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BACKGROUND OF THE INVENTION
The present invention relates to the field of solid phase polymer
synthesis. More specifically, the invention provides methods and
derivatized supports which find application in solid phase synthesis of
oligomer arrays or of single compounds on a preparative scale. The
oligomer arrays which are prepared using the derivatized supports of the
present invention may be used, for example, in screening studies for
determination of binding affinity and in diagnostic applications.
The synthesis of biological polymers such as peptides and oligonucleotides
has been evolving in dramatic fashion from the earliest stages of solution
synthesis to solid phase synthesis of a single polymer to the more recent
preparations of libraries having large numbers of diverse oligonucleotide
sequences on a single solid support or chip.
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, application Ser. Nos. 07/796,243
and 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,
describes methods for making arrays of oligonucleotide probes that can be
used to provide a partial or complete sequence of a target nucleic acid
and to detect the presence of a nucleic acid containing a specific
oligonucleotide sequence.
The control of surface properties to optimize VLSIPS.TM. substrate
performance in both chemical synthesis and bioassays has been recognized
to involve such parameters as site density for synthesis initiation,
surface wettability and the length of the linking group which attaches the
initiation site to the surface. Additionally, alternative surfaces can
lead to the use of VLSIPS.TM. technology for preparative scale synthesis.
SUMMARY OF THE INVENTION
The present invention provides a variety of derivatized supports and
methods for their preparation, which are useful in the preparation of
peptides, oligonucleotides or other small organic molecules.
Some of the methods involve substrate surface derivatization in a manner
which also expands the types of synthesis which can be performed and
provides lower density arrays of polymers for use in diagnostics.
A number of novel derivatized supports are provided which have altered
surfaces, for example polymer-coated or glycan-coated solid supports.
Other derivatized supports utilize linking groups terminating in acidic
functionalities such as carboxylic acids or sulfonic acids which are
useful in alternative synthesis strategies.
The present invention further provides methods of rendering the derivatized
supports reusable.
The present invention still further provides methods of oligomer synthesis.
Thus, according to a first aspect of the invention, a substrate surface,
useful for the preparation of diverse polymer sequences is derivatized to
control functional group spacing, improve wettability, and minimize
non-specific binding of macromolecules. In one embodiment, the substrate
surface is first derivatized with a trialkoxysilane bearing a reactive
site such as amino (--NH.sub.2), isothiocyanate (--NCS) or hydroxyl (--OH)
for the attachment of a suitable linking group. Mixtures of suitably
protected linking groups having synthesis initiation sites and an "inert"
diluent (or capping agent) are then reacted with the derivatized surface
to provide a substrate surface wherein the average spacing of synthesis
initiation sites is altered. This method provides effective control of
functional site density and can be adapted to control other surface
properties such as surface wettability and nonspecific binding of
macromolecules.
In another aspect, the present invention provides methods for the
preparation of stabilized polymer-coated supports for use in solid-phase
synthesis. These methods typically use dip coating, covalent polymer
attachment, in situ polymerization, or combinations thereof to provide the
polymer-coated support.
In yet another aspect, the present invention provides glycan-coated
supports and methods for their preparation. While similar to the
polymer-coated supports, the properties of glycan-coated supports can be
quite different and provide extremely hydrophilic surfaces which are
useful in binding assays and diagnostic applications.
In still another aspect, the present invention provides methods for the
surface-regeneration of used ligand arrays.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a doped process of substrate derivatization.
FIG. 2 illustrates a standard process of substrate derivatization.
FIG. 3 provides the structures of three polymers (polyethyleneimine,
polyacrylamide and polyallylamine) which are useful for preparing
polymer-coated glass substrates.
FIG. 4 provides the structures of a group of carboxylic acid and anhydride
polymers which are useful for the preparation of polymer-coated solid
supports.
FIG. 5 provides the structures of polyethylene glycol and poly(vinyl
alcohol) which are useful for preparing polymer-coated solid supports.
FIG. 6 is an illustration of a polymer-coated glass substrate which can be
prepared by dip coating, covalent crosslinking or in situ polymerization.
