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Description  |
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The invention relates generally to oligonucleotides and their use as probes
and therapeutic agents, and more particularly, to modified
oligonucleotides whose ends are capable of spontaneously forming a stable
ring structure whenever such oligonucleotide specifically binds to a
target polynucleotide.
BACKGROUND
The unpredictability and expense of conventional drug discovery has led to
the exploration of several drug discovery approaches that promise more
systematic and/or rapid identification candidate compounds for testing in
biological assays and disease models. Examples of such approaches include
selection of small peptides from a synthetic or recombinant peptide
libraries, e.g. Pitrung et al, U.S. Pat. No. 5,143,854; Geysen et al, J.
Immunol. Meth., 102:259-274 (1987); Lam et al, Nature, 354:82-84 (1991);
Scott et al, Science, 249:386-390 (1990); the construction and selection
of human or humanized antibodies from recombinant antibody libraries, e.g.
Riechmann et al, Nature, 332:323-327 (1988); Winter and Milstein, Nature,
349:293-299 (1991 ); selection of aptamers or ribozymes from random
sequence polynucleotide libraries, e.g. Ellington and Szostak, Nature,
346:818-822 (1990); Blackwell et al, Science, 250:1104-1110 (1990); Tuerk
et al, Science, 249:505-510 (1990); Joyce, Gene, 82:83-87 (1989); Cech et
al, U.S. Pat. No. 4,987,071; Haseloffet al, Nature, 334:585-591 (1988);
and the use of antisense oligonucleotides, e.g. Uhlmann and Peyman,
Chemical Reviews, 90:543-584 (1990); Goodchild, Bioconjugate Chemistry,
1:165-187 (1990); Helene et al, Biochim. Biophys. Acta, 1049:99-125
(1990); Cohen, Ed., Oligonucleotides: Antisense Inhibitors of Gene
Expression (Macmillan Press, New York, 1989); Crooke, Ann. Rev. Pharmacol.
Toxicol., 32:329-376 (1992); McManaway et al, Lancet, Vol. 335, pgs.
808-811 (1990); Bayever et al, Antisense Research and Development,
2:109-110 (1992); Manson et al, Lymphokine Research, Vol. 9, pgs. 35-42
(1990); Lisziewicz et al, Proc. Natl. Acad. Sci., 90:3860-3864 (1993);
Miller, Biotechnology, Vol. 9, pgs. 358-362. (1991 ); Chiang et al, J.
Biol. Chem., Vol. 266, pgs. 18162-18171 (1991); Calabretta, Cancer
Research, Vol. 51, pgs. 4505-4510 (1991); and the like.
Of the cited examples, the antisense approach presents a compelling
advantage of not requiring one or more initial screening steps to identify
candidate compounds capable of binding to a predetermined target. Specific
binding is achieved by providing an oligonucleotide or an analog thereof
capable of forming a stable duplex or triplex with a target nucleotide
sequence based on Watson-Crick or Hoogsteen binding, respectively. Thus,
as soon as the sequence of a target polynucleotide is determined, the
structure of candidate antisense compounds is also determined. The
specifically bound antisense compound then either renders the respective
targets more susceptible to enzymatic degradation, blocks translation or
processing, or otherwise blocks or inhibits the function of a target
polynucleotide.
Another advantage of the antisense approach has been the development of
reliable and convenient methods for solid phase synthesis of
polynucleotides and analogs thereof, e.g. Caruthers, Science, Vol. 230,
pgs 281-285 (1985); Beaucage et al, Tetrahedron, 48: 2223-2311 (1992); and
Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (1RL
Press, Oxford, 1991 ). In particular, the availability of synthetic
oligonucleotides and a variety of nuclease-resistant analogs, e.g.
phosphorothioates, methylphosphonates, and the like, has encouraged
investigation of antisense compounds for treating a variety of conditions
associated with the inappropriate expression of indigenous and/or
exogenous genes, such as described in the references cited above.
Notwithstanding the many hurdles that have been overcome in the course of
developing antisense compounds, several significant uncertainties still
stand in the way of their widespread adoption as drugs. One such
uncertainty concerns the degree of specificity of antisense
oligonucleotides under physiological conditions. Antisense
oligonucleotides could be non-specific in at least two Senses: (i) duplex
or triplex formation may lack specificity, e.g. non-perfectly matched
duplexes may form--leading to the unwanted inhibition of non-target
polynucleotides, and (ii) the moieties not directly involved in base
pairing, e.g. the backbone or other appendant groups, may interact
non-specifically with other cellular components leading to undesired side
effects, e.g. Woolf et al, Proc. Natl. Acad. Sci., 89:7305-7309 (1992);
Matsukura et al, Proc. Natl. Acad. Sci., 4:7706-7710 (1987); and the like.
