U.S. patent application number 09/742966 was filed with the patent office on 2002-06-27 for nucleic acid enzymes for cleaving dna.
Invention is credited to Joyce, Gerald F..
Application Number | 20020081666 09/742966 |
Document ID | / |
Family ID | 23844300 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081666 |
Kind Code |
A1 |
Joyce, Gerald F. |
June 27, 2002 |
Nucleic acid enzymes for cleaving DNA
Abstract
The present invention discloses nucleic acid enzymes capable of
cleaving single-stranded DNA in a site specific manner.
Inventors: |
Joyce, Gerald F.;
(Encinitas, CA) |
Correspondence
Address: |
THE SCRIPPS RESEARCH INSTITUTE
OFFICE OF PATENT COUNSEL, TPC-8
10550 NORTH TORREY PINES ROAD
LA JOLLA
CA
92037
US
|
Family ID: |
23844300 |
Appl. No.: |
09/742966 |
Filed: |
December 20, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09742966 |
Dec 20, 2000 |
|
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08183734 |
Jan 19, 1994 |
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Current U.S.
Class: |
435/87 ; 435/89;
435/91.1 |
Current CPC
Class: |
C12N 15/10 20130101;
C12N 2310/124 20130101; C12N 15/113 20130101 |
Class at
Publication: |
435/87 ; 435/89;
435/91.1 |
International
Class: |
C12P 019/38; C12P
019/30; C12P 019/34 |
Goverment Interests
[0002] This invention was made with government support under NASA
Grant No. NAGW-1671. The government may have certain rights in the
invention.
Claims
I claim:
1. A method for specifically cleaving a single-stranded DNA
molecule, comprising the steps of: (a) providing a first RNA
molecule that is a group I intron that cleaves a second RNA
molecule to leave a 3'-OH, said first RNA molecule having a
deoxyribonuclease activity; and (b) contacting said first RNA
molecule with said single-stranded DNA molecule under conditions
which allow said first RNA molecule to cause said single-stranded
DNA molecule to be cleaved, said conditions including providing
Mg.sup.2+ ions and guanosine or guanosine triphosphate at a pH
between about 6.0 and about 9.0 and a temperature between about
150C and about 60.degree. C.
2. The method of claim 1, further comprising providing said RNA
molecule in a reaction medium at a concentration sufficient to
cause cleavage of at least 1% of a population of the DNA molecules
in an hour.
3. The method of claim 1, further comprising providing said RNA
molecule in a reaction medium at a concentration sufficient to
cause cleavage of at least 10% of a population of the DNA molecules
in an hour.
4. The method of claim 1, wherein said RNA molecule comprises the
portions of an RNA molecule of Tetrahymena having said
deoxyribonuclease activity.
5. The method of claim 4, wherein said RNA molecule is L-19, L-21,
or an RNA molecule comprising the portions of L-19 having said
deoxyribonuclease activity.
6. The method of claim 1, wherein said RNA molecule comprises a
binding site for single-stranded DNA, which binding site is
complementary to nucleotides adjacent to a cleavage site on said
single-stranded DNA molecule.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of copending U.S.
application Ser. No. 08/007,732, filed Jan. 22, 1993, which is a
continuation of U.S. application Ser. No. 07/464,530, filed Jan.
12, 1990 (abandoned), the disclosures of which are incorporated by
reference herein.
TECHNICAL FIELD
[0003] The present invention relates to nucleic acid enzymes for
cleaving DNA.
BACKGROUND
[0004] Some genes have their coding sequences interrupted by
stretches of non-coding DNA. These non-coding sequences are termed
introns. To produce a mature transcript from these genes, the
primary RNA transcript (precursor RNA) must undergo a
cleavage-ligation reaction termed RNA splicing. This RNA splicing
produces the mature transcript of the polypeptide coding messenger
RNA (mRNA), ribosomal RNA, or transfer RNA (tRNA). Introns are
grouped into four categories (Groups I, II, III, and IV) based on
their structure and the type of splicing reaction they undergo.
[0005] Of particular interest to the present invention are the
Group I introns. Group I introns undergo an intra-molecular RNA
splicing reaction leading to cyclization that does not require
protein cofactors, Cech, Science, 236:1532-1539 (1987).
[0006] The Group I introns, including the intron isolated from the
large ribosomal RNA precursor of Tetrahymena thermophila, have been
shown to catalyze a sequence-specific phosphoester transfer
reaction involving RNA substrates. Zaug and Cech, Science,
229:1060-1064 (1985); and Kay and Inoue, Nature, 327:343-346
(1987). This sequence-specific phosphoester transfer reaction leads
to the removal of the Group I intron from the precursor RNA and
ligation of two exons in a process known as RNA splicing. Splicing
reaction catalyzed by Group I introns proceeds via a two-step
transesterification mechanism. The details of this reaction have
been recently reviewed by Cech, Science, 236:1532-1539 (1987).
[0007] The splicing reaction of Group I introns is initiated by the
binding of guanosine or a guanosine nucleotide to a site within the
Group I intron structure. Attack at the 5' splice site by the
3'-hydroxyl group of guanosine results in the covalent linkage of
guanosine to the 5' end of the intervening intron sequence. This
reaction generates a new 3'-hydroxyl group on the uridine at the 3'
terminus of the 5' exon. The 5' exon subsequently attacks the 3'
splice site, yielding spliced exons and the full-length linear form
of the Group I intron.
[0008] The linear Group I intron usually cyclizes following
splicing. Cyclization occurs via a third transesterification
reaction, involving attack of the 3'-terminal guanosine at an
interval site near the 5' end of the intron. The Group I introns
also undergo sequence specific hydrolysis reaction at the splice
site sequences as described by Inoue et al., J. Mol Biol.,
189:143-165 (1986). This activity has been used to cleave RNA
substrates in a sequence specific manner by Zaug et al., Nature,
324:429-433 (1986).
[0009] The structure of Group I introns has been recently reviewed
by J. Burke, Gene, 73:273-294 (1988). The structure is
characterized by nine base paired regions, termed P1-P9 as
described in Burke et al., Nucleic Acids Res., 15:7217-7221 (1987).
The folded structure of the intron is clearly important for the
catalytic activity of the Group I introns as evidenced by the loss
of catalytic activity under conditions where the intron is
denatured. In addition, mutations that disrupt essential
base-paired regions of the Group I introns result in a loss of
catalytic activity. Burke, Gene, 73:273-294 (1988). Compensatory
mutations or second-site mutations that restore base-pairing in
these regions also restore catalytic activity. Williamson et al.,
J. Biol. Chem., 262:14672-14682 (1987); and Burke, Gene, 73:273-294
(1988).
[0010] Several different deletions that remove a large nucleotide
segment from the Group I introns (FIG. 2) without destroying its
ability to cleave RNA have been reported. Burke, Gene, 73:273-294
(1988). However, attempts to combine large deletions have resulted
in both active and inactive introns. Joyce et al., Nucleic Acid
Res., 17:7879 (1989).
