U.S. patent application number 11/289897 was filed with the patent office on 2006-06-22 for macromolecular arrays on polymeric brushes & methods for preparing the same.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Ying Chih Chang, Curtis W. Frank, Glenn McGall.
Application Number | 20060134672 11/289897 |
Document ID | / |
Family ID | 26849044 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134672 |
Kind Code |
A1 |
Chang; Ying Chih ; et
al. |
June 22, 2006 |
Macromolecular arrays on polymeric brushes & methods for
preparing the same
Abstract
Polymeric brush substrates and methods for their preparation are
provided. Methods are also provided for preparing macromolecular
arrays on such polymeric brush substrates. Using polymeric brush
substrates allows control over functional site density as well as
wettability and porosity of the substrate. These polymeric brushes
are useful in solid-phase synthesis of arrays of peptides,
polynucleotides or small organic molecules.
Inventors: |
Chang; Ying Chih; (Atherton,
CA) ; Frank; Curtis W.; (Cupertino, CA) ;
McGall; Glenn; (Mountain View, CA) |
Correspondence
Address: |
BANNER & WITCOFF LTD.,;COUNSEL FOR AFFYMETRIX
1001 G STREET , N.W.
ELEVENTH FLOOR
WASHINGTON
DC
20001-4597
US
|
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
26849044 |
Appl. No.: |
11/289897 |
Filed: |
November 30, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09652962 |
Aug 31, 2000 |
6994964 |
|
|
11289897 |
Nov 30, 2005 |
|
|
|
60151862 |
Sep 1, 1999 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/287.2; 525/54.2 |
Current CPC
Class: |
C08F 289/00 20130101;
C40B 40/00 20130101; C08F 4/02 20130101; C08F 290/06 20130101; G01N
33/544 20130101; C07H 21/00 20130101; C08L 51/10 20130101; C07B
2200/11 20130101; C08L 51/08 20130101 |
Class at
Publication: |
435/006 ;
525/054.2; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C08G 63/91 20060101 C08G063/91; C12M 1/34 20060101
C12M001/34 |
Claims
1-8. (canceled)
9. A method for affixing functional sites to a surface of a solid
substrate, the method comprising: (a) providing a substrate to
which one or more free radical initiators are covalently attached,
wherein each free radical initiator has a radical generation site
distal to the substrate; and (b) contacting the substrate with a
mixture of linking monomers and diluent monomers under conditions
that promote free radical polymerization from the radical
generation sites of the initiators to produce a brush polymer
comprising functional sites, wherein the density of the functional
sites is determined by the ratio of functional monomers to diluent
monomers.
10. The method of claim 9, wherein the linking monomers comprise a
vinyl group.
11. The method of claim 9, wherein the linking monomers comprise at
least two different linking monomers.
12. The method of claim 9, wherein the initiator is an azo type
initiator.
13. The method of claim 9, wherein the functional sites are
selected from the group consisting of amino, hydroxyl, carboxyl or
sulfydryl.
14. The method of claim 9, wherein the ratio of linking monomers to
diluent monomers is from about 1:2 to about 1:200.
15. The method of claim 9, wherein the ratio of linking monomers to
diluent monomers is from about 1:2 to about 1:2000.
16. The method of claim 9, wherein the monomers independently have
the structure: ##STR5## wherein R1 is hydrogen or lower alkyl; and
R2 and R3 are independently hydrogen, alkyl, alkoxy, hydroxyalkyl,
polyalkylene oxide, or --Y-Z, wherein Y is linear or branched lower
alkyl, aryl, alkylaryl, or polyalkylene oxide, and Z is hydrogen,
hydroxyl, alkoxy, carboxy, amino, hydrazino, sulfydryl, or R, where
R is hydrogen, hydroxy, lower alkoxy or aryloxy.
17. The method of claim 9, wherein the substrate comprises glass or
silica.
18. A substrate capable of supporting macromolecular array
synthesis, the substrate comprising polymer brushes formed by free
radical polymerization, wherein said polymer brushes comprise
hydroxyl, amino, or carboxyl, groups or a combination thereof.
19. The substrate of claim 18, wherein the density of the polymer
brushes is 0.1 to 1000 pmoles of individual polymer chains per cm2
of substrate surface area.
20. The substrate of claim 18, further comprising an array of
macromolecules attached to polymeric brushes on the substrate.