In regard to first type of nonspecificity, it has been observed that
duplexes involving longer oligonucleotides tend to be more tolerant of
mismatches--and hence, less specific--than duplexes involving shorter
oligonucleotides, e.g. Young et al, Nucleic Acids Research, 19:2463-2470
(1991). In regard to the second type of nonspecificity, such activity may
not be surprising in view of the large body of work on the use of
polyanions, in particular homopolymeric polynucleotides, as anti-viral
compounds, e.g. Levy, Chapter 7, in Stringfellow, editor, Interferon and
Interferon Inducers (Marcel Dekker, New York, 1980). Interestingly,
increased activity--and with some polyanions increased toxicity--was
observed with increased polymer size.
The uncertainty over nonspecific binding has led to the exploration of
several ways to modify oligonucleotides to enhance duplex or triplex
stability of antisense compounds. One approach has been to couple duplex
or triplex intercalating moieties to the antisense oligonucleotide, e.g.
Park et al, Proc. Natl. Acad. Sci., 89:6653-6657 (1992); Stein et al,
Gene, 72:333-341 (1988); Mergny et al, Science, 256:1681-1684 (1992);
Miller, International application PCT/US92/03999; and the like. Another
approach involves the use of circular oligonucleotides, which are
exonuclease resistant and have been shown to melt from single-stranded
targets at substantially higher temperatures than linear oligonucleotides
when binding involves both Watson-Crick and Hoogsteen base pairing, e.g.
Prakash and Kool, J. Am. Chem. Soc., 114:3523-3527 (1992).
Additional approaches for enhancing specificity and binding strength would
be highly useful for DNA-based therapeutics and diagnostic applications of
nucleic acids.
SUMMARY OF THE INVENTION
The invention relates to compounds capable of forming stable circular
complexes and/or covalently closed macrocycles after specifically binding
to a target polynucleotide. Generally, compounds-of the invention comprise
one or more oligonucleotide moieties capable of specifically binding to a
target polynucleotide and one or more pairs of binding moieties covalently
linked to the oligonucleotide moieties. In accordance with the invention,
upon annealing of the oligonucleotide moieties to the target
polynucleotide, the binding moieties of a pair are brought into
juxtaposition so that they form a stable covalent or non-covalent linkage
or complex. The interaction of the binding moieties of the one or more
pairs effectively clamps the specifically annealed oligonucleotide
moieties to the target polynucleotide.
In one aspect, compounds of the invention comprise a first binding moiety,
a first oligonucleotide moiety, a hinge region, a second oligonucleotide
moiety, and a second binding moiety, for example, as represented by the
particular embodiment of the following formula:
X--OL1--G--OL2--Y
wherein OL1 and OL2 are the first and second oligonucleotide moieties, G is
the hinge region, X is the first binding moiety and Y is the second
binding moiety such that X and Y form a stable covalent or non-covalent
linkage or complex whenever they are brought into juxtaposition by the
annealing of the oligonucleotide moieties to a target polynucleotide, as
illustrated diagrammatically in FIG. 1a. Preferably, in this embodiment,
one of OL1 and OL2 forms a duplex through Watson-Crick type of binding
with the target polynucleotide while the other of OL1 and OL2 forms a
triplex through Hoogsteen or reverse Hoogsteen type of binding. Whenever X
and Y form a covalent linkage, the compound of the invention forms a
macrocycle of the following form:
##STR1##
wherein "XY" is the covalent linkage formed by the reaction of X and Y.
In another aspect, compounds of the invention comprise a first binding
moiety, a first, second, and third oligonucleotide moiety, a first and
second hinge region, and a second binding moiety, for example, as
represented by the particular embodiment of the following formula:
X--OL1--G.sub.1 --OL2--G.sub.2 --OL3
wherein X and Y are described as above, G.sub.1 and G.sub.2 are the first
and second hinge regions, and OL1, OL2, and OL3 are the first through
third oligonucleotide moieties. Preferably, the sequences of OL1, OL2, and
OL3 are selected so that OL1 and OL2 and OL3 form triplex structures with
the target polynucleotide, as diagrammatically illustrated in FIG. 1b.
Whenever X and Y form a covalent linkage, the compound of the invention
forms a macrocycle of the following form:
##STR2##
wherein "XY" is the covalent linkage formed by the reaction of X and Y.
