catalysts synthesized in vitro and catalysts synthesized in
vivo. In particular, "synthetic catalysts" include catalysts
produced by hosts transformed by a vector comprising a sequence
coding for the catalyst.
[0132] RNA of any length and type may be used as the substrate as
long as it contains the 5'-F.sub.1-CS-F.sub.2-3' target sequence.
In this formula, CS is the cleavage sequence (i.e., a sequence of
bases containing the site at which the catalyst cleaves the
substrate). CS is a short sequence of bases which does not base
pair with the RNA catalyst, and CS preferably has the sequence
5'-NGUC-3', wherein N is any base, and the substrate is cleaved by
the catalyst between N and G to produce a fragment having an OH at
the 5' end and a fragment having a 2,'3' cyclic phosphate at the 3'
end.
[0133] CS is flanked by two short base sequences F.sub.1 and
F.sub.2 which do base pair with the RNA catalyst. F.sub.1 is
preferably at least 3 bases in length, most preferably 4 bases in
length. F.sub.2 is also preferably at least 3 bases in length, most
preferably 6 to 12 bases in length.
[0134] Catalysts according to the invention comprise a substrate
binding portion and a "hairpin" portion. The substrate binding
portion of the catalyst has the sequence:
3'F.sub.4-L.sub.1-F.sub.3-5'
[0135] wherein,
[0136] F.sub.3 is a sequence of bases selected so that F.sub.3 is
substantially base paired with F.sub.2 when the catalyst is bound
to the substrate;
[0137] F.sub.4 is a sequence of bases selected so that F.sub.4 is
substantially base paired with F.sub.1 when the catalyst is bound
to the substrate;
[0138] the sequences of F.sub.3 and F.sub.4 being selected so that
each contains an adequate number of bases to achieve sufficient
binding of the RNA substrate to the RNA catalyst so that cleavage
of the substrate can take place; and
[0139] L.sub.1 is a sequence of bases selected so that L.sub.1 does
not base pair with CS when the catalyst is bound to the
substrate.
[0140] As used herein, "substantially base paired" means that
greater than 65% of the bases of the two RNA sequences in question
are base paired, and preferably greater than 75% of the bases are
base paired. "Substantially unpaired" means that greater than 65%
of the bases of the two sequences in question are not base paired,
and preferably greater than 75% of the bases are not paired.
[0141] F.sub.3 is preferably at least 3 bases in length, most
preferably from 6 to 12 bases in length. F.sub.4 is preferably from
3 to 5 bases in length, most preferably 4 bases in length.
[0142] L.sub.1 is a short sequence of bases which preferably has
the sequence 5'-AGAA-3' when CS has the sequence 5'-NGUC-3'.
Further, when L.sub.1 is 5'-AGAA-3' and CS is 5'-NGUC-3', then the
first base pair between F.sub.1 and F.sub.4 adjacent to CS and
L.sub.1 is preferably G:C or C:G (see FIG. 42D and Example 32).
Accordingly, a preferred target sequence in a selected substrate
contains the sequence 5'-SNGUC-3', wherein S is G or C.
[0143] The "hairpin" portion is a portion of the catalyst which
folds into a hairpin-like configuration when the substrate-catalyst
complex is modeled in two dimensions for minimum energy folding.
This is shown in FIGS. 1 and 42D for (-)sTRSV RNA. The "hairpin"
portion is not an absolute hairpin in the sense that not all bases
of the "hairpin" portion are base-paired. Indeed, it is preferable,
perhaps necessary, for the "hairpin" portion to have at least one
substantially unpaired region so that the catalyst can assume a
tertiary structure that allows for better, or optimal, catalytic
activity.
[0144] The "hairpin" portion of the catalyst preferably has the
sequence: 2
[0145] wherein,
[0146] P.sub.1 and P.sub.4 each is a sequence of bases, the
sequences of P.sub.1 and P.sub.4 being selected so that P.sub.1 and
P.sub.4 are substantially base paired;
[0147] P.sub.1 is covalently attached to F.sub.4;
[0148] S.sub.1 and S.sub.2 each is a sequence of bases, the
sequences of S.sub.1 and S.sub.2 being selected so that S.sub.1 and
S.sub.2 are substantially unpaired;
[0149] P.sub.2 and P.sub.3 each is a sequence of bases, the
sequences of P.sub.2 and P.sub.3 being selected so that P.sub.2 and
P.sub.3 are substantially base paired; and
[0150] L.sub.2 is a sequence of unpaired bases.
[0151] "Substantially base paired" and "substantially unpaired"
have the same meanings as discussed above.
[0152] P.sub.1 and P.sub.4 each is preferably from 3 to 6 bases in
length, and most preferably P.sub.1 has the sequence 5'-ACCAG-3'
and P.sub.4 has the sequence 5'-CUGGUA-3'. It has been found that
the A at the 5' end of 5'-ACCAG-3' (underlined) is not base paired
to the U at the 3' end of 5'-CUGGUA-3' (underlined), and the
unpaired A may act as a "hinge" (see Example 32). It is not known
yet, but the presence of such a "hinge" may be necessary for
catalytic activity.
[0153] S.sub.1 and S.sub.2 each preferably is from 4 to 9 bases in
length, and most preferably S.sub.1 has the sequence 5'-AGAAACA-3'
and S.sub.2 has the sequence 5'-GUAUAUUAC-3'.
[0154] P.sub.2 and P.sub.3 each is preferably from 3 to 9 bases in
length, and more preferably P.sub.2 has the sequence 5'-CAC-3' and
P.sub.3 has the sequence 5'-GUG-3'.
[0155] Finally, L.sub.2 is preferably at least 3 bases in length
and preferably has the sequence 5'-GUU-3'. Further,
5'-S.sub.1-P.sub.2-L.sub.- 2-3' preferably has the sequence
5'-AGAAACACACGUU-3'.
[0156] The specific preferred sequences set forth above for
P.sub.1, P.sub.2, S.sub.1, etc., are from the catalytic sequence of
(-)sTRSV RNA.
[0157] A preferred catalyst according to the invention contains the
sequence:
1 5'-F.sub.3-L.sub.1-F.sub.4-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGU
A-3',
[0158] and active variants thereof, wherein F.sub.3, F.sub.4 and
L.sub.1 are as defined above. As used herein "active variants"
means catalysts which, although having substitutions, deletions
and/or additions of bases as compared to the original sequence, are
still capable of cleaving an RNA substrate.
[0159] The most preferred sequence for
5'-P.sub.2-L.sub.2-P.sub.3-3' is 5'-CACGGACUUCGGUCCGUG-3' [SEQ ID
46] (see Example 32). Accordingly, the most preferred catalyst has
the sequence:
2 5'-F.sub.3-L.sub.1-F.sub.4-ACCAGAGAAACACACGGACUUCGGUCC [SEQ ID
47] GUG-GUAUAUUACCUGGUA-3'
[0160] wherein F.sub.3, F.sub.4 and L.sub.1 are as defined above
and the underlined portion is the preferred sequence for
P.sub.2-L.sub.2-P.sub.3.
[0161] Another preferred catalyst according to the invention is an
autocatalytic catalyst containing the sequence: 3
[0162] wherein, F.sub.1, F.sub.2, F.sub.3, F.sub.4, L.sub.1,
L.sub.2, S.sub.1, S.sub.2, P.sub.1, P.sub.2, P.sub.3 and P.sub.4
are as defined above. L.sub.3 is a sequence of unpaired bases, and
L.sub.3 preferably has the sequence 3'-CCUCC-5'. Thus, the molecule
contains a substrate portion (5'-F.sub.1-CS-F.sub.2-3') and a
catalytic portion
(5'-F.sub.3-L.sub.1-F.sub.4-P.sub.1-S.sub.1-P.sub.2-L.sub.2-P.sub.3-S.sub-
.2-P.sub.4-3') covalently linked together by L.sub.3 so as to
produce a synthetic autocatalytic RNA catalyst.
[0163] After being transcribed, this catalyst will spontaneously
undergo an intramolecular autocatalytic cleavage at CS. The effect
of this intramolecular cleavage is to autocatalytically terminate
any RNA transcript in which the sequence is inserted. For instance,
an engineered DNA molecule may be prepared which comprises a gene
of interest and a DNA sequence coding for the catalyst linked so
that, when transcribed, the catalyst will terminate transcription
of the messenger RNA coding for the gene product at a desired
location.
[0164] The invention further provides an engineered DNA molecule
and a vector comprising a DNA sequence coding for an RNA catalyst
of the invention. Also provided are host cells which have been
transformed with the vectors and which are capable of expressing
the RNA catalyst. Finally, the invention provides a method of
cleaving an RNA substrate which contains the sequence
5'-F.sub.1-CS-F.sub.2-3', the method comprising contacting the
substrate with a synthetic RNA catalyst according to the
invention.
[0165] The invention is further described below with particular
reference to the catalytic properties of (-)sTRSV RNA and the
structure of its catalytic complex, but the invention is not
limited to synthetic catalysts based on (-)sTSV RNA. In addition to
the particular catalytic sequences shown and described below, other
RNA molecules having catalytic activity to cleave an RNA substrate
can be readily found by applying the principles set forth in this
specification.
[0166] For example, RNA sequences having the required structural
features for cleaving an RNA substrate can be identified by
applying the Wisconsin RNA Folding Program discussed above to (1)
known sequences of molecules having catalytic or autocatalytic
activity (especially molecules in which the actual location of the
catalytic site is unknown), (2) randomly generated sequences having
the proper pairing regions and lengths, and (3) randomly modified
known catalytic sequences, while looking for known features of a
catalytic molecule, such as the "hairpin" configuration of the
catalytic complex when modeled in two dimensions. As a specific
example, information regarding known autocatalytic cleavage sites
can be used to find substrate binding sequences having the
properties described above, such as having a substrate binding
sequence adjacent a "hairpin" portion. Secondary features, such as
the two substantially paired regions in the "hairpin" portion, with
an intermediate substantially unpaired region and an appropriate
base loop can then be looked for, either by manual examination or
with automated computer programs. In order of decreasing
preference, the following features are considered important in
selecting a catalytic sequence: (1) regions that can base pair
(form helices) with the regions of the substrate RNA molecule
flanking the cleavage sequence; (2) an unpaired (loop) region
opposite the cleavage sequence of the substrate RNA; (3) two
substantially base paired regions in a "hairpin" structure near the
cleavage sequence; (4) a substantially unpaired region between
these two substantially paired regions in the "hairpin" structure;
and (5) a loop connecting the two strands of a substantially base
paired region to complete the "hairpin" structure. Standard
techniques of in vitro RNA synthesis can then be used to prepare
actual molecules having the sequence that gives the predicted
two-dimensional computer-generated structure for verification of
activity and routine testing of variation to determine optimum
sequence.
[0167] The catalysts of the present invention were developed using
a "hairpin" model or motif of RNA catalysis. According to this
model, the catalytic complex, when modeled in two dimensions for
minimum energy folding, assumes a "hairpin" configuration. This is
shown in FIGS. 1 and 42D for (-)sTRSV RNA. The catalytic complex is
a complex of the minimum, or substantially the minimum, sequence of
the catalyst necessary for activity and the minimum, or
substantially the minimum, target sequence of the substrate. The
"hairpin" configuration is not an absolute hairpin in the sense
that not all the bases that make up this "hairpin" configuration
are base-paired. Indeed, there are preferably regions of unpaired
bases and of substantially unpaired bases as discussed in detail
elsewhere in the present application.
[0168] The "hairpin" model has proved very useful in designing new
catalysts, but it is still only a computer model of the likely
secondary structure of catalytic complexes involving catalysts
according to the present invention. Also, it is ultimately the
tertiary structure of RNA catalysts that determines their activity.
For these reasons, all catalysts having the properties described
herein are considered to come within the scope of the present
invention, even if they do not form a "hairpin" configuration when
complexed with the substrate and even if they do not contain a
"hairpin" portion. For instance, it may be possible to engineer a
catalyst having the properties described herein which does not have
a loop L.sub.2. Such a catalyst would be considered to be fully
equivalent to the catalysts described and claimed herein.
[0169] As described in Example 1, a catalytic complex was
identified within the (-) strand of tobacco ringspot virus
satellite RNA (sTRSV) when the molecule was folded using computer
models to determine the minimum energy folding in two-dimensional
space. The (-) strand is a 359 base long RNA of defined sequence
and is known to have autocatalytic properties (Gerlach, W. L.,
Buzayan, J. W., Schneider, I. R. and Bruening, G. B. (1986)
Virology, 151: 172-185; Buzayan, J. M., Gerlach, W. and Bruening,
G. (1986) Nature, 323: 349-352). The (-) strand cleaves at a
defined site (ApG) into a cleavage product having an OH at the 5'
end and a 2',3' cyclic phosphate at the 3' end. Up until the
present time, however, little work had been done with the (-)
strand to find the catalytic complex and to determine the minimum
cleavage sequences because it does not fit the consensus
"hammerhead" model.
[0170] In view of the above and the fact that the catalytic center
would contain both the catalytic RNA sequence and the substrate
(target) RNA sequence and by studying the results of Example 1, a
50 nucleotide sequence between bases 175 and 224 was picked and a
14 nucleotide sequence between bases 40 and 53 was picked. Using
methodologies found in published procedures, a catalytic RNA having
a satellite RNA base sequence identical to the base sequence in
naturally-occurring (-)sTRSV between bases 175 and 224 was
transcribed from chemically synthesized DNA templates using T7 RNA
polymerase as described in Example 2. An RNA substrate having a
satellite RNA base sequence identical to the base sequence in
naturally occurring (-)sTRSV RNA between bases 40 and 53 was also
prepared in the same manner. When the newly synthesized RNAs were
mixed together under appropriate conditions as described in Example
3, the catalytic RNA cleaved the substrate RNA. As described in
Example 4, the first RNA catalyst fitting the "hairpin" motif was
discovered when the complex of the 50-base catalytic RNA and the
14-substrate RNA was modeled in two-dimensional space using
computer modeling.
[0171] The reaction of catalysts fitting the "hairpin" motif with
an appropriate substrate proved to be an excellent catalytic
reaction under physiological conditions. The reaction of the
catalyst and substrate containing the sequences of (-)sTRSV shown
in FIG. 1 gave a Km of 0.03 uM (see Example 5), which is 20 times
smaller than that of the Km for a catalyst fitting the "hammerhead"
model (Uhlenbeck, O. C. (1987) Nature 328: 596600) and accounts for
its ability to remove target RNA molecules to much lower levels (20
times lower) than that of catalysts fitting the "hammerhead" model.
In addition, the kcat for the reaction is 2.1/min at 37.degree. C.,
which is at least 6 times greater than that of a catalyst having
the "hammerhead" configuration at the same temperature (see Example
5). These reaction parameters for a catalyst that fits the
"hairpin" model can be optimized by adjusting the amount of base
pairing between the substrate and catalyst (see Example 18, 21 and
32)
[0172] Catalytic cleavage of the substrate RNA occurs over a broad
pH range, preferably 5.5 to 8.0, and in the presence of divalent
ions such as Mg++, e.g. from MgCl.sub.2. As would be expected for a
base catalyzed reaction, the rate of reaction increased with
increasing pH. The reaction rate also increased with increasing
concentration of divalent cations as shown in Example 8.
[0173] The reaction takes place at physiological temperatures,
preferably 16.degree. C. to 45.degree. C., with a temperature
optimum at 37.degree. C. as described in Example 6. Temperatures
above about 45.degree. C. inactivate the reaction. However, the
temperature optimum of the reaction is affected by the degree of
base pairing between the substrate and catalyst (see Example 18).
In particular, the length of the region of the catalyst that base
pairs with the 3' region of the substrate flanking the cleavage
sequence can be varied so that an engineered catalyst reacting at a
desired temperature can be obtained (see Example 18). Further, a
"hairpin" catalyst which is more thermal stable than the native
(-)sTRSV catalyst can be prepared by deleting the loop that closes
the "hairpin" (Loop III in FIG. 42D) and inserting therefor the
stable hairpin sequence 5-GGAC(UUCG)GUCC-3' [SEQ ID 45] (see
Example 32).
[0174] The 50 base catalytic RNA configured in the "hairpin" model
in FIGS. 1 and 42D is the minimal sequence, or substantially the
minimal sequence, of (-)sTRSV RNA necessary to achieve catalysis.