FIG. 7 illustrates one example of polymer crosslinking which uses
glutaraldehyde and polyethyleneimine.
FIGS. 8-11 illustrate a variety of methods for covalently attaching a
polymer to a derivatized solid support.
FIG. 12 illustrates in situ polymerization which can be used to prepare a
polymer-coated solid support.
FIG. 13 provides a comparison of glycine-doped and serine-doped surface
derivatization.
FIG. 14 is a graph showing contact angle data for substrates having various
linking groups.
FIG. 15 is a graph which illustrates the discrimination between specific
and non-specific binding observed in a streptavidin/biotin binding assay
as a function of surface preparation.
FIG. 16 illustrates a method for the synthesis of oligonucleotides in which
the protecting groups are cleaved and replaced as part of the synthesis
cycle.
FIG. 17 illustrates a method for the synthesis of peptides in which the
protecting groups are cleaved and replaced as part of the synthesis cycle.
DETAILED DESCRIPTION OF THE INVENTION
Contents
I. Glossary
II. General
III. Surface Engineering--The Doped Process
IV. Carboxy Chips
V. Polymer-Coated Surfaces
VI. Glycan-Coated Chips
VII. Reusable Chips
VIII. Methods for Oligomer Synthesis
IX. Examples
X. Conclusion
I. Glossary
The following abbreviations are used herein: AcOH, acetic acid; ALLOC,
allyloxycarbonyl; BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium
hexafluorophosphate; CAP, .epsilon.-aminocaproic acid; DIEA,
diisopropylethylamine; DIGLY, glycylglycine; DMF, dimethylformamide; DMT,
dimethoxytrityl; DTT, dithiothreitol; EtOAc, ethyl acetate; FMOC,
fluorenylmethoxycarbonyl; MeNPOC, .alpha.-methylnitropiperonyloxycarbonyl;
MP, melting point; NVOC, nitroveratryloxycarbonyl; OBt,
hydroxybenzotriazole radical; PBS, phosphate buffered saline; TFA,
trifluoroacetic acid; 15-ATOM-PEG, H.sub.2 N--(CH.sub.2 CH.sub.2 O).sub.2
--CH.sub.2 CH.sub.2 NHCO--(CH.sub.2).sub.3 --CO.sub.2 H; TRIGLY,
glycylglycylglycine.
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
dimethoxybenzoin, 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.
The term "protected amino acid" refers to an amino acid, typically an
.alpha.-amino acid having either or both the amine functionality and the
carboxylic acid functionality suitably protected by one of the groups
described above. Additionally, for those amino acids having reactive sites
or functional groups on a side chain (i.e., serine, tyrosine, glutamic
acid), the term "protected amino acid" is meant to refer to those
compounds which optionally have the side chain functionality protected as
well.
The term "activating agent" refers to those groups which, when attached to
a particular functional group or reactive site, render that site more
reactive toward covalent bond formation with a second functional group or
reactive site. For example, the group of activating groups which are
useful for a carboxylic acid include simple ester groups and anhydrides.
The ester groups include alkyl, aryl and alkenyl esters and in particular
such groups as 4-nitrophenyl, N-hydroxylsuccinimide and pentafluorophenol.
Other activating agents are known to those of skill in the art.
Ligand: A ligand is a molecule that is recognized by a receptor. Examples
of ligands which can be synthesized using the methods and compounds of
this invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones, opiates, steroids, peptides, enzyme substrates,
cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids,
oligosaccharides, and proteins.