In yet another aspect, the oligonucleotide clamps of the invention are
compositions of two or more components, e.g. having the form:
X--OL1--W and Y--OL2--Z
wherein X, Y, W, and Z are defined as X and Y above. In this embodiment,
the hinge region is replaced by additional complex-forming moieties W and
Z. As above, one of OL1 and OL2 undergoes Watson-Crick type of binding
while the other undergoes Hoogsteen or reverse Hoogsteen type of binding
to a target polynucleotide, as shown diagrammatically in FIG. 1c.
Similarly, whenever X and Y and W and Z form covalent linkages, compounds
X--OL1--W and Y--OL2--Z form a macrocycle of the following form:
##STR3##
depending on the selection of OL1 and OL2.
Preferably, compounds of the invention are capable of forming covalently
closed macrocycles or stable circular complexes topologically linked to a
target polynucleotide.
The invention provides compounds capable of specifically binding to
predetermined target polynucleotides with superior stability than
currently available probes and antisense compounds. Compounds of the
invention are employed either as antisense or antiogene compounds to
inhibit the function of a polynucleotide whose expression or participation
in a regulatory function is associated with a disease state or as probes
for detecting the presence of a target polynucleotide. The invention
includes the oligonucleotide clamps per se as well as pharmaceutical
compositions and kits for particular applications.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1a-c diagrammatically illustrate how three separate embodiments of
the invention bind or "clamp" a target polynucleotide.
FIG. 2a-b diagrammatically illustrates the concept of "topologically
linked."
FIG. 3 illustrates the results of the inhibition of the cytotoxic effects
of HIV in an ATH8 cell assay.
Definitions
"Topologically linked" in reference to compounds of the invention refers to
the relative configuration of target polynucleotide and oligonucleotide
clamp wherein the oligonucleotide clamp forms a closed circular complex or
macrocycle enclosing the target polynucleotide strand, as shown
diagrammatically in FIGS. 2a and 2b. In FIG. 2a, oligonucleotide clamp 20
is topologically linked to target polynucleotide 10. In FIG. 2b,
oligonucleotide 20 is topologically disjoint from target polynucleotide
10.
"Stable" in reference to the formation of a covalent linkage and/or
non-covalent complex between binding moieties means that melting
temperature of the oligonucleotide clamp incorporating the given pair(s)
of binding moieties and its target polynucleotide is increased by at least
fifty percent over the melting temperature of oligonucleotide moieties of
the clamp alone, wherein melting temperature is measured by standard
techniques, e.g. half maximum of 260 nm absorbance v. temperature as
described more fully below.
"Linkage" in reference to the reaction of binding moieties includes both
covalent linkages and non-covalent complexes.
The term "oligonucleotide" as used herein includes linear oligomers of
natural or modified monomers or linkages, including deoxyribonucleosides,
ribonucleosides, .alpha.-anomeric forms thereof, polyamide nucleic acids,
and the like, capable of specifically binding to a target polynucleotide
by way of a regular pattern of monomer-to-monomer interactions, such as
Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of
base pairing, or the like. Usually monomers are linked by phosphodiester
bonds or analogs thereof to form oligonucleotides ranging in size from a
few monomeric units, e.g. 3-4, to several hundreds of monomeric units.
Whenever an oligonucleotide is represented by a sequence of letters, such
as "ATGCCTG," it will be understood that the nucleotides are in 5'->3'
order from left to right and that "A" denotes deoxyadenosine, "C" denotes
deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine,
unless otherwise noted. Analogs of phosphodiester linkages include
phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,
phosphoramidate, and the like.
As used herein, "nucleoside" includes the natural nucleosides, including
2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker,
DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in
reference to nucleosides includes synthetic nucleosides having modified
base moieties and/or modified sugar moieties, e.g. described generally by
Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs
include synthetic nucleosides designed to enhance binding properties, e.g.
stability, specificity, or the like, such as disclosed by the references
cited herein.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to oligonucleotide clamps that are capable of binding
to a specific region of a target polynucleotide. The clamping aspect of
the compounds is achieved by the formation of stable linkages or complexes
between binding moieties after they are brought into proximity by specific
binding of the one or more oligonucleotide moieties to a target
polynucleotide. Preferably, oligonucleotide moieties of the compounds of
the invention are selected so that they simultaneously undergo
Watson-Crick and Hoogsteen types of binding with specific regions of a
target polynucleotide.
