 |
Description  |
|
|
BACKGROUND OF THE INVENTION
The present invention relates to the field of polymer synthesis and the use
of polymer libraries for biological screening. More specifically, in one
embodiment the invention provides arrays of diverse double-stranded
oligonucleotide sequences. In another embodiment, the invention provides
arrays of conformationally restricted probes, wherein the probes are held
in position using double-stranded DNA sequences as scaffolding. Libraries
of diverse unimolecular double-stranded nucleic acid sequences and probes
may be used, for example, in screening studies for determination of
binding affinity exhibited by binding proteins, drugs, or RNA.
Methods of synthesizing desired single stranded DNA sequences are well
known to those of skill in the art. In particular, methods of synthesizing
oligonucleotides are found in, for example, Oligonucleotide Synthesis: A
Practical Approach, Gait, ed., IRL Press, Oxford (1984), incorporated
herein by reference in its entirety for all purposes. Synthesizing
unimolecular double-stranded DNA in solution has also been described. See,
Durand, et al. Nucleic Acids Res. 18:6353-6359 (1990) and Thomson, et al.
Nucleic Acids Res. 21:5600-5603 (1993), the disclosures of both being
incorporated herein by reference.
Solid phase synthesis of biological polymers has been evolving since the
early "Merrifield" solid phase peptide synthesis, described in Merrifield,
J. Am. Chem. Soc. 85:2149-2154 (1963), incorporated herein by reference
for all purposes. Solid-phase synthesis techniques have been provided for
the synthesis of several peptide sequences on, for example, a number of
"pins." See e.g., Geysen et al., J. Immun. Meth. 102:259-274 (1987),
incorporated herein by reference for all purposes. Other solid-phase
techniques involve, for example, synthesis of various peptide sequences on
different cellulose disks supported in a column. See Frank and Doring,
Tetrahedron 44:6031-6040 (1988), incorporated herein by reference for all
purposes. Still other solid-phase techniques are described in U.S. Pat.
No. 4,728,502 issued to Hamill and WO 90/00626 (Beattie, inventor).
Each of the above techniques produces only a relatively low density array
of polymers. For example, the technique described in Geysen et al. is
limited to producing 96 different polymers on pins spaced in the
dimensions of a standard microtiter plate.
Improved methods of forming large arrays of oligonucleotides, peptides and
other polymer sequences in a short period of time have been devised. Of
particular note, Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT
Application No. WO 90/15070) and Fodor et al., PCT Publication No. WO
92/10092, all incorporated herein by reference, disclose methods of
forming vast arrays of peptides, oligonucleotides and other polymer
sequences using, for example, light-directed synthesis techniques. See
also, Fodor et al., Science, 251:767-777 (1991), also incorporated herein
by reference for all purposes. These procedures are now referred to as
VLSIPS.TM. procedures.
In the above-referenced Fodor et al., PCT application, an elegant method is
described for using a computer-controlled system to direct a VLSIPS.TM.
procedure. Using this approach, one heterogenous array of polymers is
converted, through simultaneous coupling at a number of reaction sites,
into a different heterogenous array. See, U.S. Pat. No. 5,384,261 and U.S.
application Ser. No. 07/980,523, the disclosures of which are incorporated
herein for all purposes.
The development of VLSIPS.TM. technology as described in the above-noted
U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and
92/10092, is considered pioneering technology in the fields of
combinatorial synthesis and screening of combinatorial libraries. More
recently, patent application Ser. No. 08/082,937, filed Jun. 25, 1993 now
abandoned, describes methods for making arrays of oligonucleotide probes
that can be used to check or determine a partial or complete sequence of a
target nucleic acid and to detect the presence of a nucleic acid
containing a specific oligonucleotide sequence.
A number of biochemical processes of pharmaceutical interest involve the
interaction of some species, e.g., a drug, a peptide or protein, or RNA,
with double-stranded DNA. For example, protein/DNA binding interactions
are involved with a number of transcription factors as well as tumor
suppression associated with the p53 protein and the genes contributing to
a number of cancer conditions.
SUMMARY OF THE INVENTION
High-density arrays of diverse unimolecular, double-stranded
oligonucleotides, as well as arrays of conformationally restricted probes
and methods for their use are provided by virtue of the present invention.
In addition, methods and devices for detecting duplex formation of
oligonucleotides on an array of diverse single-stranded oligonucleotides
are also provided by this invention. Further, an adhesive based on the
specific binding characteristics of two arrays of complementary
oligonucleotides is provided in the present invention.