When the 3' terminal A (base 175) or AU (bases 175 and 176) of this
sequence was removed, catalytic activity was substantially
decreased as shown in Example 10. The 5' end of the molecule cannot
be substantially changed either without affecting catalytic
activity because it is needed to provide base pairing with the
substrate. It can be shortened by at most about 3 bases (see
Example 9). Experiments which removed bases 195-203 in the center
of the catalytic RNA and ligated base 194 to base 204 produced an
inactive catalytic RNA as described in Example 11. This shows that
all or part of the region between bases 195-203 is essential for
catalytic activity. An additional mutagenesis experiment to test
the base requirement of the three A bases at positions 203, 202 and
201 was done. When these bases were changed to CGU, respectively,
as described in Example 12, the resulting catalytic RNA was
inactive.
[0175] Base changes can be made in the two base paired regions
(Helices 3 and 4 in FIG. 42D) of the "hairpin" portion of the
(-)sTRSV catalytic RNA, as long as substantial base pairing is
maintained. This is shown in Examples 22 and 32 where base changes
that destroyed base pairing in these two regions resulted in
inactive catalysts. When a second base change was made which
restored base pairing, the catalytic activity was also restored
(see FIGS. 19 and 42A-C). The one exception appears to be the first
C:G base pair at the end of Helix 3 nearest the substrate binding
portion (base pair C16:G48 in FIG. 42D). Currently available
evidence indicates that the identity of these two bases must be
maintained (see Example 32).
[0176] An active ribozyme is produced when Helix 4 is extended and
the sequence of the loop that closes the "hairpin" (Loop III in
FIG. 42D) is changed. As shown in FIG. 42A, Loop III was replaced
with the common and very stable RNA hairpin sequence
5'-GGAC(UUCG)GUCC-3'. As a result of this substitution, Helix 4 was
extended by four base pairs and the GUU sequence of Loop III was
replaced with the sequence UUCG (see FIG. 42A). The resulting RNA
catalyst was more active and, as noted above, more thermally stable
than the unmutated form (see Example 32). It was concluded from
this experiment that Loop III does not have a conserved or unique
base sequence and that Helix 4 can be extended by at least four
base pairs without loss of activity.
[0177] However, the simple replacement of the GUU sequence of Loop
III with the sequence UUCG gives an inactive ribozyme (see Example
32). It is believed that this shows that the sequence of Loop III
has an influence on the stability of Helix 4.
[0178] When Loop III is cut between U31 and U32, activity is lost
(see FIG. 42C). A likely explanation for this is that when Loop III
is cut, Helix 4 opens up and catalytic activity is, consequently,
lost.
[0179] Base changes can also be made in the two regions of the
catalytic RNA that base pair with the substrate, as long as
substantial base pairing with the substrate in the regions flanking
the cleavage sequence is maintained and base pairing with the
cleavage sequence is avoided as shown in Examples 9, 16-21 and 32.
Indeed, every base pair in Helices 1 and 2 (see FIG. 42D) can be
changed to any other base pair, except the base pair in Helix 2
adjacent to the NGUC cleavage sequence (see Example 32). This base
pair must be G:C or C:G and cannot be A:U or U:A, except it is
believed that this base pair is needed for stability and that A:U
or U:A base pairs can be used if other measures are taken to
stabilize the substrate-catalyst interaction. Indeed, it has been
found that A:U and U:A base pairs can be used at these positions in
the synthetic autocatalytic catalyst of the invention.
[0180] It is the ability to change the base pairs in Helices 1 and
2 that allows the RNA catalyst to be engineered to cut a specific
target RNA substrate having a cleavage sequence such as NGUC. This
is illustrated in Example 16 where the catalytic RNA was engineered
by changing base 214 from a G to a C resulting in a catalytic RNA
which failed to react with the substrate RNA developed from natural
(-)sTRSV RNA. Activity was restored, however, when the substrate
RNA was changed so that it could base pair with the subject
engineered catalytic RNA. Also see Examples 9, 17-21 and 32.
[0181] A type of "hinge" region, consisting of a single base, seems
to be present between Helices 2 and 3 of the (-)sTRSV catalytic RNA
(see FIG. 42D and Example 32). The A at position 15 is not paired
to the U at position 49, and the unpaired A may act as a
"hinge."
[0182] Further mutation studies have showed that the substantially
unpaired regions between Helices 3 and 4 (Loops II and IV in FIG.
42D) are larger than originally predicted by computer modeling and
energy minimization (compare FIGS. 1 and 42D). Further, some of the
bases in these loops appear to be required for activity (see
Example 32). In particular, changing bases C25 and A43 results in a
loss of activity (see FIG. 42C and Example 32; also see Example
12).
[0183] The (-)sTRSV catalytic RNA sequence has an 5'-AGAA-3'
sequence opposite the AGUC cleavage sequence of the substrate. As
shown in Examples 24 and 32, at least part of this AGAA sequence is
invariant. In particular, when the A's at the 3' and 5' ends of the
sequence (AGAA) were changed, the resulting catalysts were active
(see FIG. 42A). However, when G or A (AGAA) in the center of the
sequence was changed, the resulting catalysts were inactive (see
FIGS. 21 and 42C).
[0184] The target RNA substrate of the "hairpin" catalytic complex
shown in FIG. 1 has an AGUC cleavage loop which does not base pair
to the catalytic RNA in two-dimensional space. As shown in Examples
13-15, "A" in the AGUC cleavage sequence can be changed to any
other base without effecting the ability of the RNA catalyst to
cleave the substrate. Example 25 shows that the GUC sequence is
conserved. Thus, the only sequence requirement for the cleavage
sequence of the substrate RNA is GUC.
[0185] Although there is no base pairing between the NGUC cleavage
sequence and the AAGA sequence in the catalyst opposite from the
cleavage sequence, the C in NGUC apparently interacts with the A in
AAGA opposite to it. In particular, it has been found that the
single mutation of C-->A in the cleavage sequence NGUC (position
9 in the substrate sequence--see FIG. 42B) destroyed the ability of
the RNA catalyst to cleave the substrate. When a second mutation
was made in the ribozyme, changing the A at position 7 to a C,
partial activity was restored (see Example 32), indicating some
sort of interaction between C at position 9 in the substrate and
the A at position 7 in the catalyst (see FIG. 42B).
[0186] The cleavage sequence has four flanking bases at its 5' end
and six at its 3' end which base pair with the catalytic RNA. As
described above, the bases in the flanking regions can be changed
(with the possible exception of the G:C base pair in Helix 2
adjacent to NGUC) without affecting the ability of the catalytic
RNA to cleave the substrate, as long as sufficient base pairing
with the catalyst is maintained in the flanking regions. This would
be expected to work on RNA substrate sequences of any length as
long as these criteria are met. Indeed, lengthening the 3' region
of the substrate that base pairs with the catalyst has been found
to provide a more efficient catalytic reaction. See Examples 18, 21
and 32. However, a smaller 10 base substrate having three flanking
bases at its 5' end and three flanking bases at its 3' end did not
work as well as the 14-base substrate, as described in Example
9.
[0187] Using the "hairpin" model as a guide, RNA catalysts can be
engineered that base pair with an RNA substrate and mediate a
cleavage in the cleavage sequence. In particular, catalytic RNA can
be engineered that will cleave any RNA substrate having a cleavage
sequence, such as NGUC, and flanking regions with which the
catalyst base pairs, so that the catalytic RNA and RNA substrate
form a catalytic complex in a "hairpin" motif. To do this, the
bases flanking the cleavage sequence must be identified and the
catalytic RNA engineered so that it does not pair in
two-dimensional space with the cleavage sequence but does pair with
adequate numbers of flanking bases upstream and downstream of the
cleavage sequence. When designing a synthetic catalyst based on the
(-)sTRSV catalyst, the other principles set forth herein regarding
conserved or preferred sequences should also be taken into account
in designing the synthetic catalyst.
[0188] As shown in Examples 17-20 and 29, catalytic RNAs according
to the invention can cleave specific viral and messenger RNA
sequences. In Example 15, tobacco mosaic virus (TMV) RNA containing
the 5' coding region of the replicase gene was targeted for
specific cleavage by an appropriately engineered catalytic RNA. The
target sequence contained changes in 8 of the 14 bases of the
substrate RNA having a base sequence found within the catalytic
complex of (-)sTRSV RNA and was cleaved by the engineered RNA
catalyst under conditions near physiological. Catalytic RNAs were
also designed and synthesized using the "hairpin" model as a guide
which could cleave sequences from messenger RNA coding for
chloramphenicol acetyl transferase (Example 18) and from HIV-1
viral RNA (Examples 19, 20 and 29). In particular, a conserved
sequence in HIV-1 viral messenger RNAs has been identified, and a
"hairpin" catalytic RNA designed which cleaves this sequence (see
Example 29).
[0189] These examples demonstrate that the system can be used to
specifically cleave an RNA sequence for which an appropriately
engineered catalytic RNA base pairs at the designated flanking
regions. Suitable target RNA substrates include viral, messenger,
transfer, ribosomal, nuclear, organellar, other cellular RNA, or
any other natural RNA having a cleavage sequence, as well as RNAs
which have been engineered to contain an appropriate cleavage
sequence.
[0190] Catalysts that fit the "hairpin" catalytic RNA model are
useful in vivo in prokaryotes or eukaryotes of plant or animal
origin for controlling viral infections or for regulating the
expression of specific genes. In this case, a cleavage sequence
such as NGUC in the virus or complementary to NGUC in the gene
would need to be identified along with the flanking sequences
immediately upstream and downstream of the cleavage sequence.
Normally three to four bases on the 5' side of the cleavage
sequence and enough bases in the order of 6 to 12 on the 3' side to
provide adequate binding of the catalytic RNA and to provide
reasonable certainty that the target RNA sequence is unique in the
organism are required.
[0191] A catalytic RNA is then engineered which does not base pair
with the cleavage sequence and which does base pair to the flanking
regions on the 5' and 3' side of the cleavage sequence. A DNA
template corresponding to this catalytic RNA is then synthesized
using procedures that are well-known in the art. Such procedures
include the phosphoramidite method (see, e.g., Beaucage and
Caruthers, Tetrahedron Letters, 22, 1859 (1981); Matteucci and
Caruthers, Tetrahedron Letters, 21, 719 (1980); and Matteucci and
Caruthers, J. Amer. Chem. Soc., 103, 3185 (1981)) and the
phosphotriester approach (see, e.g., Ito et al., Nucleic Acids
Res., 10, 1755-69 (1982)).
[0192] The invention also includes an engineered DNA molecule and a
vector comprising a DNA sequence coding for the desired synthetic
RNA catalyst. The vector will have the DNA sequence coding for the
desired catalytic RNA operatively linked to appropriate expression
control sequences. Methods of effecting this operative linking,
either before or after the DNA coding for the catalyst is inserted
into the vector, are well known. Expression control sequences
include promoters, activators, enhancers, operators, stop signals,
cap signals, polyadenylation signals, and other signals involved
with the control of transcription.
[0193] The vector must contain a promoter and a transcription
termination signal, both operatively linked to the synthetic DNA
sequence, i.e., the promoter is upstream of the synthetic DNA
sequence and the termination signal is downstream from it. The
promoter may be any DNA sequence that shows transcriptional
activity in the host cell and may be derived from genes encoding
homologous or heterologous proteins and either extracellular or
intracellular proteins, such as amylase, glycoamylases, proteases,
lipases, cellulases, and glycolytic enzymes. Also, a promoter
recognized by T7 RNA polymerase may be used if the host is also
engineered to contain the gene coding for T7 RNA polymerase.
[0194] The promoter may contain upstream or downstream activator
and enhancer sequences. An operator sequence may also be included
downstream of the promoter, if desired.
[0195] Expression control sequences suitable for use in the
invention are well known. They include those of the E. coli lac
system, the E. coli trp system, the TAC system and the TRC system;
the major operator and promoter regions of bacteriophage lambda;
the control region of filamentous single-stranded DNA phages; the
expression control sequences of other bacteria; promoters derived
from genes coding for Saccharomyces cerevisiae TPI, ADH, PGK and
alpha-factor; promoters derived from genes coding for the
Aspergillus oryzae TAKA amylase and A. niger glycoamylase, neutral
alpha-amylase and acid stable alpha-amylase; promoters derived from
genes coding for Rhizomucor miehei aspartic proteinase and lipase;
mouse mammary tumor promoter; SV40 promoter; the actin promoter;
and other sequences known to control the expression of genes of
prokaryotic cells, eukaryotic cells, their viruses, or combinations
thereof.
[0196] The vector must also contain one or more replication systems
which allow it to replicate in the host cells. In particular, when
the host is a yeast, the vector should contain the yeast 2u
replication genes REP1-3 and origin of replication.
[0197] The vector should further include one or more restriction
enzyme sites for inserting the DNA template sequences into the
vector, and preferably contains a DNA sequence coding for a
selectable or identifiable phenotypic trait which is manifested
when the vector is present in the host cell ("a selection
marker").
[0198] Suitable vectors for use in the invention are well known.
They include retroviral vectors, vaccinia vectors, pUC (such as
pUC8 and pUC4K), pBR (such as pBR322 and pBR328), pTZ (such as
pTZ18R), pUR (such as pUR288), phage lambda, YEp (such as YEp24)
plasmids, and derivatives of these vectors.
[0199] The resulting vector having the engineered DNA sequence that
codes for the RNA catalyst is used to transform an appropriate
host. This transformation may be performed using methods well known
in the art.
[0200] Any of a large number of available and wellknown host cells
may be used in the practice of this invention. The selection of a
particular host is dependent upon a number of factors recognized by
the art. These include, for example, compatibility with the chosen
expression vector, toxicity to it of the catalytic RNA encoded for
by the engineered DNA sequence, rate of transformation, expression
characteristics, bio-safety and costs. A balance of these factors
must be struck with the understanding that not all hosts may be
equally effective for the expression of a particular catalytic
RNA.
[0201] Within these general guidelines, useful hosts include
bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.)
and other fungi, insects, plants, animals (including human), or
other hosts known in the art.
[0202] As an example of the general genetic engineering techniques
that are possible, mammalian vectors can be used to deliver DNA
coding for the catalytic RNAs of the invention to animal cells.
These vectors should have a suitable DNA replication signal, such
as from SV40, a promoter which may or may not be inducible, such as
the mouse mammary tumor promoter (which is induced by
dexamethasone) or the noninducible SV40 promoter. A multiple
cloning site is located after the promoter, and the DNA coding for
the engineered catalytic RNA is inserted into this multiple cloning
site using standard techniques. If necessary a suitable terminator
is inserted. The resulting vector is then put into cells using
standard techniques such as direct injection into the nucleus,
electroporation, or other well-known transformation techniques.
Once the vector is in the cell, the catalytic RNA is expressed
directly when noninducible promoters are used, or after addition of
the inducer when inducible promoters are used.
[0203] Similarly, plant vectors, such as the Ti plasmid or micro-Ti
plasmids, can be used to deliver DNA coding for a desired catalytic
RNA to plant cells. The Ti plasmids and micro-Ti plasmids may be
used as such to transform plant protoplasts using known techniques
or may be inserted into Agrobacterium tumefaciens which is then
used to transform plant tissue. Once the plasmid is in the cell,
the catalytic RNA will be expressed.
[0204] Once transformed, a host cell can express (transcribe) the
synthetic RNA catalyst. When the DNA coding for the catalyst is
transcribed, it produces catalytic RNA which will attack and cleave
the target RNA sequence or sequences for which it has been
designed, inactivating the RNA. If the RNA is necessary for the
life cycle of a virus, the virus will be eliminated and if the RNA
is the product of a specific gene, the expression of that gene will
thus be regulated. The catalytic RNA can be designed to work in
prokaryotes and within the nucleus (without poly(A) tail) or in the
cytoplasm of a eukaryotic cell (with polyadenylation signals in
place) for plants and animals.
[0205] Another potential method of using the catalytic RNAs of the
invention is to prepare stable synthetic derivatives of RNA
catalysts designed to bind and cleave a specific target RNA and to
deliver the modified catalysts directly to the cell or organism of
choice. For example, standard methods are available for making
phoshporothioate derivatives of DNA which have been shown to be
very stable in vivo and to be able to bind to a specific DNA or RNA
target in vivo (antisense method). A modification of these
procedures can be used to prepare a catalytically active derivative
of RNA catalysts prepared according to the invention. This would
entail determining which ribonucleotide regions can be altered and
then altering them with deoxy, phophorothio, or other modifications
which confer stability but do not destroy catalytic activity. This
chemically modified catalytic RNA (which may or may not have any
remaining RNA bonds) can then be injected or otherwise delivered to
an organism to control viruses or gene expression. For instance,
one of the catalytic RNAs whose preparation is described in
Examples 19-20 having specificity for sequences found within the
RNA of the HIV-1 virus that causes AIDS could be chemically
modified as described, encapsulated in a liposome coated with
monoclonal antibody directed to the CD4 receptors found on cells
susceptible to HIV-1, and injected into a host animal.