21. The substrate of claim 20, wherein the macromolecules comprise
polynucleotides.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S. patent
application Ser. No. 09/652,962, filed Aug. 31, 2000; which in turn
claims the priority benefit of U.S. Provisional Patent Application
Ser. No. 60/151,862, filed Sep. 1, 1999, the contents of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to macromolecular arrays
prepared on polymeric brush substrates and methods for preparing
such arrays. The invention transcends several scientific
disciplines such as polymer chemistry, biochemistry, molecular
biology, medicine and medical diagnostics.
BACKGROUND ART
[0003] Synthesis of high density macromolecular arrays is known.
Such high density macromolecular arrays include nucleic acid
arrays, peptide arrays, and carbohydrate arrays. See, for example,
the U.S. Pat. Nos. 5,143,854, 5,384,261, 5,405,783, and
5,424,186.
[0004] One method of preparing macromolecular arrays involves
photolithographic techniques using photocleavable protecting
groups. Briefly, the method includes attaching photoremovable
groups to the surface of a substrate, exposing selected regions of
the substrate to light to activate those regions, attaching a
monomer with a photoremovable group to the activated regions, and
repeating the steps of activation and attachment until
macromolecules of the desired length and sequence are synthesized.
See U.S. Pat. Nos. 5,324,663, 5,384, 261, 5,405,783, and
5,412,087.
[0005] Additional methods and techniques applicable to array
synthesis have been described in the U.S. Pat. Nos. 5,424,186,
5,445,934, 5,451,683, 5,482,867, 5,489,678, 5,491,074, 5,510,270,
5,527,681, 5,550,215, 5,571,639, 5,593,839, 5,599,695, 5,624,711,
5,631,734, 5,677,195, 5,744,101, 5,744,305, 5,753,788, 5,770,456,
5,831,070, and 5,856,011.
[0006] Traditional substrates used in array synthesis consist of
flat two-dimensional surfaces or three-dimensional surfaces such as
a porous matrix or a cross-linked polymer gel. While these
substrates have been satisfactory in general, as the density of the
array increased, signal to noise ratio under assay conditions
decreased due to crowding, resulting often in decreased
performance. This crowding and performance issues become more
important as more applications for high density macromolecular
arrays are being developed. Thus, there is a need for high density
macromolecular arrays with good or improved performance under assay
conditions. The present invention meets this need.
SUMMARY OF THE INVENTION
[0007] In one embodiment, a method of preparing a polymeric brush
substrate for use in solid-phase synthesis of macromolecules is
provided, which method comprises:
[0008] (a) providing a substrate to which one or more free radical
initiators are covalently attached, wherein each free radical
initiator has a radical generation site distal to the substrate;
and
[0009] (b) contacting the covalently attached substrate with
monomers under conditions that promote free radical polymerization
from the radical generation sites of the initiators to form a
polymeric brush.
[0010] The polymerization in the above method may be accomplished
by using free radical polymerization. The substrate in the above
method comprises, in some aspects, glass or silica. The monomers in
one embodiment comprise a vinyl group. In one embodiment, the
monomers may be include for example at least two different
monomers. An exemplary monomer is vinyl acetate.
[0011] The monomers in one embodiment may have the following
structure: ##STR1##
[0012] wherein R.sub.1 is hydrogen or lower alkyl; and
[0013] R.sub.2 and R.sub.3 are independently hydrogen, or --Y-Z,
wherein Y is lower alkyl, and Z is hydroxyl, amino, or C(O)--R,
where R is hydrogen, lower alkoxy or aryloxy.
[0014] In another embodiment, R.sub.2 and R.sub.3 are independently
hydrogen, alkyl, alkoxy, hydroxyalkyl, polyalkylene oxide, or
--Y-Z, wherein Y is linear or branched lower alkyl, aryl,
alkylaryl, or polyalkylene oxide, and Z is hydrogen, hydroxyl,
alkoxy, carboxy, amino, hydrazino, sulfydryl, or C(O)--R, where R
is hydrogen, hydroxy, lower alkoxy or aryloxy.
[0015] The polymer formed on the support may have, for example,
hydroxyl, amino, or carboxyl groups or any combination thereof.