In embodiments where triplex formation is desired, there are constraints on
the selection of target sequences. Generally, third strand association via
Hoogsteen type of binding is most stable along homopyrimidine-homopurine
tracks in a double stranded target. Usually, base triplets form in T-A*T
or C-G*C motifs (where "-" indicates Watson-Crick pairing and "*"
indicates Hoogsteen type of binding); however, other motifs are also
possible. For example, Hoogsteen base pairing permits parallel and
antiparallel orientations between the third strand (the Hoogsteen strand)
and the purine-rich strand of the duplex to which the third strand binds,
depending on conditions and the composition of the strands. There is
extensive guidance in the literature for selecting appropriate sequences,
orientation, conditions, nucleoside type (e.g. whether ribose or
deoxyribose nucleosides are employed), base modifications (e.g. methylated
cytosine, and the like) in order to maximize, or otherwise regulate,
triplex stability as desired in particular embodiments, e.g. Roberts et
al, Proc. Natl. Acad. Sci., 88:9397-9401 (1991); Roberts et al, Science,
258:1463-1466 (1992); Distefano et al, Proc. Natl. Acad. Sci.,
90:1179-1183 (1993); Mergny et al, Biochemistry, 30:9791-9798 (1991);
Cheng et al, J. Am. Chem. Soc., 114:4465-4474 (1992); Beal and Dervan,
Nucleic Acids Research, 20:2773-2776 (1992); Beal and Dervan, J. Am.
Chem. Soc., 114:4976-4982 (1992); Giovannangeli et al, Proc. Natl. Acad.
Sci., 89: 8631-8635 (1992); Moser and Dervan, Science, 238:645-650 (1987);
McShan et al, J. Biol. Chem., 267:5712-5721 (1992); Yoon et al, Proc.
Natl. Acad. Sci., 89:3840-3844 (1992); Blume et al, Nucleic Acids
Research, 20:1777-1784 (1992); and the like. Generally, after one of the
oligonucleotide moieties forms a Watson-Crick duplex with a
pyrimidine-rich or purine-rich track in a target polynucleotide, the
remaining oligonucleotide components bind to the major groove of the
duplex to form a triplex structure.
Selection of particular oligonucleotide sequences for triplex formation can
also be carded out empirically, for example, through aptamer screening, or
like process, where candidate oligonucleotide moieties are selected on the
basis of binding strength to an immobilized double stranded target, e.g.
Ellington and Szostak, Nature, 346:818-822 (1990); Toole et al,
International application PCT/US92/01383; and the like.
Target polynucleotides may be single stranded or double stranded DNA or
RNA; however, single stranded DNA or RNA target polynucleotides are
preferred.
Preferably, stability of oligonucleotide clamp/target polynucleotide
complexes are determined by way of melting, or strand dissociation,
curves. The temperature of fifty percent strand dissociation is taken as
the melting temperature, T.sub.m, which, in turn, provides a convenient
measure of stability. T.sub.m measurements are typically carried out in a
saline solution at neutral pH with target and clamp concentrations at
between about 1.0-2.0 .mu.M. Typical conditions are as follows: 150 mM
NaCl and 10 mM MgCl.sub.2 in a 10 mM sodium phosphate buffer (pH 7.0) or
in a 10 mM Tris-HCl buffer (pH 7.0); or like conditions. Data for melting
curves are accumulated by heating a sample of the oligonucleotide
clamp/target polynucleotide complex from room temperature to about
85.degree.-90.degree. C. As the temperature of the sample increases,
absorbance of 260 nm light is monitored at 1.degree. C. intervals, e.g.
using a Cary (Australia) model 1E or a Hewlett-Packard (Palo Alto, Calif.)
model HP 8459 UV/VIS spectrophotometer and model HP 89100A temperature
controller, or like instruments.
The oligonucleotide moieties of the invention are synthesized by
conventional means on a commercially available automated DNA synthesizer,
e.g. an Applied Biosystems (Foster City, Calif.) model 380B, 392 or 394
DNA/RNA synthesizer. Preferably, phosphoramidite chemistry is employed,
e.g. as disclosed in the following references: Beaucage and Iyer,
Tetrahedron, 48:2223-2311 (1992); Molko et al, U.S. Pat. No. 4,980,460;
Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos.
4,415,732; 4,458,066; and 4,973,679; and the like. For therapeutic use,
nuclease resistant backbones are preferred. Many types of modified
oligonucleotides are available that confer nuclease resistance, e.g.
phosphorothioate, phosphorodithioate, phosphoramidate, or the like,
described in many references, e.g. phosphorothioates: Stec et al, U.S.
Pat. No. 5,151,510; Hirschbein, U.S. Pat. No. 5,166,387; Bergot, U.S. Pat.