[0206] The "hairpin" catalytic RNA model of the present invention
may also be of possible interest to molecular biologists in
exploring the genetic blueprints of plants and animals. This would
be done by sending randomly constructed DNA reverse transcripts of
catalytic RNA into the DNA of the organism and waiting to see which
gene or genes were inactivated. Other techniques could be applied
to determine where those genes resided on the organism's
chromosomes, thereby greatly accelerating gene mapping.
[0207] Finally, a synthetic autocatalytic RNA catalyst has been
developed. The synthesis of one such catalyst based on the (-)sTRSV
RNA catalytic and substrate sequences is described in Example 23.
When the DNA coding for this catalyst was transcribed in vitro,
autocatalytic cleavage was observed. Further, in Example 26, DNA
coding for this catalyst was linked to the gene coding for
chloramphenicol acetyl transferase (CAT) in vector pHC-CAT. This
vector was then used to transform Escherichia coli. Northern blot
analysis of the RNA produced by the E. coli hosts indicated that
autocatalytic cleavage occurred in vivo under standard E. coli
growth conditions. Similar results were obtained when mammalian and
plant cells were transformed with a vector coding for this
autocatalytic RNA linked to either the gene coding for CAT
(mammalian cells) or to DNA coding for cauliflower mosaic virus
(plants) (see Examples 27 and 28). The autocatalytic RNA catalyst
has also been used to properly terminate other engineered RNA
catalysts after they were transcribed. In this manner, the
engineered RNA catalysts were liberated from the RNA transcript and
acted in trans in vivo (see Example 31).
EXAMPLES
[0208] The following examples further illustrate the invention.
Example 1
[0209] The (-) sense sequence of satellite RNA from the budblight
strain of tobacco ringspot virus as shown in FIG. 2 was folded
using the Wisconsin RNA folding program to identify the location of
a possible catalytic complex accounting for its ability to self
cleave (University of Wisconsin Genetics Computer Group, Program
FOLD May 6, 1986) (Zucker, M. and Stiegler, P. (1981) Nucleic Acids
Res., 9: 133-148; Devereux, J., Haeberli, P. and Smithies, O.
(1984) Nucleic Acids Res., 12: 387-395). Base numbers correspond to
(+)sTRSV RNA (Buzayan, J. M., Gerlach, W. L., Bruening, G. B.,
Keese, P. and Gould, A. R. (1986) Virology, 151: 186-199). With
this numbering scheme the 5'-31 direction of the molecule is with
decreasing base number.
[0210] The minimum catalytic complex, or active site of the
molecule, is identified in FIG. 2. The folding identified regions
of expected base pairing which are in classical double-helical or
stem regions. The folding also identified expected non-base pairing
loops at or near the site of cleavage. This model does not preclude
higher order interactions occurring between non-adjacent portions
of the catalytic center.
Example 2
[0211] A 50 nucleotide sequence between bases 175 and 224 was
picked and a 14 nucleotide sequence between bases 40 and 53 was
picked from the catalytic complex identified in Example 1. A
catalytic RNA (R51) with the 50 base sequence shown in FIG. 1 plus
one additional vector base (G at the 5' end) and a substrate RNA
(S17) with the 14 base sequence shown in FIG. 1 plus three vector
bases (GCG at the 5' end) were transcribed using T7 RNA polymerase
from synthetic DNA templates double stranded at the promoter site
(Milligan, J. F., Groebe, D. R., Witherell, G. W. and Uhlenbeck, O.
C. (1987) Nuc. Acids Res., 15: 8783-8798). The synthetic DNA
templates were made using phosphoramidite chemistry on an Applied
Biosystems 381A DNA synthesizer. The template DNAs were: catalytic
RNA R51:
3'-ATTATGCTGAGTGATATCTTTGTCTCTTCAGTT-GGTCTCTTTGTGTGCAACACCATATAATGGA-
CCAT-5' and substrate RNA S17:
3'-ATTATGCTGAGTGATATCGCACTGTCAGGACAAA-5'.
[0212] Before transcription, a 18mer or 16 mer DNA complement to
the promoter for T7 RNA polymerase on the noncoding strand was
hybridized by heating an equimolar amount of template DNA with
promoter complement to 65.degree. C. for 3 min. then placing in
ice. A typical transcription reaction used 8 ng/ul DNA template,
0.5 mM each NTP, 2 mM spermidine, 40 mM Tris pH 7.5, 4%
polyethylene glycol 6,000, 6 mM MgCl.sub.2, 4 mM NaCl, 10 mM
dithiothreitol, 0.01% Triton X-100, 2.4 units/ul RNasin, 1.8 uCi/ul
P.sup.32 CTP and 3 units/ul T7 RNA polymerase (US Biochemical) and
was run at 37.degree. C. for 90 min.
[0213] All in vitro transcribed RNAs were isolated on 7M urea,
15-20% acrylamide gels, bands cut out and isolated. All RNAs were
sequenced using standard methods (Donis-Keller, H., Maxam, A. M.
and Gilbert, W. (1980) Nucleic Acids Res., 4: 2527-2538); a method
which also gave the 5' terminal base. Terminal bases at the 3' end
were determined by ligation of the RNA to 5' P.sup.32 pCp using T4
RNA ligase (BRL methods manual), nuclease T2 digestion, and
separation of labelled bases by PEI thin layer chromatography in
0.3M LiC1 with appropriate standards. All RNA sequences
corresponded to that expected from the DNA template.
Example 3
[0214] The catalytic RNA R51 was added to the substrate RNA S17 at
a ratio of 1:30 and the time course of substrate RNA cleavage was
studied. The reaction was carried out at 37.degree. C. in 12 mM
MgCl.sub.2, 40 mM Tris pH 7.5 and 2 mM spermidine over a time
period of 150 min and is summarized as follows: 4
[0215] The reaction products were separated on polyacrylamide/urea
gels by electrophoresis and bands cut out and counted in a liquid
scintillation counter. The results are shown in FIG. 3. The time
periods analyzed were: lane 1, 30 sec; lane 2, 5 min; lane 3, 15
min; lane 4, 30 min; lane 5, 60 min; lane 6, 90 min and lane 7, 150
min. Beginning concentrations were as follows: R51=0.0032 uM and
S17=0.09 uM.
[0216] As shown in FIG. 3, the cleavage proceeds to virtual
completion during the course of the reaction with only 2% of the
substrate remaining after 150 minutes. This shows that, since there
was originally 30 times as much substrate RNA as catalytic RNA, the
RNA catalyst R51 of necessity interacts with multiple substrate
molecules during the course of the reaction. In addition, the
amount of catalyst remained the same and was unaltered, indicating
that R51 is truly a catalytic entity.
Example 4
[0217] After the RNA catalyst had been shown to be effective in
cleaving the RNA substrate as described in Example 3, minimum
energy folding of the 50 base sequence shown in FIG. 1, complexed
with the 14 base sequence was done using the computer methods
described in Example 1. The folded complex forms a "hairpin" model
or motif as shown in FIG. 1 with the substrate RNA sequence and the
catalytic RNA sequence identified. The arrow is at the site of
cleavage.
Example 5
[0218] Various concentrations of substrate S17 were used at
constant concentration of catalyst R51 and initial velocities of
each reaction determined. The reaction was carried out at
37.degree. C. in 12 mM MgCl.sub.2, 40 mM Tris pH 7.5 and 2 mM
spermidine. Concentrations were as follows: R51=0.0004 uM and
S17=0.125 uM (lane 1), 0.0624 uM (lane 2), 0.0417 uM (lane 3),
0.031 uM (lane 4), 0.021 uM (lane 5), 0.0156 uM (lane 6), 0.0078 uM
(lane 7) and 0.0039 uM (lane 8). Each reaction was analyzed on
polyacrylamide gels as described in Example 3 with the results
shown in FIG. 4. An Eadie Hofstee plot of catalytic RNA R51
cleavage of substrate RNA S17 is shown in FIG. 4. The reaction
proceeded according to the predictions of the Michaelis-Menten
equation indicating that it was a truly enzymatic reaction in that
as the concentration of the substrate goes down, the velocity of
the reaction goes down. From the velocity of the reaction plotted
as a function of substrate concentration, the Km calculated from
the graph was 0.03 uM and the kcat (turnover number) was 2.1/min.
This Km is 20 times smaller than the "hammerhead" catalysts (see
Uhlenbeck, O. C. (1987) Nature, 328: 596-600) indicating that lower
concentrations of substrate can be removed. The kcat is 6 times
larger than that of catalysts fitting the "hammerhead" model at
37.degree. C. (see Uhlenbeck, O. C. (1987) Nature, 328: 596-600),
meaning that the reaction is 6 times faster.
Example 6
[0219] The temperature dependence of the rate of cleavage of
substrate RNA S17 by catalytic RNA R51 was tested over a
temperature range and the reaction products analyzed on
polyacrylamide gels as described in Example 3 with the results
shown in FIG. 5. The reaction was carried out in 12 mM MgCl.sub.2,
40 mM Tris pH 7.5 and 2 mM spermidine at 45.degree. C. (lane 6),
41.degree. C. (lane 5), 37.degree. C. (lane 4), 33.degree. C. (lane
3), 27.degree. C. (lane 2) and 20.degree. C. (lane 1). The
concentrations used were: R51=0.0016 uM and S17=0.04 uM. R51 was
unlabeled. The velocities shown in the graph in FIG. 5 were
calculated by the use of time points of 8 and 16 minutes. The
separation patterns on the gel shown in FIG. 5 are for the
16-minute time point.
[0220] The reaction showed a temperature dependence similar to that
which would be expected of a reaction involving base paired RNA
molecules. The Arrhenius plot of the data shown in FIG. 5 gives a
temperature optimum of 37.degree. C. for the reaction. Higher
temperatures reduce the reaction rate with a very rapid rate
reduction above 41.degree. C. consistent with a melting out of the
catalytic RNA structure. At 50.degree. C. no reaction was
detectable. The reaction rate at temperatures below 37.degree. C.
showed a linear reciprocal temperature dependence consistent with a
classical lowering of the energy of activation for the reaction.
The slope of the line in the Arrhenius plot gave an energy of
activation of 19 Kcal/mole which is close to that found for
catalysts fitting the "hammerhead" cleavage mechanism (13.1
Kcal/mole) (Uhlenbeck, O. C. (1987) Nature, 328: 596-600).
Example 7
[0221] The rate of cleavage of a constant concentration of
substrate RNA S17 at varying concentrations of catalytic RNA R51
was tested and the reaction products analyzed on polyacrylamide
gels as described in Example 3 with the results shown in FIG. 6.
The reaction was carried out at 37.degree. C. in 12 mM MgCl.sub.2,
40 mM Tris pH 7.5 and 2 mM spermidine for 40 min (lane 1 and 2), 20
min (lane 3), 10 min (lane 4) and 5 min (lane 5). The concentration
of substrate was 0.175 uM. The results are plotted in FIG. 6 and
show that at saturating concentrations of substrate the reaction
rate is linear with increasing RNA catalyst concentrations as one
would expect for a true catalytic reaction.
Example 8
[0222] The effect of Mg.sup.++ concentration and pH on the rate of
cleavage of RNA substrate S17 by RNA catalyst R51 was determined as
shown in the following table:
3 t.sub.1/2 (min) MgCl.sub.2 (mM) 0 no detectable product 4 136 6
111 8 115 10 88 12 81 15 74 20 62 pH 5.5 330 6.0 120 6.5 67 7.0 48
7.5 42 8.0 38
[0223] In the Mg.sup.++ studies, the substrate S17 concentration
was 0.14 uM and RNA catalyst R51 concentration was 0.0015 uM. The
reactions were at 37.degree. C. in 40 mM Tris pH 7.5. In the pH
studies, the substrate S17 concentration was 0.062 uM and RNA
catalyst R51 concentration was 0.0014 uM. The reactions were at
37.degree. C. in 40 mM Tris for pH 7.0, 7.5, 8.0 and in 40 mM Pipes
for pH 5.5, 6.0 and 6.5.
[0224] The dependence of the reaction rate on Mg.sup.++ and pH are
virtually identical with those of catalysts fitting the
"hammerhead" model. The reaction rate increases with increasing pH
as one would expect for a base catalyzed reaction but the effect is
masked by the catalytic activity of the RNA. Hence a 100 fold
increase in [OH.sup.-] between pH 6.0 and 8.0 resulted in only a 3
fold increases in the reaction rate.
Example 9
[0225] A 10 base substrate (S10) was prepared by the methodology of
Example 2. When the substrate was mixed with catalytic RNA R51, the
reaction is summarized as follows: 5
[0226] The results of rate studies with substrate S10 comparable to
those described with S17 in Example 5 showed a Km=0.06 uM and a
kcat=0.8/min. These results are shown in FIG. 7 and indicate that
smaller substrates can be used, but not as efficiently.
Example 10
[0227] The 3' terminal base of the catalyst shown in the "hairpin"
model of the (-)sTRSV catalytic complex in FIG. 1 is at position
175. Two catalytic RNAs were prepared with exactly the same
sequence as R51, except that one of them did not contain the 3'
terminal "A" base (position 175) and the other one did not contain
the 3' terminal "UA" sequence (positions 176 and 175). Synthesis of
these catalytic RNAs, designated R50 and R49, respectively, was
carried out as described in Example 2. R50 or R49 catalytic RNA was
mixed with substrate RNA S17 under standard conditions of reaction
and the products analyzed as described in Example 3. The results
are given in FIG. 8 and show a 75% reduction in activity with
either R50 or R49 as compared to the activity of catalytic RNA R51
having the 3' terminal "A" at position 175 and the 3' terminal "AU"
at positions 175 and 176.
Example 11
[0228] An RNA with the same sequence as catalytic RNA R51 was
prepared, except that bases. 195-203 were omitted such that base
194 was in effect ligated to base 204. This RNA molecule was
prepared as described in Example 2 from an appropriate DNA template
containing the complementary sequence. When this RNA was mixed with
substrate RNA S17 as described in Example 3, no reaction occurred.
These results show that major elements of the "hairpin" structure
are required for RNA catalysis to occur and that removal of 9 bases
(see FIG. 9) in the middle inactivates the catalytic RNA.
Example 12
[0229] An RNA with the same sequence as R51, except that the bases
AAA at positions 203, 202 and 201 were changed to CGU,
respectively, was prepared as described in Example 2 using an
appropriate DNA primer. When this potential RNA catalyst was mixed
with substrate RNA S17 as described in Example 3, no reaction
occurred. This shows that the integrity of one or all of these
bases (see FIG. 10) is required for catalytic activity.
Example 13
[0230] A substrate RNA with the base at position 49 in FIG. 1
changed from an "A" to a "G" was prepared as described in Example
2. When this substrate was reacted with the RNA catalyst R51, no
difference in rate of reaction was seen between this substrate and
the substrate containing the "A" at position 49 (see FIG. 11A).
This shows that alterations can occur in the "A" base in the
substrate RNA AGUC loop without affecting the ability of the
catalytic RNA to cleave the substrate.
Example 14
[0231] Another substrate RNA identical to S17 but having "A"
replaced by "U" in the AGUC loop was prepared as described in
Example 2 (designated "S17(A-->U)"). This substrate RNA, at a
concentration of 0.12 uM, was reacted with the catalytic RNA R51,
at a concentration of 0.0065 uM, under standard conditions as
described in Example 3 for 60 minutes. The results are shown in
FIG. 11B where Lane 1 contains the products of the reaction of
substrate RNA S17(A-->U) with R51 catalytic RNA. No difference
in the rate of reaction was seen between S17(A-->U) substrate
RNA and substrate S17 containing the "A" base at position 49.
Example 15
[0232] Another substrate RNA identical to S17 but having "A"
replaced by "C" in the AGUC loop was prepared as described in
Example 2 (designated "S17(A-->C)"). This substrate RNA, at a
concentration of 0.08 uM, was reacted with the catalytic RNA R51,
at a concentration of 0.0065 uM, under standard conditions as
described in Example 3 for 60 minutes. The results are shown in
FIG. 11C where Lane 1 contains the products of the reaction of
S17(A-->C) substrate RNA with R51 catalytic RNA. Again, no
difference was seen in the rate of reaction using S17(A-->C) as
compared to S17 containing the "A" base at position 49. The
combined results of Examples 13-15 show that the base at position
49 in the cleavage sequence of the substrate may be any base.