[0011] To date, Group I introns have been shown to cleave
substrates containing either RNA, or RNA and DNA. Zaug et al.,
Science, 231:470-475 (1986); Sugimoto et al., Nucleic Acids Res.,
17:355-371 (1989); and Cech, Science, 236:1532-1539 (1987). A DNA
containing 5 deoxycytosines was shown not to be a cleavage
substrate for the Tetrahymena IVS, a Group I intron by Zaug et al.,
Science, 231:470-475 (1986).
BRIEF SUMMARY OF THE INVENTION
[0012] It has now been discovered that Group I introns have the
ability to cleave single-stranded DNA substrates in a site specific
manner.
[0013] Therefore the present invention provides a method of
cleaving single-stranded DNA at the 3'-terminus of a predetermined
nucleotide sequence present within single-stranded DNA. The
single-stranded DNA is treated under DNA cleaving conditions with
an effective amount of an endodeoxyribonuclease of the present
invention where the DNA cleaving conditions include the presence of
MgCl.sub.2 at a concentration of at least 20 millimolar.
[0014] The present invention also contemplates a composition
containing an endodeoxyribonuclease enzyme of the present
invention, single-stranded DNA and magnesium ion at a concentration
of greater than 20 millimolar.
[0015] The present invention further contemplates an
endodeoxyribonuclease enzyme capable of cleaving single-stranded
DNA at a predetermined nucleotide sequence where the enzyme has a
nucleotide sequence defining a recognition site that is contiguous
or adjacent to the 5'-terminus of the nucleotide sequence, a first
spacer region located 3'-terminal to the recognition site, a P3
[5'] region located 3'-terminal to the first spacer region, a
second spacer region located 3'-terminal to the P3 [5'] region, a
first stem loop located 3'-terminal to the second spacer region, a
second stem loop located 3'-terminal to the first stem loop, a
third spacer region located 3'-terminal to the second stem loop,
and a third stem loop located 3'-terminal to the third spacer
region, the third stem loop comprising a 5' stem portion
interrupted by a nucleotide sequence defining a P3 [3'] region
capable of hybridizing to the P3 [5'] region.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] In the drawings forming a portion of this disclosure:
[0017] In FIG. 1, the splicing mechanisms of the four major groups
of precursor RNAs. Wavy lines indicate introns, smooth lines
indicate flanking exons. For nuclear mRNA splicing, many components
assemble with the pre-mRNA to form the spliceosome; only two, the
U1 and U2 small nuclear ribonucleoproteins, are shown. Nuclear tRNA
splicing is described by Greer et al., TIBS 9:139-41(1984).
[0018] In FIG. 2, the secondary structure of the T. thermophila
pre-rRNA intron, with the recognition sequence and the core
structure that is the most conserved region among group I introns
shown in bold. The nomenclature used to denote various structural
features is the standard nomenclature described in Burke et al.,
Nucleic Acids Pes. 15:7217-7221 (1987). The nine conserved pairing
regions, P1-P9, and the various loops are shown. The nucleotide
sequence is numbered beginning at the 5' terminus of the
molecule.
[0019] The recognition site is located at nucleotide 19 to 27, the
first spacer region is located at nucleotides 27 to 28 and 94 to
95, the P3 [5'] region is located at nucleotides 96 to 103, the
second spacer region is located at nucleotides 104 to 106, the
first stem loop is located at nucleotides 107 to 214, the second
stem loop is located at nucleotides 215 to 258, the third spacer
region is located at nucleotides 259 to 261 and the third stem loop
is located at nucleotides 262 to 314.
[0020] In FIG. 3, the various deletions removing portions of the T.
therophili pre-rRNA intron are shown. The nomenclature used is the
same nomenclature defined in Burke et al., Nucleic Acids Research,
15:7217-7221 (1987). The nucleotide segments removed in each
deletion are shown with the greek character delta followed by the
number of the pairing region removed. Combination of deletions are
noted as Delta P 2/9 for example.
[0021] In FIG. 4, trans-splicing activity of the wild-type and
.DELTA.P9 mutant form of the Tetrahymena ribozyme using the
substrate GGCCCUCU.A.sub.3UA.sub.3UA.sub.3 (S1),
d(GGCCCTCU.A.sub.3TA.sub.3TA) (S2), or
d(GGCCCTCT.A.sub.3TA.sub.3TA) (S3) are shown.
[0022] In FIG. 5, selective amplification of the Tetrahymena
ribozyme (E) based on its ability to react with an oligonucleotide
substrate (S) is shown. Top, the L-21 form of the ribozyme binds an
oligopyrimidine-containing RNA substrate by complementary pairing.
The 3'-terminal G.sub.OH of the ribozyme attacks the phosphodiester
bond following a sequence of pyrimidines, resulting in transfer of
the 3' portion of the substrate to the 3' end of the ribozyme.
Middle, the product of the RNA-catalyzed reaction offers a unique
site for hybridization of an oligodeoxynucleotide used to initiate
cDNA synthesis. Bottom, a primer containing the T7 promoter is
hybridized to the cDNA, the second strand of the promoter is
completed, the DNA is transcribed to RNA.
[0023] In FIG. 6, selective amplification of an ensemble of
structural variants of the Tetrahymena ribozyme based on their
ability to carry out a trans-splicing reaction with a DNA
substrate. Lanes 1-3, trans-splicing with no substrate, the RNA
substrate GGCCCUCU.A.sub.3UA.sub.3UA, and the DNA substrate
d(GGCCCTCT.A.sub.3TA.sub.3TA). Lanes 4-5, selective cDNA synthesis
of trans-spliced products. Lanes 6-8, successive rounds of
transcription and reverse transcription leading to amplification of
selected materials. Materials were separated by electrophoresis in
a 5% polyacrylamide/8M urea gel, an autoradiogram of which is
shown.
DETAILED DESCRIPTION OF THE INVENTION
[0024] A. Enzymes
[0025] An endodeoxyribonuclease of the present invention is capable
of cleaving a single-stranded DNA substrate. Typically, the
endodeoxyribonuclease is also capable of cleaving a single-stranded
RNA substrate or a modified DNA substrate containing a uracil at
the cleavage site rather than a thymine.
[0026] The term ribozyme is used to describe an RNA containing
nucleic acid that is capable of functioning as an enzyme. Ribozymes
include endoribonucleases and endodeoxyribonucleases of the present
invention.
[0027] An endodeoxyribonuclease of the present invention may be
RNA, modified RNA, RNA-DNA polymer, a modified RNA-DNA polymer, a
modified DNA-RNA polymer or a modified RNA-modified DNA polymer.
RNA contains nucleotides containing a ribose sugar and adenine,
guanine, uracil or cytosine as the base at the 1' position.
Modified RNA contains nucleotides containing a ribose sugar and
adenine, thymine, guanine or cytosine and optionally uracil as the
base. A RNA-DNA polymer contains nucleotides containing a ribose
sugar and nucleotides containing deoxyribose sugar and adenine,
thymine and/or uracil, guanine or cytosine as the base attached to
the 1' carbon of the sugar. A modified RNA-DNA polymer is comprised
of modified RNA, DNA and optionally RNA. Modified DNA contains
nucleotides containing a deoxyribose sugar and nucleotides
containing adenine, uracil, guanine, cytosine and possibly thymine
as the base. A modified DNA-RNA polymer contains modified DNA, RNA
and optionally DNA. A modified RNA-modified DNA polymer contains
modified RNA-modified DNA, and optionally RNA and DNA.