[0016] A polymeric brush substrate capable of supporting
macromolecular array synthesis comprising covalently linked
monomers having for example hydroxyl, amino, sulfydryl or carboxyl
groups or any combination thereof may be formed.
[0017] Methods are also provided for using the polymeric brush
substrates of the above method. Methods are also provided for
performing assays of macromolecular arrays prepared on the
polymeric brush substrates. Such assays include hybridization
assays of polynucleotides and ligand-binding assays of
peptides.
[0018] The invention also provides polymeric brush substrates and
polymeric brush substrates comprising macromolecular arrays
prepared as disclosed herein.
[0019] A method for affixing functional sites to a surface of a
solid substrate is provided, comprising:
[0020] (a) providing a substrate to which one or more free radical
initiators are covalently attached, wherein each free radical
initiator has a radical generation site distal to the substrate;
and
[0021] (b) contacting the substrate with a mixture of linking
monomers and diluent monomers under conditions that promote free
radical polymerization from the radical generation sites of the
initiators, wherein the density of the functional sites is
determined by the ratio of functional monomers to diluent
monomers.
[0022] In one aspect, the initiator is an azo type initiator. The
functional site is for example amino, hydroxyl, or carboxyl. The
ratio of linking monomers to diluent monomers is for example from
about 1:2 to about 1:200; or in some aspects is from about 1:2 to
about 1:2000 or more.
[0023] The monomers of the method have, for example, independently
the following structure: ##STR2##
[0024] wherein R.sub.1 is hydrogen or lower alkyl; and
[0025] R.sub.2 and R.sub.3 are independently hydrogen, or --Y-Z,
wherein Y is lower alkyl, and Z is hydroxyl, amino, or C(O)--R,
where R is hydrogen, lower alkoxy or aryloxy.
[0026] In another embodiment, R.sub.2 and R.sub.3 are independently
hydrogen, alkyl, alkoxy, hydroxyalkyl, polyalkylene oxide, or
--Y-Z, wherein Y is linear or branched lower alkyl, aryl,
alkylaryl, or polyalkylene oxide, and Z is hydrogen, hydroxyl,
alkoxy, carboxy, amino, hydrazino, sulfydryl, or C(O)--R, where R
is hydrogen, hydroxy, lower alkoxy or aryloxy.
[0027] In one aspect, the monomer does not contain a free hydroxyl
group and is a diluent monomer. In some aspects, the diluent
monomer functions as a polymerization terminator.
[0028] In one aspect, a substrate is provided using the methods
disclosed herein, wherein the density of the polymer brushes is 0.1
to 1000 pmoles of individual polymer chains per cm.sup.2 of
substrate surface area.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIGS. 1A, 1B and 1C show the schematic of a polymer brush
formation through free radical methods. FIG. 1A illustrates a heat
generated surface radical (I) initiating polymerization of vinyl
monomers (M), resulting in a tethered polymer chain on surface.
FIG. 1B shows free radical polymerization on a surface, wherein
initiators are covalently attached to a surface and activated by
heat or light in the presence of monomers. FIG. 1C shows
schematically a three-dimensional distribution of hydroxyl groups
on a polymer brush substrate.
[0030] FIG. 2 show the structures and synthetic schemes for the
synthesis of silane coupling agent I.
[0031] FIG. 3 shows the structures and synthetic scheme of the
synthesis of silane coupling agent II.
[0032] FIG. 4 shows an example of a surface-bound initiator wherein
the initiator is an azo type.
[0033] FIG. 5 shows some exemplary monomers of the present
invention.
[0034] FIG. 6 shows free radical polymerization to make
hydroxylated polymer brush surfaces using monomer IV.
[0035] FIG. 7 shows a comparison of hydroxyl functional group
densities on polymeric brushes v. flat two-dimensional
substrates.
[0036] FIG. 8 shows a comparison of signal intensity from
hybridization on a polymeric brushes v. flat two-dimensional
substrates.
MODES OF CARRYING OUT THE INVENTION
[0037] A. General Techniques
[0038] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques of organic chemistry,
polymer technology, molecular biology (including recombinant
techniques), cell biology, biochemistry, and immunology, which are
within the skill of the art. Such conventional techniques include
polymer array synthesis, hybridization, ligation, detection of
hybridization using a label. Specific illustrations of suitable
techniques can be had by reference to the example hereinbelow.