Example 16
[0233] Base changes in the stem regions at the site of binding of
the substrate RNA to the catalytic RNA in the "hairpin"
configuration can be made as long as the base pairing is
maintained. The "C" base at position 50 of the substrate was
changed to a "G" using the methods in Example 2. When this
substrate RNA was reacted with the catalytic RNA R51, no cleavage
of this substrate occurred. However, when a new catalytic RNA,
containing a "C" at position 214, rather than the "G" found in R51,
was synthesized according to the methods in Example 2 and added to
this substrate, full cleavage was seen. The effect of the base
change from "C" to "G" in the substrate was to eliminate the base
pairing at this position predicted by the "hairpin" model since now
a "G" would be across from a "G". However, when a "G" to "C" base
change was made in the catalytic RNA, the base pairing was
restored, but in a reverse manner, and the integrity of the helices
in the stem regions where the substrate RNA binds to the catalytic
RNA was thus conserved restoring catalytic activity (see FIG.
12).
Example 17
[0234] An RNA sequence found within the sequence of tobacco mosaic
virus was synthesized using the methods described in Example 2.
This synthesized target RNA had the sequence 5'gAAACAGUCCCCAAC 3'.
A catalytic RNA was synthesized with the sequence
5'-GUUGGGAGAAGUUUACCA-GAGAAACACACGU- UGUGGUAUAUUACCUGGUA-31
selected so that base pairing between the substrate and the
catalytic RNA is maintained in the "hairpin" configuration (see
FIG. 13). When these two RNAs were mixed under standard catalytic
conditions as described in Example 3, the target was cleaved
demonstrating that a sequence found within a native viral RNA can
be cleaved.
Example 18
[0235] Three RNA sequences found within the sequence of the
messenger RNA for the enzyme chloramphenicol acetyl transferase
(CAT) were synthesized using the methods described in Example 2.
The synthesized substrate RNAs had the sequences (A)
gUUUCAGUCAGUUGC, (B) gUUUCAGUCAGUUGCUC; and (C)
gggUUUCAGUCAGUUGCUCAA (see FIG. 14).
[0236] Note that the latter two sequences are extensions of the
first sequence and that additional bases have been added to the 3'
end in the region that the "hairpin" model predicts will base pair
with the catalytic RNA to form Helix 1 (see FIG. 18). Also,
substrate (C) had two additional G vector bases as compared to
substrates (A) and (B). The site of cleavage after the A in the
AGUC cleavage sequence (see the arrow in FIG. 14) of the substrates
corresponds to position 320 of the CAT gene using the number system
found in the Tn9 sequence (Alton and Vapnak, Nature, 282, 864
(1979)). In FIG. 14, the open boxed bases are those which are
different from those in the native (-)sTRSV substrate RNA sequence
shown in FIG. 1.
[0237] Catalytic RNAs corresponding to substrate RNAs (A), (B) and
(C) were synthesized according to the methods described in Example
2. Their sequences were designed so that they would base pair with
the substrate RNAs in both the 3' and 5' regions flanking the AGUC
cleavage sequence. In addition, the catalytic RNAs designed to
react with substrate RNAs (A) and (B) each contained the vector
sequence GA at their 5' terminus, and the catalytic RNA designed to
react with substrate RNA (C) contained the vector sequence GGG at
its 5' terminus. Otherwise, the catalytic RNAs had the same
sequence as the (-) sTRSV catalytic RNA sequence shown in FIG.
1.
[0238] The various substrate and catalytic RNAs were reacted and
the reaction products analyzed as described in Example 3. All
reaction conditions were as described in Example 3, except for the
following. For substrates (A) and (B), reaction conditions were:
substrate RNA concentration=0.05 uM; catalytic RNA
concentration=0.005 uM; reaction run at 16.degree. C.; and reaction
time of 20 minutes. For substrate (C), the reaction conditions were
the same as for (A) and (B), except that the reaction time was 40
minutes and temperatures were 20.degree. C., 25.degree. C.,
30.degree. C. and 37.degree. C.
[0239] Cleavage of all of the substrate RNAs occurred when they
were mixed with the corresponding catalytic RNAs as is shown in
FIG. 14, demonstrating that catalytic RNAs according to the
invention can be synthesized which cleave specific RNA sequences
found within a messenger RNA. In addition, this example
demonstrates that cleavage of substrate RNA can be obtained even
though the length of the region at the 3' end of the substrate
which base pairs with the catalyst (i.e., the portion that forms
Helix 1 with the substrate according to the "hairpin" model) is
varied. Indeed, when the length of this region was extended to 10
bases in substrate (C), the reaction could then proceed at
37.degree. C., whereas for substrates (A) and (B) having shorter
sequences in this region, the reaction would proceed only at
16.degree. C.
Example 19
[0240] An RNA substrate corresponding to part of the sequence of
HIV-1, the virus which causes AIDS, was synthesized as described in
Example 2. The sequence of this substrate RNA is shown in FIG. 15.
The arrow in FIG. 15 shows the cleavage site which corresponds to
position 804 in the sequence of HIV-1 strain SF2CG
(Sanchez-Pescador et al., Science, 227, 484 (1985). The sequence
shown is found in the region of the viral RNA which specifies the
gag protein. The RNA substrate also had a GCG 5' vector sequence.
The open boxed bases in FIG. 15 are those which are different than
those of the native (-)sTRSV substrate sequence shown in FIG.
1.
[0241] A catalytic RNA was synthesized according to the methods of
Example 2. Its sequence was designed so that it would base pair
with the substrate RNA in both the 3' and 5' regions flanking the
CGUC cleavage sequence. In addition, the catalytic RNA contained
the vector sequence GGG at its 5' terminus. Otherwise, the
catalytic RNA had the same sequence as the (-)sTRSV catalytic RNA
sequence shown in FIG. 1.
[0242] The catalytic RNA and the substrate RNA were reacted and the
reaction products were analyzed as described in Example 3. The
reaction conditions were as set forth in Example 3, except that the
following temperatures were used: 20.degree. C., 25.degree. C.,
30.degree. C. and 37.degree. C. Also, the reaction was run for 60
minutes, except for 37.degree. C. which was run for 15 minutes, and
the substrate RNA concentration was 50 nM, and the catalytic RNA
concentration was 5 nM.
[0243] The catalytic RNA cleaved the substrate at the expected
position between the "C" and "G" in the CGUC cleavage sequence
found in the loop between the two flanking stem regions. Thus, a
specific sequence found in the HIV-1 viral RNA that codes for the
gag protein can be cleaved with a catalytic RNA according to the
invention. The reaction occurred with an RNA substrate having a
16-base target sequence which was longer than S17 in the region at
the 3'end which base pairs with the catalyst (i.e., the portion
that forms Helix 1 with the catalytic RNA according to the
"hairpin" model). Also, the reaction occurred at physiological
temperature of 37.degree. C.
Example 20
[0244] A substrate RNA having a sequence found in the beginning of
the coding region for the regulatory protein tat of HIV-1 virus was
synthesized as described in Example 2. The substrate sequence is
shown in FIG. 16. In addition to the HIV-derived sequence, the
substrate RNA had a GCG 5 vector sequence. The open boxes are
around bases which are different than those of the native (-)sTRSV
substrate sequence shown in FIG. 1.
[0245] A catalytic RNA having a sequence so that it would base pair
with the substrate RNA in the two regions flanking the UGUC loop
(ie., the regions that forms Helices 1 and 2 with the catalytic RNA
according to the "hairpin" model) was also synthesized as described
in Example 2. Otherwise, the catalytic RNAs had the same sequence
as the (-)sTRSV catalytic RNA sequence shown in FIG. 1, except that
it had an additional 5'G vector base.
[0246] The substrate RNA and catalytic RNA were reacted and the
reaction products were analyzed as described in Example 3. Reaction
conditions were: 37.degree. C.; reaction times of zero and 15
minutes; the concentration of substrate RNA was 100M; and the
concentration of catalytic RNA was 20 nM.
[0247] Cleavage occurred as expected between the "U" and the "G" in
the UGUC cleavage sequence located between the two stem regions of
the substrate. The cleavage site is indicated by the arrow in FIG.
16. This is position 5366 in the sequence of HIV clone h9c (Muesing
et al., Nature, 313, 450 (1985)). These results again confirm that
a catalytic RNA designed according to the "hairpin" model can
cleave a specific target sequence located in a naturally occurring
RNA, in this case a key regulatory region (tat) of the HIV-1 virus
which causes AIDS.
Example 21
[0248] Using the methods described in Example 2, a substrate RNA
having four non-native bases (UUUU) added to the 3' end of the
sequence of the native (-)sTRSV substrate shown in FIG. 1 and a
corresponding catalytic RNA designed to base-pair with the
substrate RNA in the 3' and 5' regions of the substrate flanking
the cleavage sequence (i.e., the portions that form Helices 1 and 2
with the catalyst according to the "hairpin" model) were made.
Thus, the substrate RNA contained a total of 18 bases, including a
four-base extension of the 3' region that base pairs with the
catalyst. The substrate RNA also had an additional GCG vector
sequence at the 5'end. The catalyst had the same sequence as the
(-)sTRSV catalytic RNA sequence shown in FIG. 1, except that it had
four additional AAAA bases at the 5' end designed to base pair with
the added UUUU bases of the substrate and had an additional "G"
vector base at the 5' terminus beyond the 18 base recognition
site.
[0249] The substrate and catalytic RNAs were reacted at standard
conditions and the reaction products were analyzed as described in
Examples 3 and 5. Catalytic RNA concentration was 0.00033 uM, and
substrate RNA concentration was 0.1 uM (Lane 1), 0.05 uM (Lane 2),
0.033 uM (Lane 3), 0.025 uM (Lane 4), 0.016 uM (Lane 5), 0.012 uM
(Lane 6), 0.006 uM (Lane 7), and 0.003 uM (Lane 8). The results are
shown in FIG. 17. Cleavage rates at each concentration of substrate
were determined by cutting out the bands, counting radioactivity in
a liquid scintillation counter and plotting the data using
Michaelis-Menton procedures to calculate Km and kcat (see Example
5).
[0250] The data show that an extension of the length of the region
of base pairing between the substrate and catalyst (i.e., those
regions of the catalyst and substrate that form Helix 1 according
to the "hairpin" model) can improve the catalytic properties of the
reaction. Cleavage of the 18-base RNA substrate occurred at the
expected site, but at an increased rate as compared to the cleavage
of S17 by R51. The kcat or turnover number of the reaction was
7/minute. This means that each molecule of catalytic RNA cleaved 7
molecules of substrate RNA per minute during the reaction. The kcat
for S17 cleavage by R51 was 2.1/minute. The Km of the reaction was
the same as for S17 cleavage by R51 (30 nM). This shows that by
optimizing the length of the region of the catalyst that base pairs
to the substrate in the 3' region flanking the cleavage sequence
(i.e., by optimizing the length of Helix 1 predicted by the
"hairpin" model), the catalytic properties of the native reaction
can be improved.
Example 22
[0251] A series of catalytic RNAs were prepared using the methods
described in Example 2 having certain base substitutions as
compared to the native (-)sTRSV catalytic RNA sequence shown in
FIG. 1. The substitutions, which are illustrated in FIG. 19, were
the following: (1) At nucleotide 35, G was replaced by C
(G35-->C) (count bases from the 5' end of the catalytic RNA
sequence shown); (2) A double mutant, having the G at position 35
replaced by C and the C at position 27 replaced by G (G35-->C;
C27-->G); (3) At nucleotide 47, G was replaced by C
(G47-->C); and (4) A double mutant having the G at position 47
replaced by C and the C at position 17 replaced by G (G47-->C;
C17-->G). All catalytic RNAs had an additional "G" vector base
at their 5' end.
[0252] The various catalytic RNAs were reacted with substrate S17
and the reaction products were analyzed as described in Example 3.
The results are shown in FIG. 19 where Lanes 1, 3, 5, 7, 9 and 11
are at zero time, and Lanes 2, 4, 6, 8, 10 and 12 are 15 minutes
incubation under standard cleavage conditions (see Example 3). The
concentration of catalytic RNA was 0.0065 uM, and the concentration
of substrate RNA was 0.17 uM. The temperature was 37.degree. C. The
control, lanes 1, 2, 7 and 8, was cleavage of the native substrate
RNA S17 by catalytic RNA R51 which has the native (-)sTRSV sequence
(see FIG. 1).
[0253] As shown in FIG. 19, the catalytic RNA G35-->C had no
catalytic activity (see Lanes 3 and 4 of FIG. 19, where this
catalytic RNA is designated "mismatch" since the base substitution
at position at position 35 results in a loss of base pairing). The
double mutant catalytic RNA G35-->C; C27-->G showed restored
catalytic activity (see Lanes 5 and 6 of FIG. 19 where this
catalytic RNA is designated "substitute b.p." since the net effect
of the two base substitutions is to create a base pair, but a base
pair different than the one found in the native (-)sTRSV catalytic
RNA). The catalytic RNA G47-->C was also inactive (see Lanes 9
and 10 of FIG. 19), while the double mutant, with the second
mutation C17-->G, showed restored activity (see Lanes 11 and 12
of FIG. 19).
[0254] The results of these mutagenesis studies provide
confirmation for the existence of Helices 3 and 4 (see FIG. 19) of
the "hairpin" catalytic RNA model proposed herein. The results show
that when mutagenesis was carried out which caused a base pair
mismatch in the region of proposed base pairing, the catalytic RNA
was inactive. However, when a second mutation was carried out so
that the base pair was restored, catalytic activity was restored,
even though the new base pair was different than the original base
pair. Such results are considered evidence for the existence of a
helix (Fox and Woese, Nature, 256, 505 (1975). These results also
show that a variety of catalytic RNAs having sequences different
from the native (-)sTRSV sequence are catalytically active if they
are designed so that they follow certain rules derived from the
"hairpin" model, such as maintenance of substantial base pairing in
regions of predicted helices.
Example 23
[0255] A synthetic "hairpin" autocatalytic cassette was prepared.
The RNA sequence of the cassette is shown in FIG. 20. Several bases
have been added at the 5' end of the catalyst as compared to the
native (-)sTRSV sequence shown in FIG. 1. The effect is to close
the open end of the "hairpin" and to provide a substrate sequence
(i.e., a cleavage sequence and upstream and downstream flanking
regions) which can base pair with the substrate binding portion of
the catalyst.
[0256] The cassette was prepared by making an appropriate synthetic
DNA template that would yield an RNA with the sequence shown in
FIG. 20 and using the DNA template to transcribe RNA as described
in Example 2, with the following differences. After synthesizing
the DNA template, it was inserted into the vector pTZ18R (US
Biochemical) into which a new multiple cloning site had been
inserted. The new multiple cloning site was a construct containing
sites, in 5'->31 order, for cleavage by the following enzymes:
BamHI, XhoI, ApaI, SacII, NaeI, StuI, KpnI, MluI. The new multiple
cloning site was inserted into vector pTZ18R by cleaving the vector
with BamHI and SalI and then ligating the multiple cloning site to
the vector using T4 ligase. Located 5' to the inserted multiple
cloning site is the T7 RNA polymerase promoter. The vector
containing the multiple cloning site was digested with MluI and
SalI, and the DNA template coding for the autocatalytic cassette
was ligated into the vector using T4 ligase. The resultant vector
was then linearized with HindIII, and transcription carried out as
described in Example 2. All restriction enzymes and the T4 ligase
were obtained from IBI and used according to manufacturer's
instructions.
[0257] After being transcribed, the cassette spontaneously
underwent an intramolecular autocatalytic cleavage at the expected
site to give the appropriate 3'F and 5'F products (see FIG. 20).
Note that the effect of this is to autocatalytically terminate a
transcript. For example, the 5'F is in itself a transcript which
has been terminated at its 3' end by the autocatalytic reaction.
Further note that this termination only leaves five essential bases
at the 3' end of this 5'F These are UGACA which are boxed in FIG.
20. Thus, it is possible to very efficiently terminate
transcription with this autocatalytic cassette and leave only a
very short 3' end to the transcript (i.e., the 5' fragment of the
autocatalytic cleavage).
Example 24
[0258] A catalytic RNA was prepared as described in Example 2. Its
sequence was identical to that of R51, except that the base at
position 217 in the AGAA loop was changed from a G to a C. The AGAA
loop of the catalyst is opposite the cleavage sequence of the
substrate when the substrate and catalyst are complexed (see FIG.