No. 5,183,885; phosphoramidates: Froehler et al, International application
PCT/US90/03138; and for a review of additional applicable chemistries:
Uhlmann and Peyman (cited above). In some embodiments it may be desirable
to employ P-chiral linkages, in which case the chemistry disclosed by Stec
et al, European patent application 92301950.9, may be appropriate.
The length of the oligonucleotide moieties is sufficiently large to ensure
that specific binding will take place only at the desired target
polynucleotide and not at other fortuitous sites. The upper range of the
length is determined by several factors, including the inconvenience and
expense of synthesizing and purifying oligomers greater than about 30-40
nucleotides in length, the greater tolerance of longer oligonucleotides
for mismatches than shorter oligonucleotides, and the like. Preferably,
the oligonucleotide moieties have lengths in the range of about 6 to 40
nucleotides. More preferably, the oligonucleotide moieties have lengths in
the range of about 12 to 25 nucleotides.
Hinge regions consist of nucleosidic or non-nucleosidic polymers which
preferably facilitate the specific binding of the monomers of the
oligonucleotide moieties with their complementary nucleotides of the
target polynucleotide. Generally, the oligonucleotide moieties may be
connected to hinge regions and/or binding moieties in either 5'->3' or
3'->5' orientations. For example, in the embodiment described above
comprising a first binding moiety, a first oligonucleotide moiety, a hinge
region, a second oligonucleotide moiety, and a second binding moiety, the
oligonucleotide moieties may have any of the following orientations:
X--(5')N.sub.1 N.sub.2 N.sub.3 --. . . --N.sub.j (3')--G--(5')N.sub.1
N.sub.2 N.sub.3 --. . . --N.sub.k (3')--Y
OR
X--(5')N.sub.1 N.sub.2 N.sub.3 --. . .--N.sub.j
(3')--G--(3')--G--(3')N.sub.k N.sub.k--1 N.sub.k--2 --. . .--N.sub.1
(5')--Y
OR
X--(3')N.sub.j N.sub.j--1 N.sub.j--2 --. . .--N.sub.1 (5')--G--(5')N.sub.1
N.sub.2 N.sub.3 --. . .--N.sub.k (3')--Y
OR
X--(3')N.sub.j N.sub.j--1 N.sub.j--2 --. . .--N.sub.1 (5')--G--(3')N.sub.k
N.sub.k--1 N.sub.k--2 --. . .--N.sub.1 (5')--Y
wherein N.sub.1 N.sub.2 N.sub.3 --. . . --N.sub.k and N.sub.1 N.sub.2
N.sub.3 --. . .--N.sub.j are k-mer and j-mer oligonucleotide moieties in
the indicated orientations.
Preferably, the hinge region has the general form:
--(M--L).sub.n --
wherein M may be an inert non-sterically hindering spacer moiety serving to
connect the oligonudeotide moieties, wherein the M's and L's in any given
chain may be the same or different. Alternatively, one or more of monomers
M may contain reactive functionalities for attaching labels;
oligonucleotides or other binding polymers for hybridizing or binding to
amplifier strands or structures, e.g. as described by Urdea et al, U.S.
Pat. No. 5,124,246 or Wang et al, U.S. Pat. No. 4,925,785; "hooks", e.g.
as described in Whiteley et al, U.S. Pat. No. 4,883,750; or other groups
for affecting solubility, cellular delivery, promotion of duplex and/or
triplex formation, such as intercalators, alkylating agents, and the like.
Preferably, the hinge regions provide a spacer of about 16-28 angstroms
between the termini of the oligonucleotides.
Preferably, L is a phosphorus(V) linking group which may be phosphodiester,
phosphotfiester, methyl or ethyl phosphonate, phosphorothioate,
phosphorodithioate, phosphoramidate, or the like. Generally, linkages
derived from phosphoramidite precursors are preferred so that compounds of
the invention can be conveniently synthesized with commercial automated
DNA synthesizers, e.g. Applied Biosystems, Inc. (Foster City, Calif.)
model 394, or the like.
n may vary significantly depending on the nature of M and L. Generally, n
will vary from 1 for M comprising alkyl, alkenyl, and/or ethers containing
10 or more carbon atoms, e.g. Salunkhe et al, J. Am. Chem. Sot.,
114:8768-8772 (1992), to about 10 for M comprising alkyl, alkenyl, and/or
ethers containing 2-3 carbon atoms. Preferably, for a hinge moiety
consisting entirely of an alkyl chain (and linkage moieties), such alkyl
chain contains form 8 to 15 carbon atoms, and more preferably, from 9 to
12 carbon atoms. Preferably, for nucleoside-sized monomers, n varies
between about 3 and about 10; and more preferably, n varies between about
4 and 8.