However, other equivalent conventional procedures can, of course,
also be used. Such conventional techniques can be found in standard
laboratory manuals such as Genome Analysis: A Laboratory Manual
Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells:
A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular
Cloning: A Laboratory Manual (all from Cold Spring Harbor
Laboratory Press), all of which are herein incorporated in their
entirety by reference.
[0039] B. Definitions
[0040] As used herein, certain terms may have the following defined
meanings.
[0041] As used in the specification and claims, the singular forms
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "an array" may
include a plurality of arrays unless the context clearly dictates
otherwise.
[0042] An "array" is an intentionally created collection of
molecules which can be prepared either synthetically or
biosynthetically. The molecules in the array can be identical or
different from each other. The array can assume a variety of
formats, e.g., libraries of soluble molecules; libraries of
compounds tethered to resin beads, silica chips, or other solid
supports.
[0043] The term "molecule" generally refers to a macromolecule or
polymer as described herein. However, arrays comprising single
molecules, as opposed to macromolecules or polymers, are also
within the scope of this invention.
[0044] "Predefined region" refers to a localized area on a solid
support which is, was, or is intended to be used for formation of a
selected molecule and is otherwise referred to herein in the
alternative as a "selected" region. The predefined region may have
any convenient shape, e.g., circular, rectangular, elliptical,
wedge-shaped, etc. For the sake of brevity herein, "predefined
regions" are sometimes referred to simply as "regions." In some
embodiments, a predefined region and, therefore, the area upon
which each distinct molecule is synthesized is smaller than about 1
cm.sup.2 or 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. 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.
[0045] In some aspects, a predefined region can be achieved by
physically separating the regions (i.e., beads, resins, gels, etc.)
into wells, trays, etc.
[0046] "Solid support", "support", and "substrate" refer to a
material or group of materials having a rigid or semi-rigid surface
or surfaces. In some aspects, at least one surface of the solid
support will be substantially flat, although in some aspects it may
be desirable to physically separate synthesis regions for different
molecules with, for example, wells, raised regions, pins, etched
trenches, or the like. In certain aspects, the solid support(s)
will take the form of beads, resins, gels, microspheres, or other
geometric configurations.
[0047] A "protective group" is a moiety which is bound to a
molecule and which may be spatially removed upon selective exposure
to an activator. Several examples of protective groups are known in
the literature. Activators include electromagnetic radiation. ion
beams, electric fields, magnetic fields, electron beams, x-ray, and
the like.
[0048] "Activating group" 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, activating
groups which can be used in the place of a hydroxyl group include
--O(CO)Cl; --OCH.sub.2Cl; --O(CO)OAr, where Ar is an aromatic
group, preferably, a p-nitrophenyl group; --O(CO)(ONHS); and the
like. Activating groups which are useful for a carboxylic group
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 groups are known to those of
skill in the art.
[0049] A "channel block" is 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.
[0050] The terms "photolabile" and "photocleavable" are used
interchangeably throughout this application.
[0051] The term "optionally substituted" refers to the presence or
lack thereof of a substituent on the group being defined.
[0052] A "polymeric brush" ordinarily refers to polymer films
comprising chains of polymers that are attached to the surface of a
substrate. The polymeric brushes of this invention are
functionalized polymer films which comprise functional groups such
as hydroxyl, amino, carboxyl, thiol, amide, cyanate, thiocyanate,
isocyanate and isothio cyanate groups, or a combination thereof, on
the polymer chains at one or more locations. The polymeric brushes
of this invention are capable of attachment or stepwise synthesis
of macromolecules thereon.
[0053] A "free radical initiator" or "initiator" is a compound that
can provide a free radical under certain conditions such as heat,
light, or other electromagnetic radiation, which free radical can
be transferred from one monomer to another and thus propagate a
chain of reactions through which a polymer may be formed. Several
free radical initiators are known in the art, such as azo type or
nitroxide type, or those comprising multi-component systems. One
example of a multi-component system is an alkyl or aryl metal and a
binding ligand and a stable oxy free radical. See U.S. Pat. No.
5,312,871.
[0054] "Living free radical polymerization" is defined as a living
polymerization process wherein chain initiation and chain
propagation occur without significant chain termination reactions.