[0028] An endodeoxyribonuclease of the present invention is capable
of cleaving DNA 3' of a predetermined base sequence. In addition,
an endodeoxyribonuclease of this invention is characterized by a
nucleotide sequence defining a recognition site that is contiguous
or adjacent to the 5' terminus of the nucleotide sequence, a first
spacer region located 3'-terminal to the recognition site, a P3
[5'] region located 3'-terminal to the IS first spacer region, a
second spacer region located 3'-terminal to the P3 [5'] region, a
first stem loop located 3'-terminal to the second spacer region, a
second stem loop located 3'-terminal to the first stem loop, a
third spacer region located 3'-terminal to the second stem loop,
and a third stem loop located 3'-terminal to the third spacer
region, the third stem loop comprising a 5' stem portion defining a
P3 [3'] region capable of hybridizing to the P3 [5'] region.
[0029] The recognition site of an endodeoxyribonuclease of the
present invention contains a sequence of at least 2 to about 8
bases preferably about 4 to about 7 bases, capable of hybridizing
to a complementary sequence of bases within the substrate nucleic
acid giving the endodeoxyribonuclease its high sequence
specificity. For example, an endodeoxyribonuclease of the present
invention with a recognition site base sequence of 5'-GGAGG-3' is
able to recognize the base sequence 5'-CCCTCT-3' present within the
single-stranded DNA substrate (see Example 2). This same
recognition site also allows the endodeoxyribonuclease to cleave
modified DNA substrates with high sequence specificity (see Example
2.)
[0030] The exact bases present in the recognition site determine
the base sequence at which cleavage will take place. Cleavage of
the substrate nucleic acid occurs immediately 3' of the substrate
cleavage sequence, the substrate nucleotide sequence that
hybridizes to the recognition site. This cleavage leaves a 3'
hydroxyl group on the substrate cleavage sequence and a 5'
phosphate on the nucleotide that was originally immediately 3' of
the substrate cleavage sequence in the original substrate. Cleavage
can be redirected to a site of choice by changing the bases present
in the recognition sequence (internal guide sequence). See Murphy
et al., Proc. Natl. Acad. Sci., USA, 86:9218-9222 (1989). (The
disclosures of all references cited within this document are
incorporated by reference.) In addition, any combination of bases
may be present in the recognition site if a polyamine is present.
See, for example, Doudna et al., Nature, 339:519-522 (1989).
Typically, the polyamine is either spermidine, putrescine or
spermine. A spermidine concentration of about 5 mM was shown to be
effective. The recognition site may also be provided as a separate
nucleic acid, an external recognition site not covalently coupled
to the rest of the endodeoxynuclease. External recognition sites
have been shown to direct endoribonuclease cleavage at a specific
base sequence by Doudna et al., Nature, 339:519-522 (1989). If an
external recognition site is used, the endodeoxyribonuclease used
with it would not contain a recognition site but would comprise a
P3 [5'] region, a second spacer region, a first stem loop, a second
stem loop, a third spacer region and a third stem loop where the
third stem loop comprises a 5' stem portion defining a P3 [3']
region capable of hybridizing to said P3 [5'] region.
[0031] Use of an endodeoxyribonuclease of the present invention
with an external recognition site would allows the target sequence
to be changed by merely changing the external recognition site
sequence. Use of a plurality of different external recognition
sequences with an endodeoxyribonuclease of the present invention
allows the substrate nucleic acid to be cleaved at each of the
different base sequences encoded by the external recognition
sequences.
[0032] First spacer regions typically contain a sequence of
nucleotides about 3 bases to about 7 bases, preferably about 5,
bases in length. Preferably, the nucleotides making up the first
spacer have the sequence 5'-NNNNA-3', where N represents the
presence of any nucleotide at that position. More preferably, the
first spacer region is defined by the sequence 5'-AACAA-3'.
[0033] In other preferred embodiments, the first spacer region is
comprised of a nucleotide sequence defining two spacer stem loops.
Preferably, the first spacer stem loop is 25 nucleotides in length,
and the second spacer stem loop is 36 bases in length. More
preferably, the first spacer stem loop has the base sequence,
5'-AGUUACCAGGCAUGCACCUGGUAG- UCA-3', and the second spacer stem
loop has the base sequence,
5'-GUCUUUAAACCAAUAGAUU-GGAUCGGUUUAAAAGGC-3'.
[0034] A stem loop is a secondary structure formed by a nucleotide
sequence that has "folded over on itself". A stem loop comprises a
5' nucleotide sequence portion, designated a 5' paring segment
(P[5']) that is capable of hybridizing to a nucleotide sequence
located 3' of the P[5'] and is designated the 3' pairing segment
(P[3']). In a stem loop, the P[5'] and P[3'] are connected by a
nucleotide sequence called a loop. The P[5'] and P[3'] hybridize
and form a nucleic acid duplex. The nucleic acid duplex formed by
the P[5'] and P[3'] does not have to be a perfect duplex and may
contain stretches of nucleotides that are either unpaired or paired
to a sequence outside the stem loop.
[0035] In preferred embodiments, the P3 [5'] region is an eight
nucleotide sequence. The eight nucleotides present in the P3 [5']
region may be any eight nucleotides as long as the P3 [5'] region
is capable of hybridizing with the P3 [3'] region to form the third
pairing segment or P3 shown in FIG. 2. The formation of P3 by P3
[5'] region and P3 [3'] region are required for catalytic activity
as has been recently reviewed by John Burke, Gene, 73:273-294
(1988).
[0036] More preferably, the P3 [5'] has the nucleotide sequence,
5'-GACCGUCA-3'. However, changes in the P3 [5'] region nucleotide
sequences may be made as long as P3 is still able to form or if
compensating changes in the P3 [3'] region nucleotide sequence have
been made as has been demonstrated by Williamson et al., J. Biol.
Chem., 262:14672-14682 (1987).
[0037] In preferred embodiments, the second spacer region is about
three nucleotides in length. Typically, any three nucleotides may
make up the second space region as long as the ribozyme containing
this spacer has the desired catalytic activity.
[0038] More preferably, the second spacer region has the nucleotide
sequence, 5'-AAU-3'. However, this sequence may be changed as long
as the desired catalytic activity is retained.
[0039] In preferred embodiments, the first stem loop is about 108
nucleotides in length. More preferred, the first stem loop
corresponds to nucleotides 107 to 214 of FIG. 2. The ten most
5'-terminal nucleotides (nucleotides 107 to 117 of FIG. 2) and the
ten most 3'-terminal nucleotides (nucleotides 204 to 214 of FIG. 2)
of the first stem loop are the most critical for known catalytic
functions. See, for example, John Burke, Gene, 73:273-294
(1988).
[0040] In other preferred embodiments, the first stem loop is about
39 nuclectides in length. More preferred, the first stem loop
corresponds to nucleotides 107 to 126 and nucleotides 196 to 214 in
FIG. 2, where nucleotide 126 is directly linked to nucleotide
196.