According to one aspect of the present invention, libraries of
unimolecular, double-stranded oligonucleotides are provided. Each member
of the library is comprised of a solid support, an optional spacer for
attaching the double-stranded oligonucleotide to the support and for
providing sufficient space between the double-stranded oligonucleotide and
the solid support for subsequent binding studies and assays, an
oligonucleotide attached to the spacer and further attached to a second
complementary oligonucleotide by means of a flexible linker, such that the
two oligonucleotide portions exist in a double-stranded configuration.
More particularly, the members of the libraries of the present invention
can be represented by the formula:
Y--L.sup.1 --X.sup.1 --L.sup.2 --X.sup.2
in which Y is a solid support, L.sup.1 is a bond or a spacer, L.sup.2 is a
flexible linking group, and X.sup.1 and X.sup.2 are a pair of
complementary oligonucleotides.
In a specific aspect of the invention, the library of different
unimolecular, double-stranded oligonucleotides can be used for screening a
sample for a species which binds to one or more members of the library.
In a related aspect of the invention, a library of different
conformationally-restricted probes attached to a solid support is
provided. The individual members each have the formula:
--X.sup.11 --Z--X.sup.12
in which X.sup.11 and X.sup.12 are complementary oligonucleotides and Z is
a probe having sufficient length such that X.sup.11 and X.sup.12 form a
double-stranded oligonucleotide portion of the member and thereby restrict
the conformations available to the probe. In a specific aspect of the
invention, the library of different conformationally-restricted probes can
be used for screening a sample for a species which binds to one or more
probes in the library.
According to yet another aspect of the present invention, methods and
devices for the bioelectronic detection of duplex formation are provided.
According to still another aspect of the invention, an adhesive is provided
which comprises two surfaces of complementary oligonucleotides.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A to 1F illustrate the preparation of a member of a library of
surface-bound, unimolecular double-stranded DNA as well as binding studies
with receptors having specificity for either the double stranded DNA
portion, a probe which is held in a conformationally restricted form by
DNA scaffolding, or a bulge or loop region of RNA.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Abbreviations
The following abbreviations are used herein: phi, phenanthrenequinone
diimine; phen', 5-amido-glutaric acid-1,10-phenanthroline; dppz,
dipyridophenazine.
Glossary
The following terms are intended to have the following general meanings as
they are used herein:
Chemical terms: As used herein, the term "alkyl" refers to a saturated
hydrocarbon radical which may be straight-chain or branched-chain (for
example, ethyl, isopropyl, t-amyl, or 2,5-dimethylhexyl). When "alkyl" or
"alkylene" is used to refer to a linking group or a spacer, it is taken to
be a group having two available valences for covalent attachment, for
example, --CH.sub.2 CH.sub.2 --, --CH.sub.2 CH.sub.2 CH.sub.2 --,
--CH.sub.2 CH.sub.2 CH(CH.sub.3)CH.sub.2 -- and --CH.sub.2 (CH.sub.2
CH.sub.2).sub.2 CH.sub.2 --. Preferred alkyl groups as substituents are
those containing 1 to 10 carbon atoms, with those containing 1 to 6 carbon
atoms being particularly preferred. Preferred alkyl or alkylene groups as
linking groups are those containing 1 to 20 carbon atoms, with those
containing 3 to 6 carbon atoms being particularly preferred. The term
"polyethylene glycol" is used to refer to those molecules which have
repeating units of ethylene glycol, for example, hexaethylene glycol
(HO--(CH.sub.2 CH.sub.2 O).sub.5 --CH.sub.2 CH.sub.2 OH). When the term
"polyethylene glycol" is used to refer to linking groups and spacer
groups, it would be understood by one of skill in the art that other
polyethers or polyols could be used as well (i. e, polypropylene glycol or
mixtures of ethylene and propylene glycols).
The term "protecting group" as used herein, refers to any of the groups
which are designed to block one reactive site in a molecule while a
chemical reaction is carried out at another reactive site. More
particularly, the protecting groups used herein can be any of those groups
described in Greene, et al., Protective Groups In Organic Chemistry, 2nd
Ed., John Wiley & Sons, New York, N.Y, 1991, incorporated herein by
reference. The proper selection of protecting groups for a particular
synthesis will be governed by the overall methods employed in the
synthesis. For example, in "light-directed" synthesis, discussed below,
the protecting groups will be photolabile protecting groups such as NVOC,
MeNPOC, and those disclosed in co-pending Application PCT/US93/10162
(filed Oct. 22, 1993), incorporated herein by reference. In other methods,
protecting groups may be removed by chemical methods and include groups
such as FMOC, DMT and others known to those of skill in the art.