Monomer: A monomer is a member of the set of small molecules which are or
can be joined together to form a polymer or a compound composed of two or
more members. The present invention is described herein in terms of
compositions and methods which are useful in solid phase synthesis. In a
number of applications, solid phase methods are used for the preparation
of biological polymers such as peptides, proteins and nucleic acids. For
these biological polymers, the set of monomers includes but is not
restricted to, for example, the set of common L-amino acids, the set of
D-amino acids, the set of synthetic and/or natural amino acids, the set of
nucleotides and the set of pentoses and hexoses. The particular ordering
of monomers within a biological polymer is referred to herein as the
"sequence" of the polymer. As used herein, monomers refers to any member
of a basis set for synthesis of a polymer. For example, dimers of the 20
naturally occurring L-amino acids form a basis set of 400 monomers for
synthesis of polypeptides. Different basis sets of monomers may be used at
successive steps in the synthesis of a polymer. Furthermore, each of the
sets may include protected members which are modified after synthesis. The
invention is described herein primarily with regard to the preparation of
molecules containing sequences of monomers such as amino acids, but could
readily be applied in the preparation of other polymers. Such polymers
include, for example, both linear and cyclic 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, polynucleotides, polyurethanes,
polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or other
polymers which will be apparent upon review of this disclosure. Such
polymers are "diverse" when polymers having different monomer sequences
are formed at different predefined regions of a substrate. Methods of
cyclization and polymer reversal of polymers are disclosed in copending
application U.S. Ser. No. 08/351,058 which is a CIP of U.S. Ser. No.
07/978,940 which is a CIP of U.S. Pat. No. 5,242,974, entitled "POLYMER
REVERSAL ON SOLID SURFACES," incorporated herein by reference for all
purposes.
In certain embodiments of the invention, polymer-coated supports are
described. The polymers used for coating a solid support include, but are
not limited to polyurethanes, polyesters, polycarbonates, polyureas,
polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes,
polyacrylamides, polyimides, polyacetates, or other polymers which will be
apparent upon review of this disclosure. The polymers used to coat a solid
support are typically repeats of a single monomers which is crosslinked
with a second molecule to provide structural integrity to the polymer.
Peptide: A peptide is a polymer in which the monomers are amino acids and
are joined together through amide bonds, alternatively referred to as a
polypeptide. When the amino acids are .alpha.-amino acids, either the
L-optical isomer or the D-optical isomer may be used. Additionally,
unnatural amino acids, for example, .beta.-alanine, phenylglycine and
homoarginine are also meant to be included. Peptides are two or more amino
acid monomers long and are 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.
Receptor: A receptor is a molecule that has an affinity for a ligand.
Receptors may be naturally-occurring or manmade molecules. They can be
employed in their unaltered 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, viruses,
cells, drugs, polynucleotides, nucleic acids, peptides, cofactors,
lectins, sugars, polysaccharides, 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.
Substrate: As used herein, the term "substrate" or "support" refers to a
material having a rigid or semi-rigid surface. In many embodiments, at
least one surface of the substrate will be substantially flat, although in
some embodiments it may be desirable to physically separate synthesis
regions for different polymers with, for example, wells, raised regions,
etched trenches, or the like. In some embodiments, the substrate itself
contains wells, trenches, flow through regions, etc. which form all or
part of the synthesis regions. According to other embodiments, small beads
may be provided on the surface, and compounds synthesized thereon may be
released upon completion of the synthesis.
Channel Block: A material having a plurality of grooves or recessed regions
on a surface thereof. The grooves or recessed regions may take on a
variety of geometric configurations, including but not limited to stripes,
circles, serpentine paths, or the like. Channel blocks may be prepared in
a variety of manners, including etching silicon blocks, molding or
pressing polymers, etc.
Predefined Region: A predefined region is a localized area on a substrate
which is, was, or is intended to be used for formation of a selected
polymer and is otherwise referred to herein in the alternative as
"reaction" region, a "selected" region, or simply a "region." The
predefined region may have any convenient shape, e.g., circular,
rectangular, elliptical, wedge-shaped, etc. In some embodiments, a
predefined region and, therefore, the area upon which each distinct
polymer sequence is synthesized is smaller than about 1 cm.sup.2, more
preferably less than 1 mm.sup.2, and still more preferably less than 0.5
mm.sup.2. In most preferred embodiments the regions have an area less than
about 10,000 .mu.m.sup.2 or, more preferably, less than 100 .mu.m.sup.2.
Within these regions, the polymer synthesized therein is preferably
synthesized in a substantially pure form. Additionally, multiple copies of
the polymer will typically be synthesized within any preselected region.
The number of copies can be in the thousands to the millions.