Preferably, hinge moieties are synthesized using conventional
phosphoramidite and/or hydrogen phosphonate chemistries. The following
references disclose several phosphoramidite and/or hydrogen phosphonate
monomers suitable for use in the present invention and provide guidance
for their synthesis and inclusion into oligonucleotides: Newton et al,
Nucleic Acids Research, 21:1155-1162 (1993); Griffin et al, J. Am. Chem.
Sot., 114:7976-7982 (1992); Jaschke et al, Tetrahedron Letters, 34:301-304
(1992); Ma et at, International application PCT/CA92/00423; Zon et al,
International application PCT/US90/06630; Durand et at, Nucleic Acids
Research, 18:6353-6359 (1990); Salunkhe et al, J. Am. Chem. Soc.,
114:8768-8772 (1992); and the like.
In a preferred embodiment, M is a straight chain, cyclic, or branched
organic molecular structure containing from 1 to 20 carbon atoms and from
0 to 10 heteroatoms selected from the group consisting of oxygen,
nitrogen, and sulfur. More preferably, M is alkyl, alkoxy, alkenyl, or
aryl containing from 1 to 16 carbon atoms; heterocyclic having from 3 to 8
carbon atoms and from 1 to 3 heteroatoms selected from the group
consisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl. Most
preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 8
carbon atoms; glycosyl; or nucleosidyl.
A variety of binding moieties are suitable for use with the invention.
Generally, they are employed in pairs, which for convenience here will be
referred to as X and Y. X and Y may be the same or different. Whenever the
interaction of X and Y is based on the formation of stable hydrophobic
complex, X and Y are lipophilic groups, including alkyl groups, fatty
acids, fatty alcohols, steroids, waxes, fat-soluble vitamins, and the
like. Further exemplary lipophilic binding moieties include glycerides,
glyceryl ethers, phospholipids, sphingolipids, terpenes, and the like. In
such embodiments, X and Y are preferably selected from the group of
steroids consisting of a derivatized perhydrocyclopentanophenanthrene
nucleus having from 19 to 30 carbon atoms, and 0 to 6 oxygen atoms; alkyl
having from 6 to 16 carbon atoms; vitamin E; and glyceride having 20 to 40
carbon atoms. Preferably, a perhydrocyclopentanophenanthrene-based moiety
is attached through the hydroxyl group, either as an ether or an ester, at
its C3 position. It is understood that X and Y may include a linkage group
connecting it to an oligonucleotide moiety. For example, glyceride
includes phosphoglyceride, e.g. as described by MacKellar et al, Nucleic
Acids Research, 20:3411-3417 (1992), and so on. It is especially preferred
that lipophilic moieties, such as perhydrocyclopentanophenanthrene
derivatives, be linked to the 5' carbon and/or the 3' carbon of an
oligonucleotide moiety by a short but flexible linker that permits the
lipophilic moiety to interact with the bases of the oligonucleotide
clamp/target polynucleotide complex or a lipophilic moiety on the same or
another oligonucleotide moiety. Such linkers include phosphate (i.e.
phosphodiester), phosphoramidate, hydroxyurethane, carboxyaminoalkyl and
carboxyaminoalkylphosphate linkers, or the like. Preferably, such linkers
have no more than from 2 to 8 carbon atoms.
Binding moieties can be attached to the oligonucleotide moiety by a number
of available chemistries. Generally, it is preferred that the
oligonucleotide be initially derivatized at its 3' and/or 5' terminus with
a reactive functionality, such as an amino, phosphate, thiophosphate, or
thiol group. After derivatization, a hydrophilic or hydrophobic moiety is
coupled to the oligonucleotide via the reactive functionality. Exemplary
means for attaching 3' or 5' reactive functionalities to oligonucleotides
are disclosed in Fung et al, U.S. Pat. No. 5,212,304; Connolly, Nucleic
Acids Research, 13: 4485-4502 (1985); Tino, International application
PCT/US91/09657; Nelson et al, Nucleic Acids Research, 17:7187-7194 (1989);
Stabinsky, U.S. Pat. No. 4,739,044; Gupta et al, Nucleic Acids Research,
19:3019 (1991 ); Reed et al, International application PCT/US91/06143;
Zuckerman et al, Nucleic Acids Research, 15:5305 (1987); Eckstein, editor,
Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,
1991 ); Clontech 1992/1993 Catalog (Clontech Laboratories, Palo Alto, CA);
and like references.