1)
[0259] Another catalytic RNA was prepared as described in Example
2. Its sequence was also identical to R51, except that the base at
position 216 was changed from A to U.
[0260] These catalysts were reacted with the RNA substrate S17
under standard conditions as described in Example 3 at 37.degree.
C. for 0 and 15 minutes. The control was the reaction of substrate
S17 with catalyst R51. The concentration of the three catalytic
RNAs was 0.007 uM, and the concentration of substrate RNA was 0.7
uM.
[0261] The reaction products were analyzed as described in Example
3, and the results are shown in FIG. 21, where the first lane in
each gel is zero time and the second lane is 15 minutes of
reaction. As shown in FIG. 21, changes in either one of these two
bases (G217-->C and A216-->U) in the loop opposite the
cleavage sequence of the substrate destroyed the activity of the
catalyst. These results indicate that these two bases are likely
invariant in the native (-)sTRSV catalytic sequence.
[0262] A third catalytic RNA was prepared as described in Example
2. Its sequence was identical to R51, except that the base at
position 218 was changed from A to C. This catalyst was also
reacted with substrate S17 as described above. Cleavage of the
substrate with this catalyst was observed, but only at about 47% of
the level achieved with R51 (data not shown).
Example 25
[0263] Substrate RNAs identical to S17, but with one of the bases
at positions 46, 47 or 48 (i.e., bases GUC of the cleavage
sequence) changed to a different base, were prepared as described
in Example 2. When these substrates were reacted with the RNA
catalyst R51 under standard conditions for 60 minutes as described
in Example 3, no cleavage of the substrates occurred. This shows
conservation of the GUC sequence of the cleavage sequence of
(-)sTRSV RNA. The results of these experiments combined with the
results of Examples 13-15 show that the cleavage sequence of
(-)sTRSV RNA is NGUC, where N is any base.
Example 26
[0264] The vector prepared in Example 23 (hereinafter referred to
as "pHC") containing the "hairpin" autocatalytic cassette was
tested for activity in vivo as follows. First, the CAT gene was
removed from plasmid pMAMNEO-CAT (purchased from Clontech Inc.)
with SmaI and XhoI. It was then ligated using T4 ligase into pHC
which had been cut with SmaI and XhoI to produce vector
pHC-CAT.
[0265] The original vector used in these constructions (pTZ18R; see
Example 23) contains an inducible promotor (lacZ) and, as a result
of the steps described in Example 23 and immediately above in this
example, the CAT gene and the "hairpin" autocatalytic cassette were
placed in this inducible region (see FIG. 22A). Also, the CAT gene
and "hairpin" autocatalytic cassette were linked so that the
expected transcript would be as shown in FIG. 22A (the
"CAT-cassette RNA") and so that the RNA sequence coded for by the
"hairpin" autocatalytic cassette would be expected to serve as a
chain terminator for the CAT transcript by cleaving at the
indicated cleavage site (see FIG. 22A). The expected 5' fragment of
such a cleavage is also shown in FIG. 22A ("Cleaved CAT-cassette
RNA").
[0266] Next, pHC-CAT was transfected into Escherichia coli strain
JM109 (widely available from a number of commercial sources and
from the American Type Culture Collection) with calcium chloride
and heat shock using standard procedures as described in Maniatis
et al., Molecular Cloning (1983). Transformed cells containing
pHC-CAT were selected on the basis of ampicillin resistance by
plating on double concentration YT medium containing 100 ug/ml
ampicillin. After selection, the transformed cells were grown
overnight in LB broth at 37.degree. C. A fresh culture of these
cells was then grown for 5 hours in LB broth at 37.degree. C.,
after which the cells were induced with 1 mM
isopropyl-beta-D-thiogalacto- side (IPTG) for one hour to allow
expression of the lacZ region, including the CAT gene-cassette RNA
transcript.
[0267] At the end of this time, RNA was isolated by incubating the
cells in 50 mM Tris, pH 8.0, 50 mM ethylenediaminetetraacetic acid
(EDTA), 1 mg/ml lysozyme at room temperature for 10 minutes to lyse
the cells. The lysate was made to 0.5% sodium dodecyl sulfate
(SDS), and then centrifuged to remove cell debris. Phenol was added
to the supernatant at a ratio of 1:1, and the supernatant was
centrifuged to remove the precipitate. This procedure was repeated,
and the resulting aqueous phase was treated with an equal volume of
isopropanol at -20.degree. C. for 20 minutes to precipitate the
RNA. The precipitate was collected by centrifugation, dried, and
redissolved in water.
[0268] This isolated RNA was electrophoresed on 1.2% agarose gels
containing formaldehyde as described in Current Protocols In
Molecular Biology (Greene 1989). After electrophoresis, Northern
blots were carried out using published methods (GeneScreen Plus,
DuPont, July 1985). Two DNA probes were used for blotting the gels.
The CAT probe was prepared by primer extension of the CAT gene
using the Klenow fragment of DNA polymerase I and dATP labelled
with alpha P.sup.32 The "hairpin" autocatalytic cassette probe was
prepared by kinasing the DNA complement to the entire "hairpin"
autocatalytic cassette RNA sequence shown in FIG. 20 with dATP
labelled with gamma P.sup.32. The expected positions of binding of
these probes to the CAT-cassette RNA and expected 5' fragment are
shown in FIG. 22A.
[0269] The results of the Northern blot test are shown in FIG. 22B.
As shown there, when the CAT probe was used, the full length
CAT-cassette RNA transcript and the expected 5' fragment were
detected on the gel, indicating that cleavage had taken place in
vivo.
[0270] When the "hairpin" autocatalytic cassette probe was used,
only the full length CAT-cassette RNA transcript was detected (see
FIG. 22B). The fact that the 5' fragment did not hybridize with
this probe was to be expected, since most of the "hairpin"
autocatalytic cassette transcript would be in the 3' fragment after
cleavage. Although it would be expected that the "hairpin"
autocatalytic cassette probe would hybridize to the 3' fragment,
the fact that the 3' fragment was not detected by Northern blot
analysis is not surprising. The 5' terminus of the 3' fragment
would contain a 5'-OH and not the 5'-ppp which is ordinarily seen
in RNA transcripts. Thus, the 3' fragment would be expected to be
very labile in vivo and was likely degraded immediately after the
autocatalytic cleavage.
[0271] The mobilities of the CAT-cassette RNA and the Cleaved
CAT-cassette 5' fragment on the gel corresponded exactly to the
predicted length of the transcripts. The standards run on the gel
were E. coli 16S and 23S RNA.
[0272] An important control was to determine whether cleavage of
the target RNA occurred during isolation of the RNA. Intact,
uncleaved "hairpin" autocatalytic RNA prepared and separated on an
acrylamide gel as described in Example 23 was isolated from the gel
using conditions similar to those described above (i.e., no
divalent cation, but in the presence of EDTA and SDS). Only intact,
uncleaved RNA was obtained when the catalyst was re-electrophoresed
on an acrylamide gel.
Example 27
[0273] Mammalian expression vector pMHC-CAT containing the
"hairpin" autocatalytic cassette linked to the CAT gene was
constructed as follows. First, the "hairpin" autocatalytic cassette
was removed from the vector pHC (prepared as described in Example
23) by digesting pHC with SmaI/SalI and ligating the resulting
fragment to the mammalian vector pMSG (purchased from Pharmacia) at
the SmaI/XhoI sites to give pMHC. This operation removed the XhoI
site used to clone the "hairpin" autocatalytic cassette insert, but
introduced another XhoI site near the 5'end of the insert.
[0274] Next, the CAT gene was removed from pMAM-NEO-CAT (Clontech,
Palo Alto, Calif.) with SmaI/XhoI and cloned into pMHC at the
SmaI/XhoI sites to give the final vector pMHC-CAT which is shown in
FIG. 23. Note that the CAT gene and the "hairpin" autocatalytic
cassette are both driven by the MMTV LTR promoter which is
dexamethasone inducible.
[0275] The vector pMHC-CAT was co-transfected into Chinese hamster
ovary cells (CHO) by the calcium phosphate method along with the
vector pMSG-dhfr (a gift from Dr. Ed Prochownick of the University
of Michigan, Ann Arbor, Mich.) which contained a mutant dhfr gene.
The isolation of this mutant dhfr gene is described in Simonson and
Levinson, Proc. Nat'l Acad. Sci. 80, 2495-99 (1983), and pMSG is
available from Pharmacia (see above).
[0276] Transfectants were selected using gpt medium (Molec. &
Cell. Biol., 3, 1421-29 (1983)) which kills non-transfected cells.
They were next amplified with methotrexate (which amplifies the
mutant dhfr gene and associated DNA) to give CHO cells which
contained the "hairpin" autocatalytic cassette linked to the CAT
gene stably integrated into the genome. Dexamethasone (1 uM) was
added to induce expression of the CAT gene and "hairpin"
autocatalytic cassette, and RNA was isolated from the individual
cells by the guanidinium isothiocyanate procedure (Current
Protocols in Molecular Bioloqy).
[0277] Next, a nuclease protection assay was performed to identify
the nature of the transcripts and cleavage products, if any,
produced by transcription of the "hairpin" autocatalytic
cassette-CAT gene fusion. The probe used in the S1 nuclease assay
was prepared from the vector pHC19R. This vector was prepared by
removing the "hairpin" autocatalytic cassette from pHC with
BamHI/SalI and ligating this fragment into the BamHI/SalI sites of
the vector PTZ19R (purchased from US Biochemical) as shown in FIG.
24. Vector pHC19R was linearized with BamHI, transcribed with T7
RNA polymerase using P.sup.32-CTP as described in Example 2, and
the transcript was isolated from 7M urea gels, also as described in
Example 2. This RNA probe is 148 nt long and is the complement to
the "hairpin" autocatalytic cassette RNA.
[0278] The probe was then hybridized in buffer (40 mM Pipes, pH6,
100 mM NaCl, 20% formamide, 1 mM ZnSO.sub.4) to RNA (5 ug) isolated
from CHO cells that had been transfected with vector pMHC-CAT, and
the hybridized RNA was digested with S1 nuclease (100 units, 1 hr.,
37.degree. C.). S1 nuclease will digest all single-stranded RNA,
but not double-stranded RNA. A negative control was RNA from
transfected CHO cells which had not been induced. A positive
control was RNA isolated from the pHC plasmid which had been
transcribed in vitro with T7 RNA polymerase as described in Example
2.
[0279] The S1 digests were electrophoresed on 10% acrylamide-7M
urea gels. The results are shown in FIG. 25 where: Lane 1 contains
the RNA probe alone; Lane 2 contains the probe digested with S1
nuclease; Lane 3 contains in vitro transcribed "hairpin"
autocatalytic cassette RNA, 5'F and 3'F that have been hybridized
to probe and digested with S1 nuclease (positive control); Lane 4
contains RNA isolated from CHO cells that had been transfected with
pMHC-CAT but were not induced by dexamethasone, hybridized to the
probe and digested with S1 nuclease; and Lane 5 contains RNA from
CHO cells that had been transfected with pMHC-CAT and induced with
dexamethasone, hybridized to the probe and digested with S1
nuclease. All mobilities were as expected, and, as can been seen in
Lane 5, RNA transcribed from the "hairpin" autocatalytic
cassette-CAT gene fusion was cleaved in vivo into the expected
products.
[0280] Another control was uncleaved "hairpin" autocatalytic
cassette RNA hybridized to the probe, S1 nuclease digested and
electrophoresed under the same conditions. No cleavage products
were seen (data not shown). This control shows that cleavage of the
"hairpin" autocatalytic cassette RNA did not occur as a result of
the analysis conditions and, therefore, that the cleavage products
seen in Lane 5 must have been produced as a result of in vivo
cleavage of the hybrid CAT-"hairpin" autocatalytic RNA.
Example 28
[0281] The following example illustrates in vivo activity of the
"hairpin" catalytic RNA in plants. The "hairpin" autocatalytic
cassette (see Example 23) was ligated to cauliflower mosaic virus
(CMV) in a viral vector. Plants were then transformed with the
resulting vector and, during replication of the virus, the
"hairpin" autocatalytic cassette RNA cleaved the viral RNA
intramolecularly. The viral RNA serves as a template for viral
replication and for attenuation of the virus. Since cleavage levels
of the "hairpin" autocatalytic cassette were about 50-60% in vitro,
attenuated viral infection in these plants would be expected if the
"hairpin" autocatalytic cassette RNA linked to the CMV RNA did
cleave in vivo in the plants, and this was what was observed.
[0282] The constructions tested had the "hairpin" autocatalytic
cassette in the sense and antisense orientation. They are shown in
FIG. 26.
[0283] These constructions were made by removing the "hairpin"
autocatalytic cassette from vector pHC (prepared as described in
Example 23) with XhoI/SalI and ligating this fragment into the
unique XhoI site of CMV plasmid pCS101 (a gift from Dr. Art Hunt,
University of Kentucky). Plasmid pCS101 contains the entire CMV
sequence. The XhoI site is located in gene II of CMV and was chosen
because DNA can be cloned into this site without subsequently
interfering with plant infection. Another plasmid identical to
pCS101, except having a pBR322 bacterial replicon, is available
from American Type Culture Collection, and can be used in its place
as a source of CMV. The "hairpin" autocatalytic cassette could be
inserted in either of two orientations (sense or antisense) since
XhoI and SalI ends are compatible.
[0284] The resulting constructions were grown in XL-1 blue E. coli
(Stratagene), and clones of the "hairpin" autocatalytic cassette
insert were isolated and identified by cleaving with KpnI and by
electrophoresing the insert on 1% agarose gels. The isolated clones
were pCS101HC7 and pCS101HC9 which had the "hairpin" autocatalytic
cassette in the sense and antisense orientation, respectively.
These constructions are shown in FIG. 26. Construction pCS101HC7
was designed so that CMV 35S RNA containing the "hairpin"
autocatalytic cassette would be transcribed from it and cleaved if
the "hairpin" autocatalytic cassette was active in vivo.
[0285] After being grown in E. coli, the constructions were cut
with SalI, and were rubbed onto 10 turnip (Brassica campestis)
plants (0.5 ug DNA/plant) which were three weeks old. Four groups
of plants were treated as follows:
[0286] A. Control-mock inoculated (no virus)
[0287] B. Virus control--no "hairpin" autocatalytic cassette
insert--pCS101
[0288] C. Virus with the sense "hairpin" autocatalytic
cassette--pCS101HC7
[0289] D. Virus with the antisense "hairpin" autocatalytic
cassette--pCS101HC9
[0290] The plants were checked weekly for viral symptoms. Within
six weeks, plants treated with wild-type virus were observed to
have viral symptoms, including vein clearing, leaf wrinkling and a
yellow mosaic pattern on the leaf. In turnips treated with
pCS101HC7, the onset of symptoms was delayed 7-10 days as compared
to turnips infected with wild-type virus, and the severity of the
symptoms never reached the levels attained with the wild-type
virus. The following was observed when the plants were nine weeks
old:
4 Treatment Symptoms A No symptoms B Viral symptoms, worst case,
most yellowing (chlorosis) and most lesions. C Viral symptoms,
distinct chlorosis and lesions, but much less than in the virus
control. D Viral symptoms, less than viral control, but not as much
yellowing as the virus with the sense "hairpin" autocatalytic
cassette. Some plants looked uninfected.
[0291] These results indicate that a "hairpin" catalytic RNA
according to the invention can cleave viral RNA in which it is
inserted so that the viral infection is attenuated (Treatment C).
Surprisingly, viral attenuation was also obtained with the
"hairpin" autocatalytic cassette linked to the viral DNA in the
antisense direction (Treatment D). This is believed to be due to
the deletion or disabling of the viral construct containing the
antisense catalyst, so that viral replication is diminished
compared to virus control (Treatment B).
[0292] Next, RNA was isolated from the leaves of the nine-week old
plants, a probe was prepared and hybridized to the isolated RNA, S1
nuclease digestion was carried out, and the S1 digests separated on
10% acrylamide-7M urea gels and analyzed by autoradiography, all as
described in Example 27. The probe used was P.sup.32-labelled
autocatalytic cassette RNA. The results are shown in FIG. 27A,
where the 3'F and 5'F resulting from in vivo RNA cleavage were
found only in plants infected with pCS101HC7 having the "hairpin"
autocatalytic cassette inserted in the sense direction (see Lane
5). None of the other control plants, including those infected with
the antisense construct pCS101HC9, showed these cleavage products,
(see Lanes 3, 4 and 6).