[0041] In another preferred embodiment, the first stem loop is 20
nucleotides in length. More preferred, the first stem loop
corresponds to nucleotides 107 to 116, and nucleotides 205 to 214
of FIG. 2, where nucleotide 116 is directly linked to nucleotide
205.
[0042] In preferred embodiments, the second stem loop is about 44
nucleotides in length. More preferred, the first stem loop
corresponds to nucleotides 215 to 258 in FIG. 2. The 5 most
5'-terminal nucleotides (nucleotides 215 to 219 in FIG. 2) and the
5 most 3'-terminal nucleotides (nucleotides 254 to 258 in FIG. 2)
of the second stem loop are the most critical For known catalytic
functions. See for example, John Burke, Gene, 73:273-294
(1988).
[0043] In other preferred embodiments, the second stem loop is
about 25 nucleotides in length. More preferably, the second stem
loop nucleotide sequence substantially corresponds to nucleotides
215 to 227 and nucleotides 247 to 258 in FIG. 2, where nucleotides
227 and 247 are directly linked. In another preferred embodiment,
the second stem loop is about 9 nucleotides in length. More
preferably, the second stem loop substantially corresponds to
nucleotides 215 to 220 and 256 to 258 in FIG. 2 where nucleotides
220 and 256 are directly linked.
[0044] In preferred embodiments, the third spacer region is about 3
nucleotides in length. More preferably, the third spacer region
corresponds to nucleotides 259 to 261 of FIG. 2. Some nucleotide
changes can be made in the third spacer region while still
preserving the desired catalytic activity. See for example,
Williamson et al., J. Biol. Chem., 262:14672-14682 (1987), where
the nucleotide at position 259 was changed to A and the nucleotide
at 261 was changed to a C while maintaining splicing activity. The
nucleotide at position 260 (FIG. 2) was changed to either G, A or U
while preserving the desired catalytic activity as reported in John
Burke, Gene, 73:273-294 (1988).
[0045] In preferred embodiments, the third stem loop is about 51
nucleotides in length. These 51 nucleotides are divided into a 5'
stem portion defining a P3 [3'] region that is capable of
hybridizing to the P3 [5'] region, a loop and a 3' stem portion.
Preferably, the P3 [3'] region is about 8 nucleotides in length.
However, the length of the P3 [3'] may vary to correspond with the
length of the P3 [5'] region. Preferably, the P3 [3'] region begins
about 10 nucleotides from the 5' end of the third stem loop.
[0046] More preferably, the third stem loop is 5 nucleotides in
length and those nucleotides substantially correspond to
nucleotides 262 to 312 in FIG. 2. Preferably, the P3 [3'] region is
about 8 nucleotides in length and those nucleotides substantially
correspond to nucleotides 271 to 278 in FIG. 2. Changes, including
deletions, mutations, reversions and insertions, can be made within
the third stem loop and the P3 [3'] region and still maintain the
desired catalytic activity. See, for example, Burke et al., Cell,
45:167-176 (1986) and Williamson et al., J. Biol. Chem.,
262:14672-14682 (1987), where nucleotide 266 was changed to G and a
compensatory mutation changing nucleotide 309 to C was made while
maintaining the desired catalytic activity. Other mutations,
including changing nucleotide 268 to C and at the same time
changing nucleotide 307 to C, and changing nucleotide 268 to U and
at the same time changing nucleotide 307 to A, were also shown to
maintain the desired catalytic activity.
[0047] Other changes in the nucleotide sequence of the third stem
loop are also contemplated by the present invention. Changing
nucleotides 301 to C (FIG. 2), 302 to 6, and 303 to C has been
shown to eliminate transesterification activity, but does not
eliminate site specific cleavage or GTP binding by Williamson et
al., J. Biol. Chem., 262:14672-14682 (1987).
[0048] Charges in nucleotides 280 and 282 (FIG. 2) together with
compensatory changes in nucleotides 296 and 298 have been shown to
preserve the desired catalytic function as reported in J. Burke,
Gene, 73:273-294 (1988). Mutations such as these preserve a given
secondary structure and these and similar mutations would be
expected to maintain the desired catalytic activity.
[0049] Changes in the P3 [3'] region, nucleotides 272 and 274 (FIG.
2), along with compensatory changes in nucleotides 100 and 102,
were made while maintaining the desired catalytic activity by
Williamson et al., J. Biol. Chem., 262:14672-14682 (1987) and Inoue
et al., Cell, 43:431-437 (1985). Changes similar to those changes
made above will maintain the desired catalytic activity as long as
the particular secondary structure such as a stem loop, pairing
region or spacer is maintained.
[0050] An endodeoxyribonuclease of the present invention may also
include additional stem loops located 3'-terminal to the third stem
loop. These additional stem loops may contain any number of stem
loops as long as the desired catalytic activity is maintained.
Preferably, any additional stem loops have a nucleotide sequence
that substantially corresponds to nucleotides 316 to 402 of FIG.
2.
[0051] In preferred embodiments, an endodeoxyribonuclease of the
present invention may combine one or more of the mutations
described above. Typically, these deletions change the length of or
alter the nucleotide sequence of a stem loop, the P3 [5'], the P3
[3'] region, a spacer region or the recognition sequence. The
mutation within one catalytically active endodeoxyribonuclease may
be combined with the mutation within a second catalytically active
endodeoxyribonuclease to produce a new endodeoxyribonuclease
containing both mutations.
[0052] In other preferred embodiments, an endodeoxyribonuclease of
the present invention may have random or defined mutations
introduced into it using a variety of methods well known to those
skilled in the art. For example, the method described by Joyce et
al., Nucleic Acids Research, 17:711-712 (1989), involves excision
of template (coding) strand of double-stranded DNA, reconstruction
of the template strand with inclusion of mutagenic
oligonucleotides, and subsequent transcription of the
partially-mismatched template. This allows the introduction of
defined or random mutations at any position in the molecule by
including polynucleotides containing known or random nucleotide
sequences at selected positions. Alternatively, mutations may be
introduced into the endodeoxyribonuclease by substituting 5-Br dUTP
for TTP in the reverse transcription reaction. 5-Br dU can pair
with dG in the "wobble" position as well as dA in the standard
Watson-Crick position, leading to A to G and G to A transitions.
Similarly, substituting 5-Br UTP for UTP in the forward
transcription reaction would lead to C to U and U to C transitions
in the subsequent found of RNA synthesis.
[0053] B. Methods
[0054] The method of the present invention is useful for cleaving
any single-stranded nucleic acid including single-stranded DNA,
modified DNA, RNA and modified RNA. The single-stranded nucleic
acid must only be single-stranded at or near the substrate cleavage
sequence so that an enzyme of the present invention can hybridize
to the substrate cleavage sequence by virtue of its recognition
sequence.
[0055] A single-stranded nucleic acid that will be cleaved by a
method of this invention may be chemically synthesized,
enzymatically produced or isolated from various sources such as
phages, viruses or cells, including plant cells, eukaryotic cells,
yeast cells and bacterial cells. Chemically synthesized
single-stranded nucleic acids are commercially available from many
sources including, Research Genetics, Huntsville, Alabama.