Complementary or substantially complementary: Refers to the hybridization
or base pairing between nucleotides or nucleic acids, such as, for
instance, between the two strands of a double stranded DNA molecule or
between an oligonucleotide primer and a primer binding site on a single
stranded nucleic acid to be sequenced or amplified. Complementary
nucleotides are, generally, A and T (or A and U), or C and G. Two single
stranded RNA or DNA molecules are said to be substantially complementary
when the nucleotides of one strand, optimally aligned and compared and
with appropriate nucleotide insertions or deletions, pair with at least
about 80% of the nucleotides of the other strand, usually at least about
90% to 95%, and more preferably from about 98 to 100%.
Alternatively, substantial complementary exists when an RNA or DNA strand
will hybridize under selective hybridization conditions to its complement.
Typically, selective hybridization will occur when there is at least about
65% complementary over a stretch of at least 14 to 25 nucleotides,
preferably at least about 75%, more preferably at least about 90%
complementary. S. ee, M. Kanehisa Nucleic Acids Res. 12:203 (1984),
incorporated herein by reference.
Stringent hybridization conditions will typically include salt
concentrations of less than about 1M, more usually less than about 500 mM
and preferably less than about 200 mM. Hybridization temperatures can be
as low as 5.degree. C., but are typically greater than 22.degree. C., more
typically greater than about 30.degree. C., and preferably in excess of
about 37.degree. C. Longer fragments may require higher hybridization
temperatures for specific hybridization. As other factors may affect the
stringency. of hybridization, including base composition and length of the
complementary strands, presence of organic solvents and extent of base
mismatching, the combination of parameters is more important than the
absolute measure of any one alone.
Epitope: The portion of an antigen molecule which is delineated by the area
of interaction with the subclass of receptors known as antibodies.
Identifier tag: A means whereby one can identify which molecules have
experienced a particular reaction in the synthesis of an oligomer. The
identifier tag also records the step in the synthesis series in which the
molecules experienced that particular monomer reaction. The identifier tag
may be any recognizable feature which is, for example: microscopically
distinguishable in shape, size, color, optical density, etc.; differently
absorbing or emitting of light; chemically reactive; magnetically or
electronically encoded; or in some other way distinctively marked with the
required information. A preferred example of such an identifier tag is an
oligonucleotide sequence.
Ligand/Probe: A ligand is a molecule that is recognized by a particular
receptor. The agent bound by or reacting with a receptor is called a
"ligand," a term which is definitionally meaningful only in terms of its
counterpart receptor. The term "ligand" does not imply any particular
molecular size or other structural or compositional feature other than
that the substance in question is capable of binding or otherwise
interacting with the receptor. Also, a ligand may serve either as the
natural ligand to which the receptor binds, or as a functional analogue
that may act as an agonist or antagonist. Examples of ligands that can be
investigated by this invention include, but are not restricted to,
agonists and antagonists for cell membrane receptors, toxins and venoms,
viral epitopes, hormones (e.g., opiates, steroids, etc.), hormone
receptors, peptides, enzymes, enzyme substrates, substrate analogs,
transition state analogs, cofactors, drugs, proteins, and antibodies. The
term "probe" refers to those molecules which are expected to act like
ligands but for which binding information is typically unknown. For
example, if a receptor is known to bind a ligand which is a peptide
.beta.-turn, a "probe" or library of probes will be those molecules
designed to mimic the peptide .beta.-turn. In instances where the
particular ligand associated with a given receptor is unknown, the term
probe refers to those molecules designed as potential ligands for the
receptor.
Monomer: Any member of the set of molecules which can be joined together to
form an oligomer or polymer. The set of monomers useful in the present
invention includes, but is not restricted to, for the example of
oligonucleotide synthesis, the set of nucleotides consisting of adenine,
thymine, cytosine, guanine, and uridine (A, T, C, G, and U, respectively)
and synthetic analogs thereof. As used herein, monomers refers to any
member of a basis set for synthesis of an oligomer. Different basis sets
of monomers may be used at successive steps in the synthesis of a polymer.
Oligomer or Polymer: The oligomer or polymer sequences of the present
invention are formed from the chemical or enzymatic addition of monomer
subunits. Such oligomers include, for example, both linear, cyclic, and
branched polymers of nucleic acids, polysaccharides, phospholipids, and
peptides having either .alpha.-, .beta.-, or .omega.-amino acids,
heteropolymers in which a known drug is covalently bound to any of the
above, polyurethanes, polyesters, polycarbonates, polyureas, polyamides,
polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides,
polyacetates, or other polymers which will be readily apparent to one
skilled in the art upon review of this disclosure. As used herein, the
term oligomer or polymer is meant to include such molecules as .beta.-turn
mimetics, prostaglandins and benzodiazepines which can also be synthesized
in a stepwise fashion on a solid support.