[0293] RNA isolated from the leaves of the nine-week old plants was
also subjected to Northern blot analysis. Total RNA was prepared by
the guanidium thiocyanate method (Chirgwin et al., Biochemistry,
18, 5294-99 (1979)) followed by pelleting through a 5.7 M CsCl step
gradient. Then, 2.5 ug of total RNA was denatured at 65.degree. C.
for 5 min. with formaldehyde and formamide. The denatured RNA was
then electrophoresed on a 1% agarose/formaldehyde gel, and the RNA
was transferred to a Duralon-UV nylon filter (Stratagene) in
10.times. standard sodium citrate (SSC) (0.15 M). After UV
crosslinking, the filter was prehybridized in 6.times.SSC and
0.05.times. BLOTTO (Sambrook, Pritsch and Maniatis, Molecular
Cloning .sctn. 1.102 (2nd ed. 1989)) for 3 hrs at 68.degree. C. The
"hairpin" autocatalytic cassette fragment was labeled with P.sup.32
using the oligolabeling procedure of Feinberg and Vogelstein, Anal.
Biochem., 132, 6-9 (1983). Then it was heated to 100.degree. C. for
5 min. and cooled on ice. This probe was added to the
prehybridization mix, and incubation was continued at 68.degree. C.
overnight. The nylon filter was washed once in 2.times.SSC, 0.1%
SDS for 20 min. at room temperature, followed by washing with
1.times.SSC, 0.1% SDS for 60 min. at 68.degree. C. and
0.1.times.SSC, 0.1% SDS for 60 min. at 68.degree. C. The filter was
then exposed overnight to Kodak X-OMAT film using two lightning
plus screens. The results are shown in FIG. 27B. Of interest is the
detection of viral RNA transcripts of about the size expected
before cleavage (8 Kb) and after cleavage (6 Kb and 2 Kb) following
infection with pCS101HC7 (Lane 3).
[0294] Some reproducible size heterogenity was observed in the S1
fragments and in the Northern blots. This may be due to the rapid
degradation by plant nucleases of these RNA fragments which contain
either a 2'-3' cyclic phosphate or a 5' hydroxyl after ribozyme
cleavage. The 5' hydroxyl could mimic naturally occurring RNA
degradation signals. After infecting turnips with pCS101HC7, RNAs
of the sizes expected before and after RNA cleavage were detected,
but the amount of the 6 Kb fragment detected was somewhat less than
the 2 Kb fragment. This could be due to enhanced stability of 2 Kb
transcript which contained a cyclic phosphate over the 6 Kb
transcript which contained the 5' hydroxyl.
[0295] To determine if the DNA encoding the "hairpin" autocatalytic
ribozyme was stable in vivo, total DNA was isolated from the leaves
of mock-inoculated turnips, turnips inoculated with wild-type CMV
(pCS101) and turnips inoculated with pCS101HC7. DNA was isolated
from plants essentially as described in Murray and Thompson,
Nucleic Acids Research, 8, 4321-25 (1980). Oligo-nucleotide primers
homologous to domains 5' and 3' to the XhoI site of the parental
CMV clone pCS101 were used as primers to amplify DNA sequences
between these domains by polymerase chain reaction (PCR). Primer 1,
which includes an EcoRI site, hybridizes 63 bases upstream from the
XhoI site and has the following sequence:
[0296] 5'-GGAATTCACC CGTCAGTTTT TAATACTGC-3' [SEQ ID 1]
[0297] Primer 2 includes a BamHI site and hybridizes 54 bases
downstream from the XhoI site and has the following sequence:
[0298] 5'-TGGATCCATT CTAGTATTTTG AGCTTCT-3' [SEQ ID 2]
[0299] The primers were synthesized on an Applied Biosystem 391
PCR-MATE using phosphoramidite chemistry. PCR was performed as
described by the vendor of TaQ polymerase (Perkin-Elmer). Briefly,
PCR conditions were 94.degree. C. for 1 min., 55.degree. C. for 2
min. and 72.degree. C. for 3 min. for 35 cycles. After PCR
amplification, the amplified DNA was size fractionated on a 2%
agarose gel and stained with ethidium bromide.
[0300] The results are shown in FIG. 27C. When total plant DNA from
mock-infected turnips was fractionated, no bands were observed
(FIG. 27C, lane 1). Total DNA isolated from plants infected with
wild-type CMV gave an expected band of about 123 bp (FIG. 27C, lane
2). DNA isolated from plants infected with pCS101HC7 gave an
expected band of 225 bp (FIG. 27C, lane 3). In the latter two
cases, the amplified DNA was the same size as those bands amplified
using the intact plasmids pCS101 and pCS101HC7 (FIG. 27C, lanes 4
and 5). These results indicate that at least the majority of the
DNA coding for the "hairpin" autocatalytic ribozyme was retained
intact in the viral genome after infection.
[0301] Finally, protein extracts were prepared from plants that
were mock-inoculated, inoculated with wild-type CMV and inoculated
with pCS101HC7, and the levels of CMV coat protein in the extracts
determined by Western immunoblot. The extracts were prepared by
homogenizing turnip leaf tissue in an equal volume of phosphate
buffered saline using a mortar and pestle. Samples were boiled for
10 min. and then spun down in a microfuge for 10 min. at 4.degree.
C. Protein concentrations of the supernatants were determined by
the method of Bradford, Anal. Biochem., 72, 248-54 (1976), and the
extracts were diluted with an equal volume of 4.times. Laemmli
sample buffer and denatured at 100.degree. C. for 5 min. (Laemmli,
Nature, 227, 680-85 (1970)). The samples were next electrophoresed
on 10% SDS-polyacrylamide gels with 5% stacking gel (id.), followed
by electrotransfer of the proteins to nitrocellulose at 45 volts,
1.25 hours, in transfer buffer as described in Towbin et al., Proc.
Natl. Acad. Sci. USA, 76, 4350-54 (1979). Prestained molecular
weight markers (BRL) were used to confirm protein transfer.
Nonspecific antibody binding was avoided by using blocking solution
(Johnson et al., Gene Anal. Techn., 1, 3-8 (1984)). All antibody
incubations were done at 22.degree. C. with gentle agitation. The
primary antibody was rabbit antiserum to CMV coat protein (1:1000
dilution in blocking solution) provided by Dr. R. Shepherd,
University of Kentucky. An anti-rabbit IgG alkaline phosphatase
conjugate (Sigma, St. Louis) was used as the secondary antibody
(1:1000 dilution in blocking solution). Visual detection of
proteins was accomplished using BCIP/NBT (Sambrook et al.,
Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring Harbor
Laboratory 1989)).
[0302] The results are presented in FIGS. 27D and 27E. No coat
protein was observed in extracts from mock-inoculated plant tissues
one or two months after infection (FIGS. 27D and 27E, lane 1). In
extracts from plants inoculated with wild-type virus, coat protein
was found in significant quantities at one month and two months
post infection (FIG. 27D and FIG. 27E, lane 2). One month after
infection with the ribozyme construct pCS101HC7, very little coat
protein was detected (FIG. 27D, lane 3). Two months after infection
with pCS101HC7, increased levels of coat protein approaching those
found after wild-type CMV infection were detected (FIG. 27E, lane
3).
[0303] The combined results of the pathological observations and
the S1 nuclease, Northern blot, PCR amplification and Western
immunoblot assays provide conclusive evidence that the "hairpin"
catalytic RNA cleaved viral RNA in vivo in plants.
Example 29
[0304] A highly conserved sequence in HIV-1 viral RNA has been
discovered which has favorable properties as a potential target
site for a suitably engineered "hairpin" catalytic RNA. The 16-base
sequence is shown in FIG. 28. Cleavage occurs between the two bases
found at positions 111/112 as counted from the 5' cap site (all
HIV-1 sequences are from the HIV Sequence Data Base, prepared and
distributed by Gerald Myers et al., Los Alamos National Laboratory,
Los Alamos, N.Mex., telephone (505) 665-0480).
[0305] When various HIV-1 isolates were compared, only two isolates
showed variations in the sequence, and the variations were, in each
case, only a single base change (see FIG. 29) (sequences shown in
FIG. 29 are from the HIV Sequence Data Base). The conserved target
site sequence is found in the 5' leader region of all nine HIV-1
mRNAs (see FIG. 28). Thus, a catalytic RNA engineered to cleave the
conserved target sequence should cleave all of these mRNAs and,
thereby, prevent or reduce the spread of the virus.
[0306] A "hairpin" catalytic RNA was designed according to the
principles set forth herein to cleave the conserved target
sequence. The catalytic RNA sequence, designated herein as "RHIV",
is shown in FIG. 30. RHIV was designed so that it would base pair
with the target sequence in the two regions flanking the CGUC
cleavage sequence (see FIG. 30). The "hairpin" portion of RHIV has
the same sequence as the "hairpin" portion of the (-)sTRSV
catalytic RNA sequence shown in FIG. 1. Bases 3'-CUGAGGG-5' at the
5' end of RHIV are vector bases and are not necessary for activity
of the catalyst. RHIV was synthesized as described in Example
2.
[0307] A substrate RNA containing the conserved 16-base target site
sequence shown in FIG. 28 plus additional GCG vector bases at its
5' end was also synthesized as described in Example 2. This
substrate RNA is designated herein as "SHIV". The sequence of SHIV
is shown in FIG. 30.
[0308] The substrate RNA SHIV and catalytic RNA RHIV were then
reacted under standard conditions as described in Example 3 over a
period of time from 0 to 280 minutes. The concentration of
substrate SHIV was 0.025 uM, and the concentration of RHIV was
0.005 uM. Cleavage of substrate SHIV (which contains the conserved
HIV-1 sequence) by the engineered "hairpin" catalytic RNA RHIV was
obtained (see FIG. 31).
[0309] The kinetics of the reaction between SHIV and RHIV were
investigated using the methods described in Example S. The results
are shown in FIG. 32. The time of incubation was 5 minutes, and the
concentration of RHIV was 0.005 uM. The concentration of SHIV was:
Lane 1--0.10 uM; Lane 2--0.05 uM; Lane 3--0.025 uM; Lane 4--0.012
uM; Lane 5--0.006 uM; and Lane 6--0.025 uM (this is the control
lane at zero time).
[0310] The rate of cleavage (turnover number, kcat) was determined
to be 1.6/minute (see FIG. 32). This is a very rapid rate when
compared to any other catalytic RNA under the mild conditions of
temperature, pH and salt concentration used. The Km was determined
to be 100 nM (see FIG. 32), which is also very small for an RNA
catalyst under these conditions. The low Km indicates that the
reaction proceeds very well at extremely low concentrations of
substrate.
[0311] Next, the ability of the catalytic RNA RHIV to cleave a long
HIV-1 transcript was tested. The RNA used as a target sequence was
a 183 nt-long transcript containing 107 nt of HIV-1 sequence. The
target transcript was made by T7 RNA polymerase transcription (as
described in Example 2) of the HaeII-linearized plasmid pROS. This
plasmid was obtained from Dr. John Rossi of the City of Hope
Medical Center, Duarte, Calif. Plasmid pROS has the 5'-HindIII
fragment of the HIVHXB2 isolate cloned into plasmid pBluescript II
(Stratagene, LaJolla, Calif.). The 5'-HindIII fragment of HIVHXB2
contains the sequence (+)77 to (+)635 from the 5' cap site
(sequence obtained from the HIV Sequence Data Base). The 183 nt
transcript contains 76 nt of vector sequence at the 5' end,
followed by 107 nt of HIV-1 sequence (the sequence from (+)77 to
(+)183 from the 5' cap site).
[0312] This 183 nt transcript (5 nM) was incubated with catalytic
RNA RHIV (25 nM) under standard conditions for 60 minutes as
described in Example 3. When the products were analyzed on 6%
acrylamide-7M urea gels, cleavage was seen to have occurred (see
FIG. 33). All mobilities in FIG. 33 were as expected. This result
shows that RHIV, a "hairpin" catalytic RNA according to the
invention, can cleave a long HIV-1 RNA transcript in vitro under
conditions near physiological for mammalian cells.
Example 30
[0313] As a prerequisite for in vivo experiments, it was necessary
to develop a system for the proper termination of the "hairpin"
catalytic RNA RHIV when it was transcribed in vivo. Such a system
was developed by cloning DNA coding for RHIV into the BamHI/MluI
sites of the vector pHC (prepared as described in Example 23) to
give plasmid pHR (see FIG. 34). In plasmid pHR, DNA coding for RHIV
is 5' to DNA coding for the autocatalytic cassette RNA (see FIG.
20).
[0314] Plasmid pHR was transcribed with T7 RNA polymerase and the
resulting 101 nt RNA (see FIG. 34) isolated, all as described in
Example 2. The catalytic activity of this 101 nt RNA transcript
(designated herein as "PRHIV") was assayed against the substrate
SHIV (see FIG. 30) as described in Example 3. The concentration of
catalytic RNA (RHIV or PRHIV) was 0.025 uM, and the concentration
of SHIV was 0.10 uM. The 101 nt PRHIV had the same catalytic
activity as the shorter RHIV (see FIG. 35 where R designates RHIV
and PR designates PRHIV), indicating that the extra sequence at the
3' and 5' ends (see FIG. 34) did not interfere with cleavage of the
substrate.
[0315] Next, a mammalian expression vector was prepared by removing
DNA coding for RHIV and the autocatalytic cassette RNA from pHR
with SmaI/SalI (see FIG. 34). The resulting fragment was cloned
into the mammalian vector pMSG (see Example 27) cut with SmaI/SalI
to give pMSGRHIV (see FIG. 36). In pMSGRHIV, the catalytic RNA is
driven by the MMTV promoter which is dexamethasone inducible.
[0316] Vector pMSGRHIV has been used to transfect human cells which
were then infected with HIV-1. Preliminary results show that the
amount of tat and gag mRNAs produced in transfected cells was lower
than the amount produced in control cells.
Example 31
[0317] In this example, evidence is presented showing the in vivo
down regulation of two genes in mammalian cells by using "hairpin"
RNA catalysts in trans. The two genes studied were hypoxanthine
guanosine phosphoribosyl transferase (HGPRT) and chloramphenicol
acetyl transferase (CAT).
[0318] A. In Vivo Inhibition of HGPRT
[0319] A 15 nt target sequence in the HGPRT gene of the hamster was
identified. The sequence is:
5 AUUCC*GUCAUGGCGA [SEQ ID 3]
[0320] A "hairpin" catalytic RNA was designed according to the
principles set forth herein to cleave this target sequence. The
catalytic RNA sequence, designated "RHGPT," is shown in FIG. 37.
RHGPT base pairs with the target sequence in the two regions
flanking the CGUC cleavage sequence, and the "hairpin" portion of
RHGPT has the same sequence as the "hairpin" portion of the
(-)sTRSV catalytic RNA sequence shown in FIG. 1. Additional vector
bases coding for restriction sites are present at the 3' and 5'
ends of RHGPT.
[0321] RHGPT and the target sequence were synthesized as described
in Example 2 and tested in vitro as described in Example 3. The
results of the test showed that the target sequence was cleaved in
vitro by the engineered "hairpin" ribozyme RHGPT at the * in the
sequence given above (which is after nt87 in the sequence of the
HGPRT gene).
[0322] Next, DNA coding for ribozyme RHGPT was cloned into the
BamHI/MluI sites of the mammalian expression vector pHC (prepared
as described in Example 23). The DNA coding for RHGPT and HC was
excised from the resulting plasmid with SmaI/SalI and cloned into
SmaI/SalI digested pMSG (Example 27) to give plasmid pMRHPT, a map
of which is shown in FIG. 37. The DNA coding for RHGPT is located
downstream of the dexamethasone-inducible MMTV promoter on pMRHPT
and upstream of the "hairpin" autocatalytic cassette (HC) which is
included so that RHGPT will be properly terminated at the 3' end
after the RNA is transcribed.
[0323] Another plasmid, pMR2HPT, was also prepared. This plasmid is
identical to pMRHPT, except that a G35-->C mutation in RHGPT was
made (numbering according to FIG. 42D). Accordingly, the resulting
ribozyme produced by this plasmid would be inactive (see Example
22).
[0324] Chinese hamster ovary cells (CHO) were cotransfected with
one of these plasmids and plasmid pMSG-dhfr. The method of
transfection and plasmid pMSG-dhfr are described in Example 27.
Transfectants were selected and amplified with methotrexate, also
as described in Example 27. A schematic drawing of the selection
scheme is presented in FIG. 38.