Single-stranded phages such as the M13 cloning vectors described by
Messing et al., Proc. Natl. Acad. Sci., USA, 74:3642-3646 (1977),
and Yanisch-Perron et al., Gene, 33:103-119 (1985). Bacterial cells
containing single-stranded phages would also be a ready source of
suitable single-stranded DNA. Viruses that are either
single-stranded DNA viruses such as the parvoviruses or are
partially single-stranded DNA viruses such as the hepatitis virus
would provide single-stranded DNA that could be cleaved by a method
of the present invention. Single-stranded RNA cleavable by a method
of the present invention could be provided by any of the RNA
viruses such as the picornaviruses, togaviruses, orthomyxoviruses,
paramyxoviruses, rhabdoviruses, coronaviruses, arenaviruses or
retroviruses.
[0056] The methods of this invention may be used on single-stranded
nucleic acid that are present inside a cell, including eucaryotic,
procaryotic, plant, mammalian, yeast or bacterial cell. Under these
conditions a method of the present invention could act as an
anti-viral agent or a regulatory of gene expression.
[0057] The method of the present invention cleaves single-stranded
DNA at the 3'-terminus of a predetermined base sequence. This
predetermined base sequence or substrate cleavage sequence may
contain from 2 to 8 nucleotides. The method allows cleavage at any
nucleotide sequence by altering the nucleotide sequence of the
recognition site of the endodeoxyribonuclease. This allows cleavage
of single-stranded DNA in the absence of a restriction endonuclease
site at that position.
[0058] Cleavage at the 3'-terminus of a predetermined base sequence
produces a single-stranded DNA, containing the substrate cleavage
sequence, with a 3'-terminal hydroxyl group. In addition, the
cleavage joins the remainder of the original single-stranded DNA
substrate with the endodeoxyribonuclease enzyme. This cleavage
reaction and products produced from this cleavage reaction are
analogous to the cleavage reaction and cleavage products produced
by the Tetrahymena ribozyme described by Zaug and Cech, Science,
231:470-475 (1986) and reviewed by T. R. Cech, Annual Rev. of
Biochem., 59:(1990). The endodeoxyribonuclease of the present
invention may be separated from the remainder of single-stranded
DNA substrate by site-specific hydrolysis at the phosphodiester
bond following the 3'-terminal guanosine of the
endodeoxyribonuclease similar to the site-specific cleavage at this
position described for the ribozyme acting on RNA by Inoue et al.,
J. Mol. Biol., 189:143-165 (1986). Separation of the
endodeoxyribonuclease from the substrate allows the
endodeoxyribonuclease to carry out another cleavage reaction.
[0059] Single-stranded DNA is treated under DNA cleaving conditions
with an effective amount of an endodeoxyribonuclease of the present
invention, where the DNA cleaving conditions include the presence
of MgCl.sub.2 at a concentration of at least 20 millimolar.
[0060] An effective amount of an endodeoxyribonuclease is the
amount endodeoxyribonuclease required to cleave a predetermined
base sequence present within the single-stranded DNA. Preferably,
the endodeoxyribonuclease is present at a molar ratio of
endodeoxyribonuclease to substrate cleavage sites of 1 to 20. This
ratio may vary depending on the length of treating and efficiency
of the particular endodeoxyribonuclease under the particular DNA
cleavage conditions employed.
[0061] Treating typically involves admixing, in aqueous solution,
the single-stranded DNA, the enzyme and the MgCl.sub.2 to form a
DNA cleavage admixture, and then maintaining the admixture thus
formed under DNA cleaving conditions for a time period sufficient
for the endodeoxyribonuclease to cleave the single-stranded DNA at
any of the predetermined nucleotide sequences present in the
single-stranded DNA.
[0062] Preferably, the amount of time necessary for the
endodeoxyribonuclease to cleave the single-stranded DNA has been
predetermined. The amount of time is from about 5 minutes to about
24 hours and will vary depending upon the concentration of the
reactants, and the temperature of the reaction. Usually, this time
period is from about 30 minutes to about 4 hours such that the
endodeoxyribonuclease cleaves the single-stranded DNA at any of the
predetermined nucleotide sequences present.
[0063] Preferably, the DNA cleaving conditions include the presence
of MgCl.sub.2 at a concentration of at least 20 mM. Typically, the
DNA cleaving conditions include MgCl.sub.2 at a concentration of
about 20 mM to about 150 mM. The optimal MgCl.sub.2 concentration
to include in the DNA cleaving conditions can be easily determined
by determining the amount of single-stranded DNA cleaved at a given
MgCl.sub.2 concentration. One skilled in the art will understand
that the optimal MgCl.sub.2 concentration may vary depending on the
particular endodeoxyribonuclease employed.
[0064] Preferably, the DNA cleaving conditions are at from about pH
6.0 to about pH 9.0. One skilled in the art will understand that
the method of the present invention will work over a wide pH range
so long as the pH used for DNA cleaving is such that the
endodeoxyribonuclease is able to remain in an active conformation.
An endodeoxyribonuclease in an active conformation is easily
detected by its ability to cleave single-stranded DNA at a
predetermined nucleotide sequence. More preferably, the DNA
cleaving conditions are at from about pH 7.0 to about pH 8.0. Most
preferred are DNA cleaving conditions at about pH 7.5.
[0065] Preferably, the DNA cleaving conditions are at from about
15.degree. C. to about 60.degree. C. More preferably, the DNA
cleaving conditions are from about 30.degree. C. to about
56.degree. C. The temperature of the DNA cleaving conditions are
constrained only by the desired cleavage rate and the stability of
that particular endodeoxyribonuclease at that particular
temperature. Most preferred are DNA cleavage conditions from about
37.degree. C. to about 50.degree. C.
[0066] In other preferred methods the present invention
contemplates DNA cleaving conditions including the presence of a
polyamine. Polyamines useful practicing the present invention
include spermidine, putrescine, spermine and the like. Preferably,
the polyamine is spermidine and it is present at a concentration of
about 1 mM to about 15 mM. More preferably, spermidine is present
at a concentration of about 1 mM to about 10 mM. Most preferred,
are DNA cleavage conditions including the presence of spermidine at
a concentration of about 2 mM to about 5 mM.
[0067] The present invention also contemplates a method of
producing a nucleic acid having a predetermined activity.
Preferably, the desired activity is a catalytic activity.
[0068] A population of Group I nucleic acids is subjected to
mutagenizing conditions to produce a diverse population of mutant
nucleic acids.
[0069] In preferred embodiments, the population of Group I nucleic
acids is made up of at least 2 Group I nucleic acids. Group I
nucleic acids are nucleic acid molecules having at least a nucleic
acid sequence defining a recognition site that is contiguous or
adjacent to the 5'-terminus of the nucleotide sequence, a first
spacer region located 3'-terminal to the recognition site, a P3
[5'] region located 3'-terminal to the first spacer region, a
second spacer region located 3'-terminal to the P3 [5'] region, a
first stem loop located 3'-terminal to the second spacer region, a
second stem loop located 3'-terminal to the first stem loop, a
third spacer region located 3'-terminal to the second stem loop,
and a third stem loop located 3'-terminal to the third spacer
region, the third stem loop comprising a 5' stem portion defining a
P3 [3'] region capable of hybridizing to the P3 [5'] region.