Each initiator molecule produces a growing monomer chain which
continuously propagates until all the available monomer has been
reacted. Living free radical polymerization differs from
conventional free radical polymerization where chain initiation,
chain propagation and chain termination reactions occur
simultaneously and polymerization continues until the initiator is
consumed. See U.S. Pat. No. 5,677,388. Living free radical
polymerization facilitates control of molecular weight and
molecular weight distribution. Living free radical polymerization
techniques, for example, involve reversible end capping of growing
chains during polymerization. One example is atom transfer radical
polymerization (ATRP).
[0055] A "radical generation site" is a site on an initiator
wherein free radicals are produced in response to heat or
electromagnetic radiation. For example, in the case of an azo-type
initiator, as shown in FIG. 2, a radical generation site exists on
the carbon atom on each side of the --N.dbd.N-- moiety.
[0056] A "polymerization terminator" is a compound that prevents a
polymer chain from further polymerization. These compounds may also
be known as "terminators," or "capping agents" or "inhibitors."
Various polymerization terminators are known in the art. In one
aspect, a monomer that has no free hydroxyl groups may act as a
polymerization terminator.
[0057] The term "capable of supporting macromolecular array
synthesis" refers to a polymeric brush that is functionalized with
functional groups such as hydroxyl, amino, carboxyl etc. These
functional groups permit macromolecular synthesis by acting as
"attachment points." For the purposes of the present invention,
those polymeric brushes that comprise functional groups only at the
terminal points are not capable of supporting macromolecular array
synthesis.
[0058] The term "conditions that promote free radical
polymerization" refers to those conditions, including the presence
of heat or electromagnetic radiation, or solvents, cosolvents, etc
which allow free radical formation and propagation. Such conditions
are well-known in the art, and are further described below.
[0059] A "macromolecule" or "polymer" comprises two or more
monomers covalently joined. The monomers may be joined one at a
time or in strings of multiple monomers, ordinarily known as
"oligomers." Thus, for example, one monomer and a string of five
monomers may be joined to form a macromolecule or polymer of six
monomers. Similarly, a string of fifty monomers may be joined with
a string of hundred monomers to form a macromolecule or polymer of
one hundred and fifty monomers.
[0060] The monomers in a given polymer or macromolecule can be
identical to or different from each other. A monomer can be a small
or a large molecule, regardless of molecular weight. Furthermore,
each of the monomers may be protected members which are modified
after synthesis. The particular ordering of monomers within a
macromolecule may be referred to herein as the "sequence" of the
macromolecule.
[0061] "Monomer" as used herein refers to those monomers that are
used to form polymers of the polymeric brush as well as those
monomers that are used to form macromolecules on the polymeric
brush. However, the meaning of the monomer will be clear from the
context in which it is used. The monomers for forming the polymers
of the polymeric brush have for example the general structure:
##STR3##
[0062] wherein R.sub.1 is hydrogen or lower alkyl; R.sub.2 and
R.sub.3 are independently hydrogen, or --Y-Z, wherein Y is lower
alkyl, and Z is hydroxyl, amino, or C(O)--R, where R is hydrogen,
lower alkoxy or aryloxy.
[0063] The term "alkyl" refers to those groups such as methyl,
ethyl, propyl, butyl etc, which may be linear, branched or
cyclic.
[0064] The term "alkoxy" refers to groups such as methoxy, ethoxy,
propoxy, butoxy, etc., which may be linear, branched or cyclic.
[0065] The term "lower" as used in the context of lower alkyl or
lower alkoxy refers to groups having one to six carbons.
[0066] The term "aryl" refers to an aromatic hydrocarbon ring to
which is attached an alkyl group. The term "aryloxy" refers to an
aromatic hydrocarbon ring to which is attached an alkoxy group. One
of ordinary skill in the art would readily understand these
terms.
[0067] In one aspect, the monomer is a "diluent" monomer when it
does not contain a free hydroxyl group.
[0068] The monomers for preparing macromolecules of the present
invention are well-known in the art. For example, when the
macromolecule is a peptide, the monomers include, but are not
restricted to, for example, the L-amino acids, the D-amino acids,
the synthetic and/or natural amino acids. When the macromolecule is
a nucleic acid, or polynucleotide, the monomers include any
nucleotide. When the macromolecule is a polysaccharide, the
monomers can be any pentose, hexose, heptose, or their
derivatives.