Peptide: A peptide is an oligomer in which the monomers are amino acids and
which are joined together through amide bonds and alternatively referred
to as a polypeptide. In the context of this specification it should be
appreciated that when .alpha.-amino acids are used, they may be the
L-optical isomer or the D-optical isomer. Other amino acids which are
useful in the present invention include unnatural amino acids such a
.beta.-alanine, phenylglycine, homoarginine and the like. Peptides are
more than two amino acid monomers long, and often more than 20 amino acid
monomers long. Standard abbreviations for amino acids are used (e.g., P
for proline). These abbreviations are included in Stryer, Biochemistry,
Third Ed., (1988), which is incorporated herein by reference for all
purposes.
Oligonucleotides: An oligonucleotide is a single-stranded DNA or RNA
molecule, typically prepared by synthetic means. Alternatively, naturally
occurring oligonucleotides, or fragments thereof, may be isolated from
their natural sources or purchased from commercial sources. Those
oligonucleotides employed in the present invention will be 4 to 100
nucleotides in length, preferably from 6 to 30 nucleotides, although
oligonucleotides of different length may be appropriate. Suitable
oligonucleotides may be prepared by the phosphoramidite method described
by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by
the triester method according to Matteucci, et al., J. Am. Chem. Soc.,
103:3185 (1981), both incorporated herein by reference, or by other
chemical methods using either a commercial automated oligonucleotide
synthesizer or VLSIPS.TM. technology (discussed in detail below). When
oligonucleotides are referred to as "double-stranded," it is understood by
those of skill in the art that a pair of oligonucleotides exist in a
hydrogen-bonded, helical array typically associated with, for example,
DNA. In addition to the 100% complementary form of double-stranded
oligonucleotides, the term "double-stranded" as used herein is also meant
to refer to those forms which include such structural features as bulges
and loops, described more fully in such biochemistry texts as Stryer,
Biochemistry, Third Ed., (1988), previously incorporated herein by
reference for all purposes.
Receptor: A molecule that has an affinity for a given ligand or probe.
Receptors may be naturally-occurring or manmade molecules. Also, they can
be employed in their unaltered natural or isolated state or as aggregates
with other species. Receptors may be attached, covalently or
noncovalently, to a binding member, either directly or via a specific
binding substance. Examples of receptors which can be employed by this
invention include, but are not restricted to, antibodies, cell membrane
receptors, monoclonal antibodies and antisera reactive with specific
antigenic determinants (such as on viruses, cells or other materials),
drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins,
sugars, polysaccharides, cells, cellular membranes, and organelles.
Receptors are sometimes referred to in the art as anti-ligands. As the
term receptors is used herein, no difference in meaning is intended. A
"ligand-receptor pair" is formed when two molecules have combined through
molecular recognition to form a complex. Other examples of receptors which
can be investigated by this invention include but are not restricted to:
a) Microorganism receptors: Determination of ligands or probes that bind to
receptors, such as specific transport proteins or enzymes essential to
survival of microorganisms, is useful in a new class of antibiotics. 0f
particular value would be antibiotics against oppormnistic fungi,
protozoa, and those bacteria resistant to the antibiotics in current use.
b) Enzymes: For instance, the binding site of enzymes such as the enzymes
responsible for cleaving neurotransmitters. Determination of ligands or
probes that bind to certain receptors, and thus modulate the action of the
enzymes that cleave the different neurotransmitters, is useful in the
development of drugs that can be used in the treatment of disorders of
neurotransmission.
c) Antibodies: For instance, the invention may be useful in investigating
the ligand-binding site on the antibody molecule which combines with the
epitope of an antigen of interest. Determining a sequence that mimics an
antigenic epitope may lead to the development of vaccines of which the
immunogen is based on one or more of such sequences, or lead to the
development of related diagnostic agents or compounds useful in
therapeutic treatments such as for autoimmune diseases (e.g., by blocking
the binding of the "self" antibodies).
d) Nucleic Acids: The invention may be useful in investigating sequences of
nucleic acids acting as binding sites for cellular proteins ("trans-acting
factors"). Such sequences may include, e.g., transcription factors,
suppressors, enhancers or promoter sequences.
e) Catalytic Polypeptides: Polymers, preferably polypeptides, which are
capable of promoting a chemical reaction involving the conversion of one
or more reactants to one or more products. Such polypeptides generally
include a binding site specific for at least one reactant or reaction
intermediate and an active functionality proximate to the binding site,
which functionality is capable of chemically modifying the bound reactant.
Catalytic polypeptides are described in, Lerner, R.A. et al., Science 252:
659 (1991), which is incorporated herein by reference.
f) Hormone receptors: For instance, the receptors for insulin and growth
hormone. Determination of the ligands which bind with high affinity to a
receptor is useful in the development of, for example, an oral replacement
of the daily injections which diabetics must take to relieve the symptoms
of diabetes, and in the other case, a replacement for the scarce human
growth hormone that can only be obtained from cadavers or by recombinant
DNA technology. Other examples are the vasoconstrictive hormone receptors;
determination of those ligands that bind to a receptor may lead to the
development of drugs to control blood pressure.
g) Opiate receptors: Determination of ligands that bind to the opiate
receptors in the brain is useful in the development of less-addictive
replacements for morphine and related drugs.