[0070] In preferred embodiments, mutagenizing conditions include
conditions that introduce either defined or random nucleotide
substitutions within the Group I nucleic acid. Examples of typical
mutagenizing conditions include conditions disclosed in other parts
of this specification and the methods described by Joyce et al.,
NAR 17:711-722(1989) and Joyce, Gene, 82:83-87(1989).
[0071] In preferred embodiments, a diverse population of mutant
nucleic acid contains at least 2 nucleic acid molecules that do not
have the exact same nucleotide sequence.
[0072] A nucleic acid having a predetermined activity is selected
from the diverse population of mutant nucleic acids on the basis of
its ability to perform the predetermined activity.
[0073] In preferred embodiments, selecting includes any means of
physically separating the mutant nucleic acids having a
predetermined activity from the diverse population of mutant
nucleic acids. Typically, selecting includes separation by size,
the presence of a catalytic activity and hybridizing the mutant
nucleic acid to another nucleic acid that is either in solution or
attached to a solid matrix. Preferably, the predetermined activity
is such that the mutant nucleic activity having the predetermined
activity becomes labelled in some fashion by virtue of the
activity. For example, the predetermined activity may be an
endodeoxyribonuclease activity whereby the activity of the mutant
nucleic acid upon its substrate causes the mutant nucleic acid to
become covalently linked to it. The mutant nucleic acid is then
selected by virtue of the covalent linkage.
[0074] In other preferred embodiment, selecting a mutant nucleic
acid having a predetermined activity includes amplification of the
mutant nucleic acid as described in Joyce, Gene,
82:83-87(1989).
[0075] D. Compositions
[0076] Also contemplated by the present invention are compositions
containing an endodeoxyribonuclease enzyme of the present
invention, single-stranded DNA and magnesium ion at a concentration
of greater than 20 millimolar.
[0077] Preferably, the endodeoxyribonuclease is present at a
concentration of about 0.05 .mu.M to about 2 mM. Typically, the
endodeoxyribonuclease is present at concentration ration of
endodeoxyribonuclease to single-stranded DNA from about 1 to 5 to
about 1 to 50. More preferably, the endodeoxyribonuclease is
present in the composition at a concentration of about 0.1 .mu.M to
about 1 .mu.M. Most preferred, are compositions containing the
endodeoxyribonuclease at a concentration of about 0.1 .mu.M to
about 0.5 .mu.M.
[0078] Preferably, single-stranded DNA is present in the
composition at a concentration of about 0.5 .mu.M to about 1000
.mu.M. One skilled in the art will understand that there are many
sources of single-stranded DNA including synthetic DNA, phage DNA,
denatured double-stranded DNA, viral DNA and cellular.
[0079] Preferably, magnesium ion is present in the composition at a
concentration of about 20 mM to about 200 mM. More preferably, the
magnesium ion is present in the composition at a concentration of
about 20 mM to about 150 mM. One skilled in the art will understand
that the magnesium ion concentration is only constrained by the
limits of solubility of magnesium in aqueous solution and a desire
to have the endodeoxyribonuclease present in the same composition
in an active conformation.
[0080] Also contemplated by the present invention are compositions
containing an endodeoxyribonuclease enzyme of the present
invention, single-stranded DNA, magnesium ion at a concentration of
greater than 20 millimolar and a polyamine.
[0081] Preferably, the polyamine is spermidine, putrescine, or
spermine. More preferably, the polyamine is spermidine and is
present at a concentration of about 2 mM to about 10 mM.
[0082] Also contemplated by the present invention are composition
containing an endodeoxyribonuclease enzyme of the present
invention, single-stranded DNA, magnesium ion at a concentration of
greater than 20 millimolar, a second single-stranded DNA molecule
ending in a 3'-terminal hydroxyl, and a third single-stranded DNA
molecule having a guanine nucleotide at its 5'-terminal end.
[0083] Also contemplated by the present invention are compositions
containing an endodeoxyribonuclease enzyme of the present
invention, singled-stranded DNA and magnesium ion at a
concentration of greater than 20 millimolar, wherein said
single-stranded DNA is greater in length than the recognition site
present on the endodeoxyribonuclease enzyme.
EXAMPLES
[0084] The following examples illustrate, but do not limit, the
present invention.
[0085] 1. Preparation of Endodeoxyribonucleases.
[0086] The wild-type and mutant ribozymes were produced by first
isolating the 443 base-pair Eco RI to Hind III restriction
endonuclease fragment from the plasmid PT7-21 described by Zaug et
al., Biochemistry, 27:8924 (1988) using the standard methods
described in Current Protocols in Molecular Biology, Ausubel et
al., eds. John Wiley and Sons, New York (1987).
[0087] This 443 base-pair fragment contains the T7 promoter
described by Dunn et al., J. Mol. Biol., 166:477-535 (1983) and
residues 22-414 of the Tetrahymena IVS and residues 1-25 of the 3'
Tetrahymena exon described by Been et al., Cell, 47:207-216 (1986).
This Eco RI and Hind III fragment was inserted into the M13 vector,
M13mp18 that is similar to the vector described by Yanisch-Perron
et al., Gene, 33:103-119 (1985), that had been previously cleaved
with Eco RI Hind III, and using standard subcloning procedures
described in Current Protocols in Molecular Biology, Ausubel et al,
eds. John Wiley and Sons, New York (1987). The resulting M13T7L-21
DNA construct was transformed into E. coli host cells according to
the transformation procedure described in Molecular Cloning: A
Laboratory Manual, Maniatis et al., eds., Cold Spring Harbor
Laboratories, Cold Spring Harbor, New York (1989). Single stranded
DNA was then prepared from the M13T7L-21 transformed cells
according to the procedures described in Current Protocols in
Molecular Biology, Ausubel et al, eds. John Wiley and Sons, New
York (1987). The accuracy of the above construction was confirmed
by DNA sequencing using the klenow fragment of E. coli DNA
polymerase I (Boehringer Mannheim Biochemicals, Indianapolis, Ind.)
and the dideoxynucleotide sequencing method described by Sanger et
al., Proc. Natl. Acad. Sci., USA, 74:5463-5467 (1977).
[0088] The wild-type and mutant ribozymes were prepared directly
from the single-stranded M13T7L-21 DNA using a modification of the
technique previously described by Joyce and Inoue, Nucleic Acid
Research, 17:711-722 (1989). The technique involves construction of
a template strand that optionally includes one or more mutagenic
oligodeoxynucleotides. The resulting partially-mismatched
double-stranded DNA is transcribed directly using T7 RNA
polymerase. Briefly, a five fold molar excess of a terminator
polynucleotide and a mutator oligo were admixed with 5 .mu.g of
single-stranded M13T7L-21 DNA and a solution containing 20 mM
tris[hydroxy-methyl]aminomethane adjusted to pH 7.5 with
HCl(Tris-HCl), 50 mM NaCl and 2 mM MgCl.sub.2. This solution was
maintained at 70 degrees centigrade (70.degree. C.) for 5 minutes
and then steadily cooled to 30.degree. C. over 40 minutes. Fifteen
units(U) of T4 DNA ligase (U.S. Biochemicals, Cleveland, Ohio) and
7.5 U of T4 DNA polymerase (U.S. Biochemicals) were admixed to the
solution together with sufficient amounts of reagents to make the
solution contain a final concentration of 20 mM Tris-HCl at pH 7.5,
50 mM NaCl, 5 mM MgCl.sub.2, 2 mM dithiothreitol (DTT), 1 mM
adenosine triphosphate (ATP) and 0.5 mM each of dGTP, dTTP, dATP
and dCTP (dNTPs). The resulting solution was maintained at
37.degree. C. for 60 minutes to complete the synthesis of the
mutant strand. The resulting DNA was purified by ethanol
precipitation and then used to direct the transcription of mutant
RNA.