II. General
The compounds, compositions and methods of the present invention can be
used in a number of solid phase synthesis applications, including
light-directed methods, flow channel and spotting methods, pin-based
methods and bead-based methods.
Light-Directed Methods
"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.
Flow Channel or Spotting Methods
Additional methods applicable to library synthesis on a single substrate
are described in co-pending applications Ser. Nos. 07/980,523, filed Nov.
20, 1992, and 07/796,243, filed Nov. 22, 1991, incorporated herein by
reference for all purposes. In the methods disclosed in these
applications, reagents are delivered to the substrate by either (1)
flowing within a channel defined on predefined regions or (2) "spotting"
on predefined regions. However, other approaches, as well as combinations
of spotting and flowing, may be employed. In each instance, certain
activated regions of the substrate are mechanically separated from other
regions when the monomer solutions are delivered to the various reaction
sites.
A typical "flow channel" method applied to the compounds and libraries of
the present invention can generally be described as follows. Diverse
polymer sequences are synthesized at selected regions of a substrate or
solid support by forming flow channels on a surface of the substrate
through which appropriate reagents flow or in which appropriate reagents
are placed. For example, assume a monomer "A" is to be bound to the
substrate in a first group of selected regions. If necessary, all or part
of the surface of the substrate in all or a part of the selected regions
is activated for binding by, for example, flowing appropriate reagents
through all or some of the channels, or by washing the entire substrate
with appropriate reagents. After placement of a channel block on the
surface of the substrate, a reagent having the monomer A flows through or
is placed in all or some of the channel(s). The channels provide fluid
contact to the first selected regions, thereby binding the monomer A on
the substrate directly or indirectly (via a spacer) in the first selected
regions.
Thereafter, a monomer B is coupled to second selected regions, some of
which may be included among the first selected regions. The second
selected regions will be in fluid contact with a second flow channel(s)
through translation, rotation, or replacement of the channel block on the
surface of the substrate; through opening or closing a selected valve; or
through deposition of a layer of chemical or photoresist. If necessary, a
step is performed for activating at least the second regions. Thereafter,
the monomer B is flowed through or placed in the second flow channel(s),
binding monomer B at the second selected locations. In this particular
example, the resulting sequences bound to the substrate at this stage of
processing will be, for example, A, B, and AB. The process is repeated to
form a vast array of sequences of desired length at known locations on the
substrate.
After the substrate is activated, monomer A can be flowed through some of
the channels, monomer B can be flowed through other channels, a monomer C
can be flowed through still other channels, etc. In this manner, many or
all of the reaction regions are reacted with a monomer before the channel
block must be moved or the substrate must be washed and/or reactivated. By
making use of many or all of the available reaction regions
simultaneously, the number of washing and activation steps can be
minimized.
One of skill in the art will recognize that there are alternative methods
of forming channels or otherwise protecting a portion of the surface of
the substrate. For example, according to some embodiments, a protective
coating such as a hydrophilic or hydrophobic coating (depending upon the
nature of the solvent) is utilized over portions of the substrate to be
protected, sometimes in combination with materials that facilitate wetting
by the reactant solution in other regions. In this manner, the flowing
solutions are further prevented from passing outside of their designated
flow paths.
The "spotting" methods of preparing compounds and libraries of the present
invention can be implemented in much the same manner as the flow channel
methods. For example, a monomer A can be delivered to and coupled with a
first group of reaction regions which have been appropriately activated.
Thereafter, a monomer B can be delivered to and reacted with a second
group of activated reaction regions. Unlike the flow channel embodiments
described above, reactants are delivered by directly depositing (rather
than flowing) relatively small quantities of them in selected regions. In
some steps, of course, the entire substrate surface can be sprayed or
otherwise coated with a solution. In preferred embodiments, a dispenser
moves from region to region, depositing only as much monomer as necessary
at each stop. Typical dispensers include a micropipette to deliver the
monomer solution to the substrate and a robotic system to control the
position of the micropipette with respect to the substrate, or an ink-jet
printer. In other embodiments, the dispenser includes a series of tubes, a
manifold, an array of pipettes, or the like so that various reagents can
be delivered to the reaction regions simultaneously.