Substrate or Solid Support: A material having a rigid or semi-rigid
surface. Such materials will preferably take the form of plates or slides,
small beads, pellets, disks or other convenient forms, although other
forms may be used. In some embodiments, at least one surface of the
substrate will be substantially flat. In other embodiments, a roughly
spherical shape is preferred.
Synthetic: Produced by in vitro chemical or enzymatic synthesis. The
synthetic libraries of the present invention may be contrasted with those
in viral or plasmid vectors, for instance, which may be propagated in
bacterial, yeast, or other living hosts.
DESCRIPTION OF THE INVENTION
The broad concept of the present invention is illustrated in FIGS. 1A to
1F. FIGS. 1A, 1B and 1C illustrate the preparation of surface-bound
unimolecular double stranded DNA, while FIGS. 1D, 1E, and 1F illustrate
uses for the libraries of the present invention.
FIG. 1A shows a solid support 1 having an attached spacer 2, which is
optional. Attached to the distal end of the spacer is a first oligomer 3,
which can be attached as a single unit or synthesized on the support or
spacer in a monomer by monomer approach. FIG. 1B shows a subsequent stage
in the preparation of one member of a library according to the present
invention. In this stage, a flexible linker 4 is attached to the distal
end of the oligomer 3. In other embodiments, the flexible linker will be a
probe. FIG. 1C shows the completed surface-bound unimolecular double
stranded DNA which is one member of a library, wherein a second oligomer 5
is now attached to the distal end of the flexible linker (or probe). As
shown in FIG. 1C, the length of the flexible linker (or probe) 4 is
sufficient such that the first and second oligomers (which are
complementary) exist in a double-stranded conformation. It will be
appreciated by one of skill in the art, that the libraries of the present
invention will contain multiple, individually synthesized members which
can be screened for various types of activity. Three such binding events
are illustrated in FIGS. 1 D, 1E and 1F.
In FIG. 1D, a receptor 6, which can be a protein, RNA molecule or other
molecule which is known to bind to DNA, is introduced to the library.
Determining which member of a library binds to the receptor provides
information which is useful for diagnosing diseases, sequencing DNA or
RNA, identifying genetic characteristics, or in drug discovery.
In FIG. 1E, the linker 4 is a probe for which binding information is
sought. The probe is held in a conformationally restricted manner by the
flanking oligomers 3 and 5, which are present in a double-stranded
conformation. As a result, a library of conformationally restricted probes
can be screened for binding activity with a receptor 7 which has
specificity for the probe.
The present invention also contemplates the preparation of libraries of
unimolecular, double-stranded oligonucleotides having bulges or loops in
one of the strands as depicted in FIG. 1F. In FIG. 1F, one oligonucleotide
5 is shown as having a bulge 8. Specific RNA bulges are often recognized
by proteins (e.g., TAR RNA is recognized by the TAT protein of HIV).
Accordingly, libraries of RNA bulges or loops are useful in a number of
diagnostic applications. One of skill in the art will appreciate that the
bulge or loop can be present in either oligonucleotide portion 3 or 5.
Libraries of Unimolecular, Double-Stranded Oligonucleotides
In one aspect, the present invention provides libraries of unimolecular
double-stranded oligonucleotides, each member of the library having the
formula:
Y--L.sup.1 --X.sup.1 --L.sup.2 --X.sup.2
in which Y represents a solid support, X.sup.1 and X.sup.2 represent a pair
of complementary oligonucleotides, L.sup.1 represents a bond or a spacer,
and L.sup.2 represents a linking group having sufficient length such that
X.sup.1 and X.sup.2 form a double-stranded oligonucleotide.
The solid support may be biological, nonbiological, organic, inorganic, or
a combination of any of these, existing as particles, strands,
precipitates, gels, sheets, tubing, spheres, containers, capillaries,
pads, slices, films, plates, slides, etc. The solid support is preferably
flat but may take on alternative surface configurations. For example, the
solid support may contain raised or depressed regions on which synthesis
takes place. In some embodiments, the solid support will be chosen to
provide appropriate light-absorbing characteristics. For example, the
support may be a polymerized Langmuir Blodgett film, functionalized glass,
Si, Ge, GaAs, GaP, SiO.sub.2, SiN.sub.4, modified silicon, or any one of a
variety of gels or polymers such as (poly)tetrafluoroethylene,
(poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations
thereof. Other suitable solid support materials will be readily apparent
to those of skill in the art. Preferably, the surface of the solid support
will contain reactive groups, which could be carboxyl, amino, hydroxyl,
thiol, or the like. More preferably, the surface will be optically
transparent and will have surface Si--OH functionalities, such as are
found on silica surfaces.