[0069] As used herein, a "polynucleotide" is a sequence of two or
more nucleotides. Polynucleotides of the present invention include
sequences of deoxyribopolynucleotide (DNA) or ribopolynucleotide
(RNA) which may be isolated from natural sources, recombinantly
produced, or artificially synthesized. A further example of a
polynucleotide of the present invention may be polyamide
polynucleotide (PNA). The polynucleotides and nucleic acids may
exist as single-stranded or double-stranded.
[0070] The term "nucleotide" includes deoxynucleotides and analogs
thereof. These analogs are those molecules having some structural
features in common with a naturally occurring nucleotide such that
when incorporated into a polynucleotide sequence, they allow
hybridization with a complementary polynucleotide in solution.
Typically, these analogs are derived from naturally occurring
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be tailor-made to
stabilize or destabilize hybrid formation, or to enhance the
specificity of hybridization with a complementary polynucleotide
sequence as desired, or to enhance stability of the
polynucleotide.
[0071] Analogs also include protected and/or modified monomers as
are conventionally used in polynucleotide synthesis. As one of
skill in the art is well aware, polynucleotide synthesis uses a
variety of base-protected nucleoside derivatives in which one or
more of the nitrogens of the purine and pyrimidine moiety are
protected by groups such as dimethoxytrityl, benzyl, tert-butyl,
isobutyl and the like.
[0072] For instance, structural groups are optionally added to the
ribose or base of a nucleoside for incorporation into a
polynucleotide, such as a methyl, propyl or allyl group at the 2'-O
position on the ribose, or a fluoro group which substitutes for the
2'-Q group, or a bromo group on the ribonucleoside base.
2'-O-methyloligoribonucleotides (2'-O-MeORNs) have a higher
affinity for complementary polynucleotides (especially RNA) than
their unmodified counterparts. 2'-O-MeORNA phosphoramidite monomers
are available commercially, e.g., from Chem Genes Corp. or Glen
Research, Inc. Alternatively, deazapurines and deazapyrimidines in
which one or more N atoms of the purine or pyrimidine heterocyclic
ring are replaced by C atoms can also be used.
[0073] The phosphodiester linkage, or "sugar-phosphate backbone" of
the polynucleotide can also be substituted or modified, for
instance with methyl phosphonates, O-methyl phosphates or
phosphororthioates. Another example of a polynucleotide comprising
such modified linkages for purposes of this disclosure includes
"peptide polynucleotides" in which a polyamide backbone is attached
to polynucleotide bases, or modified polynucleotide bases. Peptide
polynucleotides which comprise a polyamide backbone and the bases
found in naturally occurring nucleotides are commercially available
from, e.g., Biosearch, Inc. (Bedford, Mass.). See also U.S. Pat.
Nos. 5,773,571 and 5,786,461.
[0074] Nucleotides with modified bases can also be used in this
invention. Some examples of base modifications include
2-aminoadenine, 5-methylcytosine, 5-(propyn-1-yl)cytosine,
5-(propyn-1-yl)uracil, 5-bromouracil, 5-bromocytosine,
hydroxymethylcytosine, methyluracil, hydroxymethyluracil, and
dihydroxypentyluracil which can be incorporated into
polynucleotides in order to modify binding affinity for
complementary polynucleotides.
[0075] Groups can also be linked to various positions on the
nucleoside sugar ring or on the purine or pyrimidine rings which
may stabilize the duplex by electrostatic interactions with the
negatively charged phosphate backbone, or through interactions in
the major and minor groves. For example, adenosine and guanosine
nucleotides can be substituted at the N.sup.2 position with an
imidazolyl propyl group, increasing duplex stability. Universal
base analogues such as 3-nitropyrrole and 5-nitroindole can also be
included. A variety of modified polynucleotides suitable for use in
this invention are described in, e.g., "Antisense Research and
Application", S. T. Crooke and B. LeBleu (eds.) (CRC Press, 1993)
and "Carbohydrate Modifications in Antisense Research" in ACS Symp.
Ser. #580, Y. S. Sanghvi and P. D. Cook (eds.) ACS, Washington,
D.C. 1994
[0076] When the macromolecule of interest is a peptide, the amino
acids can be any amino acids, including .alpha., .beta., or
.omega.-amino acids. 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 contemplated by this
invention. These amino acids are well-known in the art. See for
example, Stryer, Biochemistry, latest edition, Chapter on amino
acids and or proteins, which is incorporated herein by
reference.