Pin-Based Methods
Another method which is useful for the preparation of compounds and
libraries of the present invention involves "pin based synthesis." This
method is described in detail in U.S. Pat. No. 5,288,514, previously
incorporated herein by reference. The method utilizes a substrate having a
plurality of pins or other extensions. The pins are each inserted
simultaneously into individual reagent containers in a tray. In a common
embodiment, an array of 96 pins/containers is utilized.
Each tray is filled with a particular reagent for coupling in a particular
chemical reaction on an individual pin. Accordingly, the trays will often
contain different reagents. Since the chemistry disclosed herein has been
established such that a relatively similar set of reaction conditions may
be utilized to perform each of the reactions, it becomes possible to
conduct multiple chemical coupling steps simultaneously. In the first step
of the process the invention provides for the use of substrate(s) on which
the chemical coupling steps are conducted. The substrate is optionally
provided with a spacer having active sites. In the particular case of
oligonucleotides, for example, the spacer may be selected from a wide
variety of molecules which can be used in organic environments associated
with synthesis as well as aqueous environments associated with binding
studies. Examples of suitable spacers are polyethyleneglycols,
dicarboxylic acids, polyamines and alkylenes, substituted with, for
example, methoxy and ethoxy groups. Additionally, the spacers will have an
active site on the distal end. The active sites are optionally protected
initially by protecting groups. Among a wide variety of protecting groups
which are useful are FMOC, BOC, t-butyl esters, t-butyl ethers, and the
like. Various exemplary protecting groups are described in, for example,
Atherton et al., Solid Phase Peptide Synthesis, IRL Press (1989),
incorporated herein by reference. In some embodiments, the spacer may
provide for a cleavable function by way of, for example, exposure to acid
or base.
Bead Based Methods
Yet another method which is useful for synthesis of polymers and small
ligand molecules on a solid support "bead based synthesis." A general
approach for bead based synthesis is described copending application
Serial Nos. 07/762,522 (filed Sep. 18, 1991); 07/946,239 (filed Sep. 16,
1992); 08/146,886 (filed Nov. 2, 1993); 07/876,792 (filed Apr. 29, 1992);
PCT/US94/12347 (filed Nov. 2, 1994) and PCT/US93/04145 (filed Apr. 28,
1993), the disclosures of which are incorporated herein by reference.
For the synthesis of molecules such as oligonucleotides on beads, a large
plurality of beads are suspended in a suitable carrier (such as water) in
a container. The beads are provided with optional spacer molecules having
an active site. The active site is protected by an optional protecting
group.
In a first step of the synthesis, the beads are divided for coupling into a
plurality of containers. For the purposes of this brief description, the
number of containers will be limited to three, and the monomers denoted as
A, B, C, D, E, and F. The protecting groups are then removed and a first
portion of the molecule to be synthesized is added to each of the three
containers (i.e., A is added to container 1, B is added to container 2 and
C is added to container 3).
Thereafter, the various beads are appropriately washed of excess reagents,
and remixed in one container. Again, it will be recognized that by virtue
of the large number of beads utilized at the outset, there will similarly
be a large number of beads randomly dispersed in the container, each
having a particular first portion of the monomer to be synthesized on a
surface thereof.
Thereafter, the various beads are again divided for coupling in another
group of three containers. The beads in the first container are
deprotected and exposed to a second monomer (D) , while the beads in the
second and third containers are coupled to molecule portions E and F
respectively. Accordingly, molecules AD, BD, and CD will be present in the
first container, while AE, BE, and CE will be present in the second
container, and molecules AF, BF, and CF will be present in the third
container. Each bead, however, will have only a single type of molecule on
its surface. Thus, all of the possible molecules formed from the first
portions A, B, C, and the second portions D, E, and F have been formed.
The beads are then recombined into one container and additional steps such
as are conducted to complete the synthesis of the polymer molecules. In a
preferred embodiment, the beads are tagged with an identifying tag which
is unique to the particular double-stranded oligonucleotide or probe which
is present on each bead. A complete description of identifier tags for use
in synthetic libraries is provided in copending application Ser. No.