Attached to the solid support is an optional spacer, L.sup.1. The spacer
molecules are preferably of sufficient length to permit the
double-stranded oligonucleotides in the completed member of the library to
interact freely with molecules exposed to the library. The spacer
molecules, when present, are typically6-50 atoms long to provide
sufficient exposure for the attached double-stranded DNA molecule. The
spacer, L.sup.1, is comprised of a surface attaching portion and a longer
chain portion. The surface attaching portion is that part of L.sup.1 which
is directly attached to the solid support. This portion can be attached to
the solid support via carbon-carbon bonds using, for example, supports
having (poly)trifluorochloroethylene surfaces, or preferably, by siloxane
bonds (using, for example, glass or silicon oxide as the solid support).
Siloxane bonds with the surface of the support are formed in one
embodiment via reactions of surface attaching portions bearing
trichlorosilyl or trialkoxysilyl groups. The surface attaching groups will
also have a site for attachment of the longer chain portion. For example,
groups which are suitable for attachment to a longer chain portion would
include amines, hydroxyl, thiol, and carboxyl. Preferred surface attaching
portions include aminoalkylsilanes and hydroxyalkylsilanes. In
particularly preferred embodiments, the surface attaching portion of
L.sup.1 is either bis(2-hydroxyethyl)-aminopropyltriethoxysilane,
2-hydroxyethylaminopropyltriethoxysilane, aminopropyltriethoxysilane or
hydroxypropyltriethoxysilane.
The longer chain portion can be any of a variety of molecules which are
inert to the subsequent conditions for polymer synthesis. These longer
chain portions will typically be aryl acetylene, ethylene glycol oligomers
containing 2-14 monomer units, diamines, diacids, amino acids, peptides,
or combinations thereof. In some embodiments, the longer chain portion is
a polynucleotide. The longer chain portion which is to be used as part of
L.sup.1 can be selected based upon its hydrophilic/hydrophobic properties
to improve presentation of the double-stranded oligonucleotides to certain
receptors, proteins or drugs. The longer chain portion of L.sup.1 can be
constructed of polyethyleneglycols, polynucleotides, alkylene,
polyalcohol, polyester, polyamine, polyphosphodiester and combinations
thereof. Additionally, for use in synthesis of the libraries of the
invention, L.sup.1 will typically have a protecting group, attached to a
functional group (i.e., hydroxyl, amino or carboxylic acid) on the distal
or terminal end of the chain portion (opposite the solid support). After
deprotection and coupling, the distal end is covalently bound to an
oligomer.
Attached to the distal end of L.sup.1 is an oligonucleotide, X.sup.1, which
is a single-stranded DNA or RNA molecule. The oligonucleotides which are
part of the present invention are typically of from about 4 to about 100
nucleotides in length. Preferably, X.sup.1 is an oligonucleotide which is
about 6 to about 30 nucleotides in length. The oligonucleotide is
typically linked to L.sup.1 via the 3'-hydroxyl group of the
oligonucleotide and a functional group on L.sup.1 which results in the
formation of an ether, ester, carbamate or phosphate ester linkage.
Attached to the distal end of X.sup.1 is a linking group, L.sup.2, which is
flexible and of sufficient length that X.sup.1 can effectively hybridize
with X.sup.2. The length of the linker will typically be a length which is
at least the length spanned by two nucleotide monomers, and preferably at
least four nucleotide monomers, while not be so long as to interfere with
either the pairing of X.sup.1 and X.sup.2 or any subsequent assays. The
linking group itself will typically be an alkylene group (of from about 6
to about 24 carbons in length), a polyethyleneglycol group (of from about
2 to about 24 ethyleneglycol monomers in a linear configuration), a
polyalcohol group, a polyamine group (e.g., spermine, sperrnidine and
polymeric derivatives thereof), a polyester group (e.g., poly(ethyl
acrylate) having of from 3 to 15 ethyl acrylate monomers in a linear
configuration), a polyphosphodiester group, or a polynucleotide (having
from about 2 to about 12 nucleic acids). Preferably, the linking group
will be a polyethyleneglycol group which is at least a
tetraethyleneglycol, and more preferably, from about 1 to 4
hexaethyleneglycols linked in a linear array. For use in synthesis of the
compounds of the invention, the linking group will be provided with
functional groups which can be suitably protected or activated. The
linking group will be covalently attached to each of the complementary
oligonucleotides, X.sup.1 and X.sup.2, by means of an ether, ester,
carbamate, phosphate ester or amine linkage. The flexible linking group
L.sup.2 will be attached to the 5'-hydroxyl of the terminal monomer of
X.sup.1 and to the 3'-hydroxyl of the initial monomer of X.sup.2.