Preferably, whenever X and Y form a covalent linkage, X and Y pairs must
react specifically with each other when brought into juxtaposition, but
otherwise they must be substantially unreactive with chemical groups
present in a cellular environment. In this aspect of the invention, X and
Y pairs are preferably selected from the following group: when one of X or
Y is phosphorothioate, the other is haloacetyl, haloacyl, haloalkyl, or
alkylazide; when one of X or Y is thiol, the other is alkyl iodide,
haloacyl, or haloacetyl; when one of Y or Y is phenylazide the other is
phenylazide. More preferably, when one of X or Y is phosphorothioate, the
other is haloacetyl, haloacyl, or haloalkyl, wherein said alkyl, acetyl,
or acyl moiety contains from one to eight carbon atoms.
Most preferably, when one of X or Y is phosphorothioate, the other is
haloacetyl. Most preferably, whenever one of X or Y is phosphorothioate,
the other is bromoacetyl. These binding moieties form a covalent
thiophosphoylacetylamino bridge, as shown below, selectively and
efficiently at low concentrations, e.g. less than one .mu.M, when reacted
in an aqueous environment in the presence of a target polynucleotide:
##STR4##
wherein X is halo and N.sub.1, N.sub.2, N.sub.j and N.sub.k are
nucleotides of a j-mer and k-mer, respectively. Compound 1 can be prepared
by N-succinimidyl haloacetate in N,N-dimethylformamide (DMF) with a
3'-aminodeoxyribonucleotide precursor in a sodium borate buffer at room
temperature. After about 35 minutes the mixture is diluted (e.g. with
H.sub.2 O), desalted and, purified, e.g. by reverse phase HPLC. The
3'-aminodeoxyribonucleotide precursor can be prepared as described in
Gryaznov and Letsinger, Nucleic Acids Research, 20:3403-3409 (1992) or
Tetrahedron Letters, 34: 1261-1264 (1993). Briefly, after deprotection,
the 5' hydroxyl of a deoxythymidine linked to a support via a standard
succinyl linkage is phosphitylated by reaction with
chloro-(diisopropylethylamino)-methoxyphosphine in an appropriate solvent,
such as dichloromethane/diisopropylethylamine. After activation with
tetrazole, the 5'-phosphitylated thymidine is reacted with a
5'-trityl-O-3'-amino-3'-deoxynucleoside to form a nucleoside-thymidine
dimer wherein the nucleoside moieties are covalently joined by a
phosphoramidate linkage. The remainder of the oligonucleotide is
synthesized by standard phosphoramidite chemistry. After cleaving the
succinyl linkage, the oligonucleotide with a 3'-terminal amino group is
generated by cleaving the phosphoramidate link by acid treatment, e.g. 80%
aqueous acetic acid for 18-20 hours at room temperature.
5'-trityl-O-3'-amino-3'-deoxynucleosides may be synthesized in accordance
with Glinski et al, J. Chem. Soe. Chem. Comm., 915-916 (1970); Miller et
al, J. Org. Chem. 29:1772 (1964); Zielinki and Orgel, Nucleic Acids
Research, 13:2469-2484 (1985) and 15:169%1715 (1987); Ozols et al,
Synthesis, 7:557-559 (1980); and Azhayev et al, Nucleic Acids Research, 6:
625-643 (1979); which references are incorporated by reference.
5' monophosphorothioate 2 is formed as follows: A 5' monophosphate is
attached to the 5' end of an oligonucleotide either chemically or
enzymatically with a kinase, e.g. Sambrook et al, Molecular Cloning: A
Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory, New York,
1989). Preferably, as a final step in oligonucleotide synthesis, a
monophosphate is added by chemical phosphorylation as described by Horn
and Urdea, Tetrahedron Lett., 27:4705 (1986) (e.g. using commercially
available reagents such as 5'Phosphate-ON.sup.TM from Clontech
Laboratories (Palo Alto, California)). The 5'-monophosphate is then
sulfurized using conventional sulfurizing agents, e.g. treatment with a 5%
solution of S.sub.8 in pyridine/CS.sub.2 (1:1, v/v, 45 minutes at room
temperature); or treatment with sulfurizing agent described in U.S. Pat.
Nos. 5,003,097; 5,151,510; or 5,166,387. Preferably, the haloacetylamino
derivatized oligonucleotides are synthesized separately from unprotected
monophosphorothioate groups.