08/146,886 (filed Nov. 2, 1993) previously incorporated by reference for
all purposes.
The advent of methods for the synthesis of diverse chemical compounds on
solid supports has resulted in the genesis of a multitude of diagnostic
applications for such chemical libraries. A number of these diagnostic
applications involve contacting a sample with a solid support, or chip,
having multiple attached biological polymers such as peptides and
oligonucleotides, or other small ligand molecules synthesized from
building blocks in a stepwise fashion, in order to identify any species
which specifically binds to one or more of the attached polymers or small
ligand molecules.
For example, patent application Ser. No. 08/082,937, filed Jun. 25, 1993,
describes methods for making arrays of oligonucleotide probes that can be
used to provide the complete sequence of a target nucleic acid and to
detect the presence of a nucleic acid containing a specific
oligonucleotide sequence. Patent application Ser. No. 08/327,687, filed
Oct. 24, 1994, describes methods of making arrays of unimolecular,
double-stranded oligonucleotides which can be used in diagnostic
applications involving protein/DNA binding interactions such as those
associated with the p53 protein and the genes contributing to a number of
cancer conditions. Arrays of double-stranded oligonucleotides can also be
used to screen for new drugs having particular binding affinities.
A number of factors contribute to the successful synthesis and use of
oligomer arrays on solid supports. For example, issues of relevance to the
use of derivatized glass substrates for carrying out VLSIPS.TM. synthesis
of peptide arrays are the spacing of the synthesis initiation sites, the
wettability of the surface by organic solvents and aqueous solutions, and
the extent to which non-specific binding of receptors, antibodies or other
biological macromolecules occurs.
The spacing of the synthesis initiation sites (typically, primary amines)
is of concern since very high site densities will affect binding events
between tethered ligands and receptors. Additionally, increased yields in
synthesis can be achieved by control of phenomena such as free radical
formation during photolytic reaction, solvent accessibility and surface
electrostatic effects.
It will be apparent to those of skill in the art that the methods and
compositions of the present invention will find application in any of the
above-noted processes for solid phase synthesis of biological polymers and
other small molecule ligands. Additionally, the method of regenerating a
used ligand array surface will find application with ligand arrays
prepared by light-directed methods, bead- or pin-based methods, or flow
channel or spotting methods.
III. Surface Engineering--The Doped Process
The derivatization of supports for the preparation of ligand arrays, as
well as other forms of solid phase synthesis, must take into account
several issues relating to both the synthesis which occurs on the support
and the subsequent use of the arrays in binding studies and diagnostic
assays. Foremost among the many issues are the spacing of initiation
sites, the wettability of the surface by both organic solvents and aqueous
solutions, and the extent to which non-specific binding of receptors
occurs.
The spacing of synthesis initiation sites on a solid support can affect not
only the synthesis of the ligand array but also the binding events between
a receptor and a tethered ligand. The synthesis can be influenced through
phenomena such as free radical formation during photolytic reaction (in
light-directed synthesis), solvent accessibility and surface electrostatic
effects.
The wettability of the support, or substrate surface, is also likely to
have a direct influence on the yield of coupling reactions and subsequent
binding events. The presentation of peptides or other ligands for
recognition is expected to be a function of not only the
hydrophobicity/hydrophilicity of the peptide or ligand, but also the
physicochemical nature of the surface to which it is attached. Thus,
hydrophilic peptide sequences are expected to extend fully into the
surrounding aqueous environment, thereby maximizing their availability for
recognition and binding by receptors. In contrast, hydrophobic sequences
in the presence of a moderately hydrophobic substrate surface can collapse
onto the surface and effectively be eliminated from the pool of available
ligands presented to a receptor.
In view of the above considerations, the present invention provides a
method for affixing functional sites to the surface of a solid substrate
at a preselected density. In this method, a solid substrate is reacted
with a derivatization reagent having a substrate attaching group on one
end and a reactive site on a distal end (away from the surface) to provide
a substrate having an even distribution of reactive sites. The derivatized
substrate is then co | | |