[0089] Transcription took place either in a 10 .mu.l volume
containing 1 .mu.g of mutant DNA, 2 .mu.Ci [.alpha..sup.32P] GTP
and 50 U of T7 RNA polymerase that was prepared as previously
described by Davanloo et al., Proc. Natl. Acad. Sci., USA,
81:2035-2039 (1984), and purified according to a procedure
originally developed by Butler & Chamberlain, J. Bio. Chem.,
257:5772-5779 (1982), or in a 400 .mu.l volume containing 10 .mu.g
of mutant DNA, 40 .mu.Ci [.sup.3H] UTP and 2,400 U of T7 RNA
polymerase. In either case, the transcription mixture also
contained 40 mM Tris-HCl at pH 7.5, 15 mM MgCl.sub.2, 10 mM
dithiothreitol, 2 mM spermidine, and 1 mM (each) NTPS, and was
incubated at 37.degree. C. for 90 minutes. The T7 RNA polymerase
was extracted with phenol and the transcription products were
purified by ethanol precipitation. The mutant RNA was isolated by
electrophoresis in a 5% polyacrylamide/8 M urea gel, eluted from
the gel, and purified by ethanol precipitation and chromatography
on Sephadex G-50.
[0090] The Delta P2 mutant was prepared using the mutagenic
oligodeoxynucleotide 01 (Table 1 and FIG. 3). The
partially-randomized Delta P2 mutant was prepared using the
mutagenic oligodeoxynucleotide 02 (Table 1 and FIG. 3). The Delta
PS mutant was prepared using mutagenic oligonucleotides 03 or 04
(Table 1 and FIG. 3). The Delta P6 mutant was prepared using the
mutagenic oligodeoxynucleotide 05 (Table 1 and FIG. 3). The Delta
P6b mutant was prepared using the mutagenic oligodeoxy-nucleotide
06 (Table 1 and FIG. 3). The Delta P9 mutant was prepared using the
mutagenic oligodeoxy-nucleotide 07 (Table 1 and FIG. 3).
[0091] Wild-type and mutant RNAs other than those containing the
Delta P9 deletion were defined at their 3' end by the
oligodeoxynucleotide 08 (Table 1 and FIG. 3). Mutants containing
the Delta P9 deletion were defined by the Delta P9 mutagenic oligo
which directs a transcript that includes 10 nucleotides of the 3'
exon.
1TABLE 1 01) 5'-TTTGACGGTCTTGTTCCCTCCTATAGTGAG-3' 02)
5'-TTTGACGGTCTNNNNCCCTCCTATAGTGAG-3' 03)
5'-TGCGTGGTTACTTTCCCGCAA-3' 04) 5'-GGACTTGGCTGCGTGGTTACTTTCCCGCAA--
3' 05) 5'-TTTAGTCTGTGAACTCTTGGC-3 06)
5'-TCTGTGAACTGCATCCAAGCTTAGGACTTGG-3' 07) 5'-GGCTACCTTACGAGTACTCCG-
ACTATATCTTAT-3' 08) 5'-CGAGTACTCCAAAAC-3'
[0092] The 3' exon sequence was removed by RNA-catalyzed
site-specific hydrolysis as has been previously, Inoue et al., J.
Mol. Biol., 189:143-165 (1986). Briefly, the RNA was incubated in
the presence of 50 mM CHES at pH 9.0 and 10 mM MgCl.sub.2 at
42.degree. C. for 1 hour. Wild-type and mutant RNAs were isolated
by electrophoresis in a 5% polyacrylamide/8M urea gel, eluted from
the gel, and purified by affinity chromatography on du Pont Nensorb
(du Pont Company, Wilmington, Del.). RNAs were sequenced by primer
extension analysis using AMV reverse transcriptase (Life
Technologies, Inc., Gaithersburg, Md.) in the presence of
dideoxynucleotides, using a modification of the methods described
by Sanger et al., Proc. Natl. Acad. Sci., USA, 74:5463-5467 (1977),
except for those containing the Delta P9 deletion, which were
sequenced from the 3' end by partial RNase digestion, Donis-Keller
et al., Nucleic Acids Res., 15:8783-8798 (1987).
[0093] The RNA substrate 5'-GGCCCUCUA.sub.13-3' was prepared by in
vitro transcription using a partially single-stranded synthetic DNA
template according to the methods described by Milligan et al.,
Nucleic Acids Res., 4:2527-2538 (1977). The template contains both
strands of the promoter for T7 RNA polymerase (positions -17
through +1) followed by the single-stranded template sequence
3'-CGGGAG- AT.sub.10-5'. Run-off transcripts of the form
5'-GGCCCUCUA.sub.n-3', where n=9-16, were obtained. The resulting
products were separated by electrophoresis in a 20%
polyacrylamide/8M urea gel, eluted from the gel, purified by
affinity chromatography on du Pont Nensorb, and sequenced by
partial RNase digestion Donis-Keller et al., Nucleic Acids
Research, 15:8783-8798 (1987). RNA substrates having the sequence
5'-GGCCCUCUA.sub.13-3' were used throughout this study.
[0094] The DNA substrates were either purchased from a number of
commercial sources (i.e., Research Genetics, Huntsville, Ala.) or
synthesized using an Applied Biosystems (Foster City, Calif.)
oligonucleotide synthesizer according to the manufacture's
instructions.
[0095] 2. Cleavage of single-stranded DNA by
Endodeoxyribonuclease
[0096] The ability of the Delta P9 mutant and wild-type ribozymes
to cleave three different substrates was determined. The reactions
were carried out by admixing 0.02M of the ribozyme, 2.0 .mu.M of
either GGCCCUCU.A.sub.3UA.sub.3UA.sub.3 (S1) or
d(GGCCCTCU.A.sub.3TA.sub.3TA) (S2) or d(GGCCCTCT.A.sub.3TA.sub.3TA)
(S3), 30 mM N-[2-hydroxyethyl]-pipe- razine-N'-[3-propane-sulfonic
acid] (EPPs) at pH 7.5, 50 mM MgCl.sub.2 and 2 mM spermidine. The
resulting solution was maintained at 50.degree. C. for one hour.
The resulting reaction products were separated by electrophoresis
in a 5% polyacrylamide/8m urea gel. The gel was used to expose
x-ray film to produce an autoradiogram shown in FIG. 4.
[0097] The Delta P9 ribozyme cleaves the RNA substrate, S1 the
modified DNA substrate, S2, and the DNA substrate S3 (FIG. 4).