Compounds of the invention can be employed as diagnostic probes to detect
the presence of one or more target polynucleotides in a wide range of
samples, including environmental samples, e.g. from public water supplies,
samples from foodstuffs, and from other biological samples, such as blood,
saliva, semen, amniotic fluid, tissue homogenates of plants or animals, or
of human patients, and the like. The use of nucleic acid probes in human
diagnostics, forensics, and genetic analysis has been extensively
reviewed. For example, the following references describe many diagnostic
applications of nucleic acid probes for which the present invention can be
usefully employed: Caskey, Science 236:1223-1228 (1987); Landegren et al,
Science, 242:229-237 (1988); and Arnheim et al, Ann. Rev. Biochem.,
61:131-156 (1992). Moreover, there is extensive guidance in the literature
concerning the selection of hybridization conditions, labeling means, and
the like, which is applicable to the practice of the present invention,
e.g. Wallace et al, Nucleic Acids Research 6:3543-3557 (1979); Crothers et
al, J. Mol. Biol. 9:1-9 (1964); Gotoh, Adv. Biophys. 16:1-52 (1983);
Wetruer, Critical Reviews in Biochemistry and Molecular Biology 26:227-259
(1991 ); Breslauer et al, Proc. Natl. Acad. Sci. 83:3746-3750 (1986);
Wolfet al, Nucleic Acids Research, 15:2911-2926 (1987); McGraw et al,
Biotechniques, 8:674-678 (1990), and the like.
Oligonucleotide clamps of the invention may be used in essentially any of
the known solution or solid phase hybridization formats, such as those in
which the analyte is bound directly to a solid phase, or sandwich
hybridizations in which the analyte is bound to an oligonucleotide that
is, in turn, bound to a solid phase. Oligonucleotide clamps having an
oligonucleotide "tail" attached to a hinge region are particularly useful
in conjunction with branched polymer amplification schemes, such as those
disclosed by Urdea et al, U.S. Pat. No. 5,124,246; Wang et al, U.S. Pat.
No. 4,925,785; and the like. Urdea et al and Wang et al are incorporated
by reference for their description of such hybridization assays. In such
embodiments, the oligonucleotide clamp serves as a highly stable "capture"
probe by binding to a target polynucleotide analyte of interest. The
oligonucleotide tail then hybridizes with a directly or indirectly labeled
amplifier strand or complex. Such tails are long enough to form a stable
duplex with the amplifier strand. Preferably, such tails are between 18
and 60 nucleotides in length. Tails may also comprise a second
oligonucleotide clamp. That is, a dimer of oligonucleotide clamps having
different binding specificities can be used to tightly couple an amplifier
complex to a target polynucleotide.
Preferably, oligonucleotide tails are coupled to hinge regions at an amino
group which has been derivatized with bromoacetyl. An oligonucleotide tail
having either a 5' or 3' phosphorothioate group is then reacted with the
bromoacetyl group to form a thiophosphorylacetylamino bridge, as described
more fully above and in the Examples below. Phosphoramidate linkages are
introduced in accordance with published procedures, e.g. Letsinger, U.S.
Pat. No. 4,958,013; Agrawal et al, Nucleic Acids Research, 18:5419-5423
(1990); or the like. By a similar procedure, dimers of oligonucleotide
clamps can also be constructed.
Whenever oligonucleotide clamps of the invention are employed in diagnostic
assays, or in other processes not requiring direct contact with a patient,
a wider range of binding moieties may be employed than would be possible
for therapeutic use. In diagnostic and other non-therapeutic applications,
reaction of the binding moieties may involve an activation step wherein
one or both of the binding moieties are activated or rendered reactive
towards one another by exposure to an activating agent or condensing
agent, such as radiation, a reducing agent, an oxidizing agent, or the
like. Exemplary, binding moieties employing activating agents include
thiophosphoryl groups in the presence of K.sub.3 Fe(CN).sub.6 or KI.sub.3,
e.g. Gryaznov and Letsinger, Nucleic Acids Research, 21: 1403-1408 (1993
); phosphoryl and hydroxyl in the presence of N-cyanoimidazole, e.g.
Luebke et al, J. Am. Chem. Soc., 113:7447-7448 (1991); phosphoryl or amino
group and hydroxyl in the presence of cyanogen bromide, e.g. Sokolova et
al, FEBS Letters, 232:153-155 (1988); phosphoryl and hydroxyl groups in
the presence of spermine-5-(N-ethylimidazole)carboxamide and
cyanoimidazole, e.g. Zuber et al, J. Am. Chem. Soc., 115: 4939-4940
(1993); and the like.
Kits incorporating oligonucleotide clamps can take a variety of forms
depending on the particular embodiment, the type of assay format employed,
and the labeling scheme empl | | |