Preferred linkages are phosphate ester linkages which can be formed in the
same manner as the oligonucleotide linkages which are present in X.sup.1
and X.sup.2. For example, hexaethyleneglycol can be protected on one
terminus with a photolabile protecting group (i.e., NVOC or MeNPOC) and
activated on the other terminus with
2-cyanoethyl-N,N-diisopropylamino-chlorophosphite to form a
phosphoramidite. This linking group can then be used for construction of
the libraries in the same manner as the photolabile-protected,
phosphoramidite-activated nucleotides. Alternatively, ester linkages to
X.sup.1 and X.sup.2 can be formed when the L.sup.2 has terminal carboxylic
acid moieties (using the 5'-hydroxyl of X.sup.1 and the 3'-hydroxyl of
X.sup.2). Other methods of forming ether, carbamate or amine linkages are
known to those of skill in the art and particular reagents and references
can be found in such texts as March, Advanced Organic Chemistry, 4th Ed.,
Wiley-Interscience, New York, N.Y, 1992, incorporated herein by reference.
The oligonucleotide, X.sup.2, which is covalently attached to the distal
end of the linking group is, like X.sup.1, a single-stranded DNA or RNA
molecule. The oligonucleotides which are part of the present invention are
typically of from about 4 to about 100 nucleotides in length. Preferably,
X.sup.2 is an oligonucleotide which is about 6 to about 30 nucleotides in
length and exhibits complementary to X.sup.1 of from 90 to 100%. More
preferably, X.sup.1 and X.sup.2 are 100% complementary. In one group of
embodiments, either X.sup.1 or X.sup.2 will further comprise a bulge or
loop portion and exhibit complementary of from 90 to 100% over the
remainder of the oligonucleotide.
In a particularly preferred embodiment, the solid support is a silica
support, the spacer is a polyethyleneglycol conjugated to an
aminoalkylsilane, the linking group is a polyethyleneglycol group, and
X.sup.1 and X.sup.2 are complementary oligonucleotides each comprising of
from 6 to 30 nucleic acid monomers.
The library can have virtually any number of different members, and will be
limited only by the number or variety of compounds desired to be screened
in a given application and by the synthetic capabilities of the
practitioner. In one group of embodiments, the library will have from 2 up
to 100 members. In other groups of embodiments, the library will have
between 100 and 10000 members, and between 10000 and 1000000 members,
preferably on a solid support. In preferred embodiments, the library will
have a density of more than 100 members at known locations per cm.sup.2,
preferably more than 1000 per cm.sup.2, more preferably more than 10,000
per cm.sup.2.
Libraries of Conformationally Restricted Probes
In still another aspect, the present invention provides libraries of
conformationally-restricted probes. Each of the members of the library
comprises a solid support having an optional spacer which is attached to
an oligomer of the formula:
--X.sup.11 --Z--X.sup.12
in which X.sup.11 and X.sup.12 are complementary oligonucleotides and Z is
a probe. The probe will have sufficient length such that X.sup.11 and
X.sup.12 form a double-stranded DNA portion of each member. X.sup.11 and
X.sup.12 are as described above for X.sup.1 and X.sup.2 respectively,
except that for the present aspect of the invention, each member of the
probe library can have the same X.sup.11 and the same X.sup.12, and differ
only in the probe portion. In one group of embodiments, X.sup.11 and
X.sup.12 are either a poly-A oligonucleotide or a poly-T oligonucleotide.
As noted above, each member of the library will typically have a different
probe portion. The probes, Z, can be any of a variety of structures for
which receptor-probe binding information is sought for
conformationally-restricted forms. For example, the probe can be an
agonist or antagonist for a cell membrane receptor, a toxin, venom, vital
epitope, hormone, peptide, enzyme, collector, drug, protein or antibody.
In one group of embodiments, the probes are different peptides, each
having of from about 4 to about 12 amino acids. Preferably the probes will
be linked via polyphosphate diesters, although other linkages are also
suitable. For example, the last monomer employed on the X.sup.11 chain can
be a 5'-aminopropyl-functionalized phosphoramidite nucleotide (available
from Glen Research, Sterling, Va., USA or Genosys Biotechnologies, The
Woodlands, Tex., USA) which will provide a synthesis initiation site for
the carboxy to amino synthesis of the peptide probe. Once the peptide
probe is formed, a 3'-succinylated nucleoside (from Cruachem, Sterling,
Va., USA) will be added under peptide coupling conditions. In yet another
group of embodiments, the probes will be oligonucleotides of from 4 to
about 30 nucleic acid monomers which will form a DNA or RNA hairpin
structure. For use in synthesis, the probes can also have associated
functional groups (i.e., hydroxyl, amino, carboxylic acid, anhydride and
derivatives thereof) for attaching two positions on the probe to each of
the complementary oligonucleotides.