[0077] 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, incorporated herein by reference.
[0078] The term "hybridization" refers to the process in which two
single-stranded polynucleotides bind non-covalently to form a
stable double-stranded polynucleotide; triple-stranded
hybridization is also theoretically possible. The resulting
(usually) double-stranded polynucleotide is a "hybrid." The
proportion of the population of polynucleotides that forms stable
hybrids is referred to herein as the "degree of hybridization."
[0079] Methods for conducting polynucleotide hybridization assays
have been well developed in the art. Hybridization assay procedures
and conditions will vary depending on the application and are
selected in accordance with the general binding methods known
including those referred to in: Maniatis et al., "Molecular
Cloning: A Laboratory Manual" 2nd Ed., Cold Spring Harbor, N.Y.,
1989; Berger and Kimmel, "Methods in Enzymology," Vol. 152, "Guide
to Molecular Cloning Techniques", Academic Press, Inc., San Diego,
Calif., 1987; Young and Davis, Proc. Natl. Acad. Sci., U.S.A.,
80:1194 (1983), each of which are incorporated herein by
reference.
[0080] It is appreciated that the ability of two single stranded
polynucleotides to hybridize will depend upon factors such as their
degree of complementarity as well as the stringency of the
hybridization reaction conditions.
[0081] As used herein, "stringency" refers to the conditions of a
hybridization reaction that influence the degree to which
polynucleotides hybridize. Stringent conditions can be selected
that allow polynucleotide duplexes to be distinguished based on
their degree of mismatch High stringency is correlated with a lower
probability for the formation of a duplex containing mismatched
bases. Thus, the higher the stringency, the greater the probability
that two single-stranded polynucleotides, capable of forming a
mismatched duplex, will remain single-stranded. Conversely, at
lower stringency, the probability of formation of a mismatched
duplex is increased.
[0082] The appropriate stringency that will allow selection of a
perfectly-matched duplex, compared to a duplex containing one or
more mismatches (or that will allow selection of a particular
mismatched duplex compared to a duplex with a higher degree of
mismatch) is generally determined empirically. Means for adjusting
the stringency of a hybridization reaction are well-known to those
of skill in the art. See, for example, Sambrook, et al., "Molecular
Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor
Laboratory Press, 1989; Ausubel, et al., "Current Protocols In
Molecular Biology," John Wiley & Sons, 1987, 1988, 1989, 1990,
1991, 1992, 1993, 1994, 1995, 1996 and periodic updates; and Hames
et al., "Nucleic Acid Hybridization: A Practical Approach," IRL
Press, Ltd., 1985.
[0083] In general, conditions that increase stringency (i.e.,
select for the formation of more closely-matched duplexes) include
higher temperature, lower ionic strength and presence or absence of
solvents; lower stringency is favored by lower temperature, higher
ionic strength, and lower or higher concentrations of solvents (for
example, lower concentrations of formamide or dimethyl sulfoxide).
The duration of the hybridization reaction and the concentration of
reactants (i.e., single stranded polynucleotide) can also affect
stringency, with short reaction times and low reactant
concentrations favoring higher stringency.
[0084] Throughout this disclosure, various aspects of this
invention are presented in a range format. It should be understood
that the description in range format is merely for convenience and
brevity and should not be construed as an inflexible limitation on
the scope of the invention. Accordingly, the description of a range
should be considered to have specifically disclosed all the
possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the
breadth of the range.
[0085] C. Macromolecular Arrays on Polymeric Brushes
[0086] I. Polymeric Brushes
[0087] a) General Background
[0088] Polymeric brushes are known in the art See for example,
Prucker and Ruhe, Macromolecules, 31:592-601 (1998); Huang and
Wirth, Analytical Chemistry, 69:4577-4580 (1997); and Husseman et
al., Macromolecules, 32:1424-1431 (1999).