[0325] The poison 8-azaguanine, which is allowed into cells by the
HGPRT enzyme, was then added to the cells (80 .mu.g/ml) both in the
presence and absence of dexamethasone (1 .mu.M) which induces the
HGPRT ribozyme. In the absence of dexamethasone, cells with or
without DNA coding for RHGPT died as expected. In the presence of
dexamethasone, control cells died, but colonies of survivors were
seen for cells transfected with pMRHPT. All cells transformed with
pMR2HPT (coding for the inactive ribozyme) died.
[0326] To quantitate the rate of survival, CHO cells were grown in
gpt medium (see Example 27), except untransfected, uninduced CHO
cells which were grown in MEM. The concentration of 8-azaquanine
used was 80 .mu.g/ml, and the concentration of dexamethasone used
was 1 .mu.M. One hundred cells were plated per dish. Colonies of
cells were stained with crystal violet. The results are presented
below:
6 Cells Percent Survival Untransfected, 100%* uninduced Transfected
with 33% pMRHPT and induced Transfected with 0% pMRHPT but
uninduced Transfected with 0% pMR2HPT and induced *Results were
normalized with untransfected, uninduced cells set at 100%. Actual
survival rate for these cells was 80%.
[0327] The results are consistent with reduced levels of HGPRT due
to the cleavage of HGPRT mRNA by the engineered "hairpin" ribozyme
RHGPT after induction with dexamethasone. The in vivo activity of
the ribozyme is not likely due to antisense effects, since the
disabled ribozyme coded for by pMR2HPT was ineffective in
increasing resistance to 8-azaquanine. Note that the mutation in
pMR2HPT is not in the area of the ribozyme that base pairs to the
substrate, so binding to the substrate should occur.
[0328] Next, an S1 nuclease assay was performed to observe the in
vivo levels of mRNA coding for HGPRT. The S1 nuclease assay was
performed as described in Example 27. The 148 nt probe which was
used hybridizes to HGPRT mRNA and was prepared by transcribing
plasmid pHPTPr as described in Example 27 using P.sup.32-labelled
CTP. Plasmid pHPTPr contains the antisense HGPRT sequence from nt
20-160 (Konecki et al., Nucleic Acids Res., 10, 6763-75 (1982))
bridging the cleavage sequence at nt 87 cloned into pTZ18R (US
Biochemical) between the EcoRI and HindIII sites.
[0329] The results are shown in FIG. 39. In FIG. 39, lane 3
contains RNA from cells transfected with pMRHPT and pMSG-dhfr but
not induced, and lane 4 contains RNA from cells transfected with
pMRHPT and pMSG-dhfr which were induced with dexamethasone. A 30%
reduction in the level of mRNA was observed when the "hairpin"
ribozyme was induced (compare lanes 3 and 4). This shows that the
engineered "hairpin" ribozyme RHGPT reduces HGPRT activity by
lowering the amount of mRNA, and the likely mechanism is cleavage
of the HGPRT mRNA.
[0330] B. In Vivo Lowering of CAT mRNA Levels.
[0331] From a series of in vitro experiments using the techniques
described in Examples 2 and 3, the optimum target sequence in the
CAT gene was determined to be:
[0332] 5-UUUCA*GUCAGUUGCUCAA-3' [SEQ ID 5]
[0333] with cleavage at (*), which is nt 320 of the CAT gene. The
"hairpin" catalytic RNA designed to cleave this target sequence,
designated "RCAT," is shown in FIG. 40. RCAT was designed according
to the principles set forth herein. It base pairs with the target
sequence in the two regions flanking the AGUC cleavage sequence,
and the "hairpin" portion of RCAT has the same sequence as the
"hairpin" portion of the (-)sTRSV catalytic RNA sequence shown in
FIG. 1. Additional vector bases coding for restriction sites are
present at the 3' and 5' ends of RCAT.
[0334] DNA coding for RCAT was ligated to pHC (Example 23) which
had been cut with BamHI/MluI. The resulting plasmid was cut with
SmaI/SalI to remove the fragment coding for RCAT and HC. This
fragment was ligated to pMSG (Example 27) which had been cut with
SmaI/SalI to give pMSGRCAT. Next pCAT (Promega) was cut with
PvuI/PstI, and the fragment containing the CAT gene under the
control of the SV40 promoter and enhancer was isolated on a 1%
low-melting agarose gel (Nusieve). Then the fragment was blunt
ended with Klenow fragment and ligated into the EcoRI-cut,
blunt-ended pMSGRCAT to give the final plasmid pMCATRCAT (see FIG.
40). This plasmid contains DNA coding for RCAT under control of the
dexamethasone-inducible MMTV promoter and the CAT gene under
control of the SV40 promoter. Further, DNA coding for the
autocatalytic "hairpin" ribozyme is located downstream of DNA
coding for RCAT so that RCAT will be properly terminated at the 3'
end upon transcription of the RNA.
[0335] Plasmid pMCATRCAT was cut with NdeI and then used to
transfect CHO cells along with XhoI-cut pMSG-dhfr as described in
Example 27. Transfectants were selected and amplified and an S1
nuclease assay performed, all as described in Example 27. The probe
used for the S1 nuclease assay was RNA transcribed from the plasmid
pCATP. This plasmid contains the sequence of the CAT gene from nt
260-372 (GenBank sequence) bridging the cleavage sequence at nt 320
cloned into the EcoRI/HindIII site of pTZ18R (US Biochemical) in
the antisense direction. The probe was prepared as described in
Example 27 using .sup.32P-CTP and was a total of 119 nt long.
[0336] The results of the S1 nuclease assay are presented in FIG.
41. A reduction in CAT mRNA was observed in cells transfected with
pMCATRCAT and pMSG-dhfr and induced with dexamethasone as compared
to uninduced cells. Thus, the engineered "hairpin" catalyst RCAT
did reduce the level of CAT mRNA. However, further attempts to
locate the mRNA cleavage products have so far failed, and no
lowering of CAT enzymatic activity was seen.
Example 32
[0337] Additional mutagenesis experiments were performed changing
bases in (-)sTRSV RNA and its substrate. All substrate RNAs and
catalytic RNAs were prepared as described in Example 2. Mutagenesis
was carried out simply by making the required base change in the
synthetic DNA template. All catalytic RNAs had additional vector
bases GGG at the 5' end, and all substrate RNAs had additional
vector bases GCG at the 51 end. These bases are required for
efficient transcription (Milligan et al., Nucleic Acids Res., 15,
8783-98 (1987)), and the C near the 5' end of all substrates
ensured at least one P.sup.32-labelled C in the 5' cleavage
fragment. The reference sequences were the unmutated catalytic and
substrate sequences (see FIG. 1).
[0338] Substrate and catalytic RNAs were assayed for catalytic
activity as described in Example 3. Generally, the final
concentrations of substrate RNA was 0.1 uM, and the final
concentration of catalytic RNA was 0.01 uM. Assays were done at
37.degree. C. for times ranging from 15-30 minutes, and a zero-time
control was always included. Reaction products were analyzed on 15%
acrylamide/7M urea gels, autoradiography performed, and the bands
cut from the gels and counted. The control (unmutated
ribozyme/substrate) was assayed at the same time as all mutant
catalytic RNAs and substrates.
[0339] Nucleotide changes made in the native, unmutated sequence of
both the catalytic RNA and the substrate showed a range of
catalytic effects. The nucleotide changes shown in FIG. 42A had
very little effect on the catalytic activity of the ribozyme
(50-100% of the activity of the unmutated sequence), while other
nucleotide changes (FIG. 42B) had an intermediate effect on
catalytic activity (5-50% of the activity of the unmutated
sequence). Those mutational changes resulting in very low or no
catalytic activity (less than 5% of the activity of the unmutated
sequence) are shown in FIG. 42C. The results further define the
two-dimensional structure of the (-)sTRSV catalytic complex. A
revised "hairpin" structure for the catalytic complex is shown in
FIG. 42D. In FIGS. 42A-D, the ribozyme is numbered consecutively
1-50 nt and the substrate 1-16 nt (see FIG. 42D). Also, in FIGS.
42A-D, upper case letters are used for the ribozyme nucleotides and
lower case letters are used for the substrate nucleotides.
[0340] In summary, the results show that Helices 2 and 3 (see FIG.
42D) are not continuous but have an un-paired base between them.
Helix 4 was found to be shorter and Loops II and IV were found to
be larger than previously predicted by computer modeling. Also,
certain bases in Loops II and IV were found to be invariant. Helix
4 can be extended towards the closed end (Loop III) of the
"hairpin" to give increased stability to the ribozyme, and the
sequence of Loop III can be mutated with retention of catalytic
activity. While conventional base pairing interactions between Loop
I on the ribozyme and Loop V of the substrate were not observed, an
A-->C mutation in ribozyme Loop I partly restored activity to a
previously inactive c-->a mutation in substrate Loop V,
indicating that some type of interaction between these two bases
may be occurring. Finally, the data show that the first base pair
upstream of the N*GUC cleavage sequence cannot be an A:U or U:A, it
must be G:C or C:G. It is believed that the G:C or C:G base pair is
necessary for stability of the catalyst-substrate complex and that
A:U or U:A base pairs can be used if sufficient stability is
provided by other means such as possibly lengthening Helix 1.
Indeed, it has been found that A:U and U:A base pairs can be used
at these positions in the synthetic autocatalytic catalyst of the
invention. Accordingly, in engineering a "hairpin" catalyst based
on (-)sTRSV, the substrate RNA preferably contains the target
sequence 5'-SN*GUC-3', where S is G or C and cleavage occurs at the
*.
[0341] The results will now be discussed in detail. First, Table I
lists all substrate sequences successfully cleaved by a catalytic
RNA designed according to the "hairpin" model so that the bases
flanking the N*GUC cleavage sequence in the substrate were base
paired to the catalyst. Cleavage occurred at the *. The lower case
letters in Table I designate additional vector sequences.
7 TABLE 1 Helix2 LoopV Helix1 1 gcg UGAC A*GUC CUGUUU [SEQ ID 8] 2
gcg UGAC A*GUC CUGUUUUUUU [SEQ ID 9] 3 gcg UGAC A*GUC CUGUUUUUUUCGC
[SEQ ID 10] 4 gcg UGUC A*GUC CUGUUU [SEQ ID 11] 5 gcg UGAG A*GUC
CUGUUU [SEQ ID 12] 6 g AAAC A*GUC CCCAAC [SEQ ID 13] 7 g UUUC A*GUC
AGUUGC [SEQ ID 14] 8 gcg UUUC A*GUC AGUUGCUCAA [SEQ ID 15] 9 gcg
CCCC U*GUC CCCGAG [SEQ ID 16] 10 gcg UGGG U*GUC GACAUAgc [SEQ ID
17] 11 gcg UGAC A*GUC GUGUUU [SEQ ID 18] 12 gcg UGAC A*GUC AUGUUU
[SEQ ID 19] 13 gcg AGAG C*GUC GGUAUUAA [SEQ ID 20] 14 gcg AGAG
C*GUC GGUAUUAAGCGG [SEQ ID 21] 15 gcg AGAG C*GUC GGUAUUAAGC [SEQ ID
22] 16 gcg UUUC U*GUC GUUUAACU [SEQ ID 23] 17 gcg UGAC U*GUC CUGUUU
[SEQ ID 24] 18 gcg UGAC C*GUC CUGUUU [SEQ ID 25] 19 gcg UGAC G*GUC
CUGUUU [SEQ ID 26] 20 gcg UGCC C*GUC UGUUGUGUGA [SEQ ID 27] 21 gcg
UGCC C*GUC UGUUGUGU [SEQ ID 28] 22 gcg CCAC U*GUC GAUCGA [SEQ ID
29] 23 gcg CCAC U*GUC GAUCGAG [SEQ ID 30] 24 gcg AUUC C*GUC AUGGCGA
[SEQ ID 31] 25 gcg AUUC C*GUC AUGGC [SEQ ID 32] 26 gcg AUGC G*GUC
ACUCAUUA [SEQ ID 33] 27 gcg AUGC G*GUC ACUCAU [SEQ ID 34] 28 gcg
AUCC U*GUC CAUUCAA [SEQ ID 35] 29 gcg AUCC U*GUC CAUUCAAG [SEQ ID
36] 30 gcg UUGG U*GUC GACCUGAA [SEQ ID 37] 31 gcg ACAG C*GUC UGCUCC
[SEQ ID 38] 32 gcg UUGC G*GUC GCUACG [SEQ ID 39] 33 gcg UUGC G*GUC
GCUACGUC [SEQ ID 40] 34 gcg UCUC A*GUC ACUAUG [SEQ ID 41] 35 gcg
CACC U*GUC ACAUAA [SEQ ID 42] 36 gcg CACC U*GUC ACAUAAUU [SEQ ID
43] 37 gc GUGG U*GUC UGUGGA [SEQ ID 44]
[0342] As shown in Table I, every base pair in Helices 1 and 2 can
be changed to any other base pair, and the substrate will be
cleaved by the ribozyme, except the base pair in Helix 2 adjacent
to the N*GUC cleavage sequence (designated by S in FIG. 42D). When
base G11 in the ribozyme was changed to C, catalytic activity was
lost (see FIG. 42C). When a second mutation (c4-->g) was made in
the substrate so that base pairing was restored, catalytic activity
was also restored. However, when catalysts and substrates having
A:U base pairs at these positions were tested, no catalytic
activity was observed. A:U base pairs in this position were checked
for catalytic activity with a variety of substrates and
corresponding ribozymes, and all of them were found to be inactive.
Accordingly, this base pair must be G:C or C:G and cannot be A:U or
U:A unless, as discussed above, other measures are taken to
stabilize the substrate-catalyst complex.
[0343] As already demonstrated in Examples 18 and 21, it is
possible to adjust the length of Helix 1 to optimize the rate of
cleavage. In particular, when the native sequence was extended four
base pairs by adding four A:U base pairs to the open end of Helix
1, an increased rate of activity was seen (Example 21). However,
when three additional G:C base pairs were added in the present
experiments, a large loss of activity occurred. This phenomenon was
observed for numerous substrates and ribozymes. Accordingly, Helix
1 has an optimal length for each substrate used.
[0344] A type of "hinge" region, consisting of a single A base at
position 15, is present between Helices 2 and 3 of the catalytic
RNA (see FIG. 42D). When the A15:U49 potential base pair was
changed to the compensatory base pair U15:A49 by a double mutation,
activity remained at nearly 100% (FIG. 42A). The single U49->A49
mutation, which would lead to an A:A mismatch, also had no effect
on activity (FIG. 42A), showing that no base pair was needed at
this position. Accordingly, the results show that a base pair does
not exist between bases A15 and U49 of the ribozyme.
[0345] Proof of the base pair C17:G47 was obtained previously (see
Example 22), but the presumptive base pair next to it, C16:G48,
could not be shown to exist. Both the catalyst containing the C:C
mismatch and the catalyst containing the reverse G:C base pair were
inactive (FIG. 42C). Since this G and C are opposite each other in
this position and are adjacent a base pair, it is highly likely
they are actually base paired as well. However, the fact both the
mismatch and reverse base pair were inactive suggests that the
identity of the bases must be maintained in this position. Thus, it
is likely that this base pair exists and must be C16:G48 as
shown.
[0346] At the end of Helix 3 is the predicted base pair G19:C45.
The catalyst containing the C:C mismatch was inactive (FIG. 42C),
and the catalyst containing the reverse base pair C19:G45 was
active (FIG. 42B), showing that a base pair exists in this
position.
[0347] Base pairing between A18:U46 is shown in FIG. 42D even
though mutagenesis was not done. The existence of a base pair at
this position is likely since it would be located between two
proven G:C base pairs.
[0348] Helix 4 is a shorter helix than predicted by straightforward
computer modeling and two-dimensional energy minimization (compare
FIGS. 1 and 19 with FIG. 42D). This helix was previously shown to
exist by showing that the base pair C27:G35 actually existed (see
Example 22). The next base pair downstream, A28:U34, also exists.
Catalysts containing the single mismatch mutations, U34->A or C,
were inactive (FIG. 42C), and catalysts containing the compensatory
double mutations to form either U28:A34 or G28:C34 base pairs
restored activity (FIG. 42A).
[0349] The following results show that an active ribozyme is
produced when Helix 4 is extended and the sequence of Loop III is
changed. As shown in FIG. 42A, Loop III was replaced with the
common and very stable RNA hairpin sequence 5'-GGAC(UUCG)GUCC-3'
[SEQ ID 45] characterized by Tinoco and colleagues (Cheong et al.,
Nature, 346, 680-82 (1990)); Varani et al., Biochem., 30, 3280-89
(1991). As a result of this substitution, Helix 4 was extended by
four base pairs and the GU sequence of Loop III was replaced with
the sequence UUCG (see FIG. 42A). The resulting RNA catalyst was
active. In fact, the activity of this ribozyme was greater than
that of the unmutated form. Further, the mutant ribozyme was more
thermal stable. It remained active at 45.degree. C., while the
unmutated RNA catalyst loses most of its activity at this
temperature (see FIG. 43).