The surface of the solid support is preferably provided with a spacer
molecule, although it will be understood that the spacer molecules are not
elements of this aspect of the invention. Where present, the spacer
molecules will be as described above for L.sup.1.
The libraries of conformationally restricted probes can also have virtually
any number of members. As above, the number of members will be limited
only by design of the particular screening assay for which the library
will be used, and by the synthetic capabilities of the practitioner. In
one group of embodiments, the library will have from 2 to 100 members. In
other groups of embodiments, the library will have between 100 and 10000
members, and between 10000 and 1000000 members. Also as above, in
preferred embodiments, the library will have a density of more than 100
members at known locations per cm.sup.2, preferably more than 1000 per
cm.sup.2, more preferably more than 10,000 per cm.sup.2.
Preparation of the Libraries
The present invention further provides methods for the preparation of
diverse unimolecular, double-stranded oligonucleotides on a solid support.
In one group of embodiments, the surface of a solid support has a
plurality of preselected regions. An oligonucleotide of from 6 to 30
monomers is formed on each of the preselected regions. A linking group is
then attached to the distal end of each of the oligonucleotides. Finally,
a second oligonucleotide is formed on the distal end of each linking group
such that the second oligonucleotide is complementary to the
oligonucleotide already present in the same preselected region. The
linking group used will have sufficient length such that the complementary
oligonucleotides form a unimolecular, double-stranded oligonucleotide. In
another group of embodiments, each chemically distinct member of the
library will be synthesized on a separate solid support.
Libraries on a Single Substrate
Light-Directed Methods
For those embodiments using a single solid support, the oligonucleotides of
the present invention can be formed using a variety of techniques known to
those skilled in the art of polymer synthesis on solid supports. For
example, "light directed" methods (which are one technique in a family of
methods known as VLSIPS.TM. methods) are described in U.S. Pat. No.
5,143,854, previously incorporated by reference. The light directed
methods discussed in the '854 patent involve activating predefined regions
of a substrate or solid support and then contacting the substrate with a
preselected monomer solution. The predefined regions can be activated with
a light source, typically shown through a mask (much in the manner of
photolithography techniques used in integrated circuit fabrication). Other
regions of the substrate remain inactive because they are blocked by the
mask from illumination and remain chemically protected. Thus, a light
pattern defines which regions of the substrate react with a given monomer.
By repeatedly activating different sets of predefined regions and
contacting different monomer solutions with the substrate, a diverse array
of polymers is produced on the substrate. Of course, other steps such as
washing unreacted monomer solution from the substrate can be used as
necessary. Other techniques include mechanical techniques such as those
described in PCT No. 92/10183, U.S. Pat. No. 5,384,261 also incorporated
herein by reference for all purposes. Still further techniques include
bead based techniques such as those described in PCT US/93/04145, also
incorporated herein by reference, and pin based methods such as those
described in U.S. Pat. No. 5,288,514, also incorporated herein by
reference.
The VLSIPS.TM. methods are preferred for making the compounds and libraries
of the present invention. The surface of a solid support, optionally
modified with spacers having photolabile protecting groups such as NVOC
and MeNPOC, is illuminated through a photolithographic mask, yielding
reactive groups (typically hydroxyl groups) in the illuminated regions. A
3'-O-phosphoramidite activated deoxynucleoside (protected at the
5'-hydroxyl with a photolabile protecting group) is then presented to the
surface and chemical coupling occurs at sites that were exposed to light.
Following capping, and oxidation, the substrate is rinsed and the surface
illuminated through a second mask, to expose additional hydroxyl groups
for coupling. A second 5'-protected, 3'-O-phosphoramidite activated
deoxynucleoside is presented to the surface. The selective
photodeprotection and coupling cycles are repeated until the desired set
of oligonucleotides is produced. Alternatively, an oligomer of from, for
example, 4 to 30 nucleotides can be added to each of the preselected
regions rather than synthesize each member in a monomer by monomer
approach. At this point in the synthesis, either a flexible linking group
or a probe can be attached in a similar manner. For example, a flexible
linking group such as polyethylene glycol will typically having an
activating group (i.e., a phosphoramidite) on one end and a photolabile
protecting group attached to the other end. Suitably derivatized
polyethylene glycol linking groups can be prepared by the methods
described in Durand, et al. Nucleic Acids Res. 18:6353-6359 (1990).
Briefly, a polyethylene glycol (i. | | |