[0098] 3. Selection of Mutant Ribozymes Capable of Cleaving
DNA.
[0099] Mutant Ribozymes capable of cleaving a DNA substrate were
selected using the in vitro evolution system described by G. F.
Joyce, Gene, 82:83-87 (1989). This technique allows a structural
variant of Tetrahymena ribozyme capable of catalyzing a specific
reaction to be selectively amplified from a population of
Tetrahymena ribozyme structural variants.
[0100] This in vitro evolution technique was used to select a
Tetrahymena ribozyme structural variant that cleaves a
polydeoxyribonucleic acid (FIG. 5). The first step in this
technique is the ribozyme trans-splicing reaction involving the
attack by its 3'-terminal guanosine at a phosphodiester bond
following a sequence of pyrimidines located within a RNA substrate
previously described by G. F. Joyce, Gene, 82:83-87 (1989). The
product of the reaction is the ribozyme joined to the substrate
sequence that lies downstream from the target phosphodiester (FIG.
5, top). Selection occurs when an oligodeoxynucleotide primer is
hybridized across the ligation junction and used to initiate
synthesis of complementary using reverse transcriptase DNA (FIG. 5,
bottom). The primer ligation junction does not bind to unreacted
starting materials (<10.sup.-6 compared to reaction products, at
or below the limits of detection), and thus leads to selective
reverse transcription of reactive materials. In order to amplify
the selected materials, a primer containing one strand of a
promoter for T7 RNA polymerase is hybridized to the extreme 3' end
of the cDNA, the second strand of the promoter is completed using a
DNA-dependent DNA polymerase, and the DNA is transcribed to RNA as
has been previously described by Joyce, G. F. in Molecular Biology
of RNA UCLA Symposia on Molecular and Cellular Biology, ed., Cech,
T. R., 94:361-371, Alan R. Liss, New York, (1989) and Kwoh et al.,
Proc. Natl. Acad. Sci., USA, 86, 1173-1177 (1989).
[0101] The selected material is amplified at the transcription
level due to the high turnover of T7 RNA polymerase that has been
previously described by Chamberlin et al., in The Enzymes, ed. P.
Boyer, pp. 87-108, Academic Press, New York (1982). Mutations can
be introduced by replacing a portion of the cDNA with one or more
mutagenic oligodeoxynucleotides and transcribing the
partially-mismatched template directly as has been previously
described by Joyce et al., Nucleic Acid Research, 17:711-722
(1989). Ribozymes produced in this way can also be internally
labelled with .sup.32P-GTP.
[0102] The ability of a population of wild-type and mutant forms of
the Tetrahymena ribozyme to cleave the RNA substrate
GGCCCUCUAAAUAAAUA (S1), the modified DNA substrate
d(GGCCCTCUAAATAAATA) (S2), and the DNA substrate
d(GGCCCTCTAAATAAATA) (S3) was determined. Briefly, 1 .mu.M each of
internally labelled wild-type, Delta P6, Delta P2, Delta P9, Delta
P6/P9 and Delta P2/P9 ribozymes were admixed with 2 .mu.M of either
the S3 DNA substrate or the S1 RNA substrate, 30 mM EPPS at pH 7.5,
50 MM MgCl.sub.2 and 2 mM spermidine were admixed to form
endodeoxyribonuclease reaction admixtures. The
endodeoxyribonuclease reaction admixture was maintained at
50.degree. C. for one hour. The cleavage of substrate by each of
these ribozymes is detected as the appearance of a slower migrating
ribozyme caused by the ligation of the cleaved substrate to the
ribozyme (FIG. 6, Lanes 2 and 3).
[0103] Focusing on the DNA substrate S3, two rounds of selective
amplification were performed to recover the nucleic acid enzymes,
in this case endodeoxyribonucleases, capable of cleaving DNA from a
collection of ribozyme structural variants including, wild-type,
Delta P6, Delta P2, Delta P9, Delta P6/P6, and Delta P2/P9. These
structural variants (1 .mu.M, internally labelled with 1
.mu.Ci/nmole .sup.32P-GTP) were admixed with 2 .mu.M DNA substrate
S3, 30 mM EPPS at pH 7.5, 50 mM MgCl.sub.2 and 2 mM spermidine and
maintained at 50.degree. C. for 1 hour. The first round of cDNA
synthesis was carried out by admixing a 20-fold excess of
d(TAT.sub.3AT.sub.3CGAGT) primer, heating the solution to
65.degree. C. for 5 minutes in the presence of 50 mM Tris-HCl at pH
7.5 and 5 mM DTT and then rapidly cooling the solution to 0.degree.
C. The solution was then made to contain 6 mM MgCl.sub.2, 100 .mu.M
(each) dNTPs and 1 U/.mu.l of AMV reverse transcriptase. The
resulting solution was maintained at 37.degree. C. for 20 minutes.
A small aliquot of the solution was removed and analyzed by
electrophoresis in a 5% polyacrylamide/8 M urea gel. After the
first cDNA synthesis, the selected reverse transcriptant of the
Delta P6, Delta P2, Delta P9, and Delta P2/9 ribozymes can be seen
in Lane 5 of FIG. 6.
[0104] The RNA is destroyed by alkaline hydrolysis and the monomers
removed by ethanol precipitation. RNA was transcribed from the cDNA
by admixing a 20-fold excess of d(ATCGATAATA
CGACTCACTATAGGAGGGAAAAGTTATCAGG- C) primer, heating the resulting
solution to 65.degree. C. for 5 minutes in the presence of 50 mM
Tris-HCl at pH 7.5 and 5 mM DTT and then rapidly cooling the
solution to 0.degree. C. The solution is then made to contain 15 MM
MgCl.sub.2, 2 mM spermidine, 100 .mu.M (each) dNTPs, 2 mM (each)
NTPs 1 U/.mu.L AMV reverse transcriptase, 0.2 U/.mu.L DNA
polymerase I (Klenow fragment) and 20 U/.mu.L of T7 RNA polymerase.
The solution was maintained at 37.degree. C. for 1 hour to allow
RNA to be transcribed from the cDNA. This step results in a large
amplification of the ribozymes having the desired catalytic
activity (FIG. 6, Lane 6, {fraction (1/50)} of the material).
[0105] A second round of cDNA synthesis was performed using an
equal molar mixture of the primers d(CGAGTACTCCAAAC) and
d(CC-AGTACTCCGAC) to restore the 3' end of the RNA. The remainder
of the cDNA D synthesis was performed as above. The resulting
reaction products were analyzed by electrophoresis (FIG. 6, Lane
7).
[0106] A second round of RNA synthesis was performed using the
remaining reaction mixture using the RNA synthesis conditions
described above. A portion representing {fraction (1/50)} of the
resulting reaction products were analyzed by gel electrophoresis
and are shown in FIG. 6, Lane 8.
[0107] This system allowed the selection of a ribozyme having a
desired catalytic activity from a mixture of ribozymes.
[0108] The foregoing specification, including the specific
embodiments and examples, is intended to be illustrative of the
present invention and is not to be taken as limiting. Numerous
other variations and modifications can be effected without
departing from the true spirit and scope of the present
invention.
* * * * *