[0350] It was concluded from this experiment that Loop III does not
have a conserved or invariant base sequence and that Helix 4 can be
extended towards loop III by at least four base pairs without loss
of activity. The four additional base pairs in Helix 4 should
provide helix stabilization of this region. The secondary folding
energy of Helix 4 and Loop III in the native structure is +0.6
Kcal/mole, while that of the catalyst having the extended Helix 4
and the Loop III of the sequence UUCG was determined to be -11.1
Kcal/mole (methods described in Example 6). Thus, the presence of
the Tinoco et al. hairpin sequence increases the folding energy by
11.7 Kcal/mole.
[0351] However, the simple replacement of Loop III with the
sequence UUCG (see FIG. 42C) gives an inactive ribozyme. This is
believed to occur because the bases of Loop III help stabilize
Helix 4.
[0352] When Loop III of the native ribozyme is cut between U31 and
U32, activity is lost (see FIG. 42C). A likely explanation for this
is that when the loop is cut, Helix 4 opens up and catalytic
activity is, consequently, lost. The "cut" ribozyme was prepared by
synthesizing the ribozyme in two parts and allowing the parts to
anneal.
[0353] With the extension of Helix 4 by the Tinoco et al. hairpin
sequence, the potential base pair C29:G33 is between the two proven
bases of Helix 4 and the four base pairs of the Tinoco sequence.
Accordingly, it seemed likely that this base pair existed. However,
both the single mutation G33-->C and the double mutation
C29:G33-->G29:C33 were inactive (see FIG. 42C). Either a base
pair does not exist at this position or the identity of the bases
must be maintained. This second possibility seems the most likely
since the ribozyme likely needs to be involved in specific
three-dimensional folding to carry out catalysis, and specific
functional groups on the bases may be involved in the folding
and/or the catalysis itself.
[0354] The existence of other base pairs in Helix 4 could not be
shown. The next potential base pair upstream in Helix 4 was
C25:G36. The single mutation C25->G was inactive (FIG. 42C). The
double mutation, G25:C36, was also inactive (FIG. 42C), indicating
that a base pair does not actually exist. Although the base pair
could exist with the identity of the bases being required, this
appears unlikely since the G and C are not directly opposite each
other since there is A between the C and the next proven base pair.
By the same sort of analysis, the next potential base pair
upstream, A24:U37, was shown not to exist. When the mutation
A24:U37->G24:C37 was made, no activity was seen (FIG. 42C),
indicating that a base pair does not exist at this position.
[0355] Further mutation studies showed that Loops II and IV are
larger than originally predicted by computer modeling and energy
minimization (compare FIGS. 1 and 42D). Also, some of the bases in
these loops are required for activity.
[0356] As already shown in Example 12, when bases A22, A23 and A24
in Loop II were mutated to GUC, a totally inactive ribozyme was
obtained, indicating that one or all of these bases are essential.
The single mutation in Loop II of C25->G was inactive (see FIG.
42C). The double mutation C25->G+G36->C was also inactive
(see FIG. 42C).
[0357] In Loop IV, the single mutation U39->G was active (see
FIG. 42A), indicating that this base was not essential for
catalytic activity and was not involved in a base pair. The single
mutation A43->U was inactive, as was the double mutation
A43->U and U37->A. This result was significant because the
possibility existed for a stem to occur in Loop IV:
8 36 G U A U A U C A U 44
[0358] If this stem existed, the A43:U37 alternate base pair of
U43:A37 should have been active. Since it was not, it was concluded
that a stem in Loop IV does not exist.
[0359] In Loop V, the loop formed by the cleavage sequence in the
substrate molecule, the bases guc are invariant and are not base
paired to the catalyst (see Example 25 and FIG. 42C).
[0360] All of the bases of Loop I in the ribozyme, the loop
opposite Loop V, have been mutated. When base A7 was changed to a G
or C and when base A10 was changed to a G, the resulting catalysts
were active (FIG. 42A). However, when base G8 was changed to a U or
C or when base A9 was changed to U, inactive ribozymes were
produced (see FIG. 42C).
[0361] Potential base pairs between Loop V in the substrate and the
Loop I in the ribozyme were tested. In particular, experiments to
determine whether G8 and A9 of Loop I were base paired with the
corresponding bases in substrate Loop V were performed. Mutations
tested were G8->C:c8->g and A9->U:u7->a. These
mutations were in-active (see FIG. 42C), showing that, for the
bases checked, no base pairs existed.
[0362] While the c9-->a mutation in substrate Loop V was
inactive (FIG. 42C) and the A7-->C mutation of ribozyme Loop I
was fully active (FIG. 42A), the double mutation of c9->a and
A7->C showed partial activity (see FIG. 42B). This suggests that
some type of interaction may be occurring between base c9 of the
substrate and base A7 of the ribozyme which are opposite each other
in the loops.
[0363] A triple mutation comprising this same double mutation plus
changing base A20->C in the ribozyme showed the same level of
activity as the double mutation (FIG. 42B). The triple mutation
represents base changes in the ArMV sequence proposed to be
catalytic (Gerlach and Haseloff, Gene, 82, 43-52 (1989)) and would
suggest that a triple base interaction might occur between the
three bases. However, the catalyst containing the triple mutation
gave catalytic activity at the same level as the double mutation,
arguing against a triple base interaction. The results obtained
with the catalyst containing the double mutation, however, indicate
that an interaction of some type takes place between substrate C9
and ribozyme A7.
[0364] As various changes could be made in the above-described
products and methods without departing from the scope of the
invention, it is intended that all matter contained in the above
description or shown in accompanying drawings shall be interpreted
as illustrative and shall not be interpreted in a limiting sense.
Sequence CWU 1
1
114 1 12 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 1 ggacuucggu cc 12 2 10 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
2 gacaguccug 10 3 15 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 3 gugacagucc uguuu 15 4 13
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 4 agaaacacac guu 13 5 36 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 5
accagagaaa cacacguugu gguauauuac cuggua 36 6 18 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
6 cacggacuuc gguccgug 18 7 45 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 7 accagagaaa
cacacggacu ucgguccgug guauauuacc uggua 45 8 68 DNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
8 taccaggtaa tataccacaa cgtgtgtttc tctggttgac ttctctgttt ctatagtgag
60 tcgtatta 68 9 34 DNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 9 aaacaggact gtcacgctat
agtgagtcgt atta 34 10 17 RNA Artificial Sequence Ribozyme, portion
of ribozyme or ribozyme target substrate 10 gcgugacagu ccuguuu 17
11 15 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 11 gaaacagucc ccaac 15 12 50 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 12 guugggagaa guuuaccaga gaaacacacg uugugguaua
uuaccuggua 50 13 15 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 13 guuucaguca guugc 15 14 17
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 14 guuucaguca guugcuc 17 15 21 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
15 ggguuucagu caguugcuca a 21 16 29 DNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 16
ggaattcacc cgtcagtttt taatactgc 29 17 28 DNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 17
tggatccatt ctagtatttt gagcttct 28 18 15 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 18
auuccgucau ggcga 15 19 18 RNA Artificial Sequence Ribozyme, portion
of ribozyme or ribozyme target substrate 19 uuucagucag uugcucaa 18
20 21 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 20 gcgugacagu ccuguuuuuu u 21 21 24 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 21 gcgugacagu ccuguuuuuu ucgc 24 22 17 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 22 gcgugucagu ccuguuu 17 23 17 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
23 gcgugagagu ccuguuu 17 24 15 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 24 gaaacagucc
ccaac 15 25 15 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 25 guuucaguca guugc 15 26 21
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 26 gcguuucagu caguugcuca a 21 27 17 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
27 gcgccccugu ccccgag 17 28 19 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 28 gcgugggugu
cgacauagc 19 29 17 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 29 gcgugacagu cguguuu 17 30
17 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 30 gcgugacagu cauguuu 17 31 19 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 31 gcgagagcgu cgguauuaa 19 32 23 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
32 gcgagagcgu cgguauuaag cgg 23 33 21 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 33
gcgagagcgu cgguauuaag c 21 34 19 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 34 gcguuucugu
cguuuaacu 19 35 17 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 35 gcgugacugu ccuguuu 17 36
17 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 36 gcgugaccgu ccuguuu 17 37 17 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 37 gcgugacggu ccuguuu 17 38 21 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
38 gcgugcccgu cuguugugug a 21 39 19 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 39
gcgugcccgu cuguugugu 19 40 17 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 40 gcgccacugu
cgaucga 17 41 18 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 41 gcgccacugu cgaucgag 18 42
18 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 42 gcgauuccgu cauggcga 18 43 16 RNA
Artificial Sequence Ribozyme, portionof ribozyme or ribozyme target
substrate 43 gcgauuccgu cauggc 16 44 19 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 44
gcgaugcggu cacucauua 19 45 17 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 45 gcgaugcggu
cacucau 17 46 18 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 46 gcgauccugu ccauucaa 18 47
19 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 47 gcgauccugu ccauucaag 19 48 19 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 48 gcguuggugu cgaccugaa 19 49 17 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
49 gcgacagcgu cugcucc 17 50 17 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 50 gcguugcggu
cgcuacg 17 51 19 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 51 gcguugcggu cgcuacguc 19 52
17 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 52 gcgucucagu cacuaug 17 53 17 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 53 gcgcaccugu cacauaa 17 54 19 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
54 gcgcaccugu cacauaauu 19 55 16 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 55 gcgugguguc
ugugga 16 56 16 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 56 ugcccgucug uugugu 16 57 50
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 57 aaacagagaa gucaaccaga gaaacacacg uugugguaua
uuaccuggua 50 58 14 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 58 ugacaguccu guuu 14 59 359
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 59 uggccuacac gaaaggccag acuacucagg cacuccugcu
uuguccugac aguccaccgg 60 cuuucggugg ugcauuugau cacuuggcac
gacgcaucgc auccccagac gauggagcaa 120 ccuccaccuc uaacaucgga
agcacacccg cgccgccaca ucgaucaguu ccgcaugguc 180 cauuauaugg
uguugcacac aaagagacca acugaagaga caaacaacac aguaaccaag 240
ggccuagagc guaaucgccg cugccccaua agaguaagcu guaccuucaa acucucuggc
300 gcggagaugu gauacgcgcc ggccccgcuu agguuuaaca agaucgggcu
augggacag 359 60 50 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 60 aaacagagaa gucaaccaga
gaaacacacg uugugguaua uuaccuggua 50 61 14 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 61
ugacuguccu guuu 14 62 14 RNA Artificial Sequence Ribozyme, portion
of ribozyme or ribozyme target substrate 62 ugaccguccu guuu 14 63
50 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 63 aaacagagaa cucaaccaga gaaacacacg
uugugguaua uuaccuggua 50 64 14 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 64 ugagaguccu guuu
14 65 50 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 65 guugggagaa guuuaccaga gaaacacacg
uugugguaua uuaccuggua 50 66 14 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 66 aaacaguccc caac
14 67 16 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 67 uuucagucag uugcuc 16 68 14 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 68 uuucagucag uugc 14 69 18 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
69 uuucagucag uugcucaa 18 70 16 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 70 agagcgucgg
uauuaa 16 71 14 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 71 ugggugucga caua 14 72 18
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 72 ugacaguccu guuuuuuu 18 73 50 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
73 nnnnnnagaa nnnnaccaga gaaacacacg uugugguaua uuaccuggua 50 74 14
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 74 nnnnngucnn nnnn 14 75 85 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
75 nnnnnnnuga caguccuguu uccuccaaac agagaaguca accagagaaa
cacacguugu 60 gguauauuac cugguannnn nnnnn 85 76 133 DNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
76 ctcgagggcc cgcggccggc aggcctggta ccacgcgtga cagtcctgtt
tcctccaaac 60 agagaagtca accagagaaa cacacgttgt ggtatattac
ctggtagtcg agggatcttt 120 gtgaaggaac ctt 133 77 161 DNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
77 gggaaagctt gcatgcctgc aggtcgacta ccaggtaata taccacaacg
tgtgtttctc 60 tggttgactt ctctgtttgg aggaaacagg actgtcacgc
gtggtaccag gcctgccggc 120 cgcgggccct cgagggatcc ccgggtaccg
agctcgaatt c 161 78 16 DNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 78 tgcccgtctg ttgtgt 16 79 14
DNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 79 tgcccgtctg ttgt 14 80 16 DNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
80 tgcccgtctg ttatgt 16 81 14 DNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 81 tgcccatctg ttgt
14 82 59 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 82 gggagucaca caacaagaag gcaaccagag
aaacacacgu ugugguauau uaccuggua 59 83 19 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 83
gcgugcccgu cuguugugu 19 84 189 DNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 84 gggaattcga
gctcggtacc cggggatccc tcgaggatcc acacaacaag aaggcaacca 60
gagaaacaca cgttgtggta tattacctgg tacgcgtgac agtcctgttt cctccaaaca
120 gagaagtcaa ccagagaaac acacgttgtg gtatattacc tggtagtcga
cctgcaggca 180 tgcaagctt 189 85 159 DNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 85
cccggggatc cctcgaggat ccacacaaca agaaggcaac cagagaaaca cacgttgtgg
60 tatattacct ggtacgcgtg acagtcctgt ttcctccaaa cagagaagtc
aaccagagaa 120 acacacgttg tggtatatta cctggtagtc gacctcgag 159 86
153 DNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 86 gctagcccgg ggatcctcgc catagaagaa
taccagagaa acacacgttg tggtatatta 60 cctggtacgc gtgacagtcc
tgtttcctcc aaacagagaa gtcaaccaga gaaacacacg 120 ttgtggtata
ttacctggta gtcgacctcg agg 153 87 150 DNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 87
cccggggatc cttgagcaac tagaagaaaa ccagagaaac acacgttgtg gtatattacc
60 tggtaacgcg tgacagtcct gtttcctcca aacagagaag tcaaccagag
aaacacacgt 120 tgtggtatat tacctggtag tcgacctcga 150 88 51 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 88 nnnnnnnaga asnnnaccag agaaacacac guugugguau
auuaccuggu a 51 89 15 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 89 nnnsngucnn nnnnn 15 90 18
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 90 cacggacuuc gguccgug 18 91 7 RNA Artificial
Sequence Ribozyme, portion of ribozyme or ribozyme target substrate
91 gggaguc 7 92 5 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 92 snguc 5 93 5 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 93 accag 5 94 6 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 94 cuggua 6 95 7
RNA Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 95 agaaaca 7 96 9 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 96
guauauuac 9 97 5 RNA Artificial Sequence Ribozyme, portion
of ribozyme or ribozyme target substrate 97 ccucc 5 98 9 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 98 guauauuac 9 99 19 RNA Artificial Sequence
Ribozyme, portion of ribozyme or ribozyme target substrate 99
nngannnnnn nanauuacn 19 100 6 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 100 nngann 6 101
14 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 101 acugaagaga caaa 14 102 60 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 102 nnnnnnnaga annnnaccag agaaacacac ggacuucggu
ccgugguaua uuaccuggua 60 103 25 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 103 nngannnaga
aanannnana uuacn 25 104 25 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 104 nagaannaga
aacannnaua uuacn 25 105 25 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 105 nagagnnaga
aacannnaua uuacn 25 106 19 RNA Artificial Sequence Ribozyme,
portion of ribozyme or ribozyme target substrate 106 nngaannnnn
nanauuacn 19 107 19 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 107 nngagnnnnn nanauuacn 19
108 19 RNA Artificial Sequence Ribozyme, portion of ribozyme or
ribozyme target substrate 108 nngannnnnn nanauuacn 19 109 25 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 109 nngannnnga aacannnana uuacn 25 110 25 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 110 nngannnaga aacannnana uuacn 25 111 25 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 111 nagaannaga aacannnaua uuacn 25 112 25 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 112 nagagnnaga aacannnaua uuacn 25 113 50 RNA
Artificial Sequence Ribozyme, portion of ribozyme or ribozyme
target substrate 113 acaacaagaa ggcaaccaga gaaacacacg uugugguaua
uuaccuggua 50 114 56 RNA Artificial Sequence Ribozyme, portion of
ribozyme or ribozyme target substrate 114 gggagucaca acaagaaggc
aaccagagaa acacacguug ugguauauua ccuggu 56
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