[0089] One traditional method of preparing polymeric brushes is
known as "grafting." This method is typically used to prepare block
copolymers wherein involves adsorbing one block of the polymer is
strongly absorbed to the surface while the other block forms the
brush layer. Some of the drawbacks of this adsorption-based
grafting include desorption of the brush and the limited choice of
functional groups for the block copolymer structure. Another method
of grafting involves forming a covalent linkage between polymer
chains and the substrate. Covalent linkage can be achieved by
condensing a functionalized polymer with reactive surface groups on
the substrate. In one such methodology, an initiator, such as a
monochlorosilyl functionalized azo initiator, can be covalently
attached to the substrate surface. Chain growth can be accomplished
under ionic or traditional free radical polymerization conditions.
These methods are shown to produce covalently attached polymer
brushes with high graft densities and molecular weights. See
Husseman, supra.
[0090] However, one major problem affecting the polymerization art
is the inability to maintain narrow polydispersity (i.e., molecular
weight distribution) at relatively high molecular weights.
Moreover, obtaining the desired polymeric structure with the
desired functional groups on the polymer has been a challenge. In
response, "living polymerization" methods have been developed.
[0091] The term "living polymerization" refers to a polymerization
process where the growing polymer chains contain one or more active
sites that are capable of promoting further polymerization. See
U.S. Pat. No. 5,708,102. One general strategy for obtaining living
polymerization is to have a chemical species reversibly cap the
active center that promotes polymerization. In ionic
polymerizations initiated by anions (anionic polymerization) or
cations (cationic polymerization) the counter cation or anion
respectively functions as a capping agent. When the ions are bound
together, polymerization stops, but reversible dissociation into
ionic fragments provides a controlled source of sites that promote
further ionic polymerization. Living ionic polymerizations are
widely utilized in forming block copolymers by sequential addition
of different alkene monomers.
[0092] In contrast to living ionic polymerizations, living free
radical polymerizations utilize polymerization initiators (R--X)
that can fragment into an alkyl radical (R.) that promotes
polymerization of monomers. This process can be illustrated as
shown in FIGS. 1A-C. Heat or electromagnetic radiation can be used
to produce the radical which initiates the polymerization of
monomers. When heat is used, the initial radical can be generated
spontaneously at temperatures above 100.degree. C. or can be
generated at temperatures under 100.degree. C. by the addition of a
small amount of free radical initiator. See, for example, Hawker,
Macromolecules, 30:373-82 (1997).
[0093] At a desired stage, the polymerization is terminated by a
polymerization terminator. Such terminators are known in the art.
See Greszta et al., Macromolecules, 27:638 (1994). One approach to
terminate polymerization is to react the growing radicals
reversibly with scavenging radicals to form covalent species.
Another approach involves reacting the growing radicals reversibly
with covalent species to produce persistent radicals. Yet another
approach involves allowing the growing radicals to participate in a
degenerative transfer reaction which regenerates the same type of
radicals. See U.S. Pat. No. 4,581,429; Hawker, J. Am. Chem. Soc.,
116:11185 (1994); and Georges et al., Macromolecules, 26:2987
(1993).
[0094] Living free radical methods allow the use of block
copolymers in forming the brushes, and allow better control of
polymeric structural characteristics such as molecular weight,
polymeric density, branching, etc. Further, living free radical
polymerization methods allow the chain elongation in the presence
of different monomers such that the polymer chain can be
varied.
[0095] Various types of initiators, methods of free radical
generation, monomers, and free radical capping agents have been
described in the prior art. See, for example, U.S. Pat. Nos.
5,677,388, 5,728,747, 5,708,102, 5,807,937, and 5,852,129. A
benzoyl peroxide-chromium initiator may also be used. See Lee et
al., J. Chem. Soc. Trans. Faraday Soc. I, 74: 1726 (1978).
Additional types of initiators include .alpha.-haloester,
alkoxyamine, and halobenzyl type initiators, all of which may be
used in the present invention. See Husseman, supra and Hawker,
supra.
[0096] Examples of photoinitiators selected in various effective
amounts, such as from about 1 to about 10 weight percent based on
the total weight percent of reactants, include benzoins,
disulfides, aralkyl ketones, oximinoketones, peroxyketones, acyl
phosphine oxides, diamino ketones, such as Micher's ketones, 3-keto
courmarins, and the like, and preferably 1-hydroxycyclohexyl phenyl
ketone.
[0097] Monomers include N-carboxy anhydride (for preparing
peptides), styrene, and vinyl compounds. Examples of initiators
include: azo-type, nitroxide type etc. An example of a terminator
is a stable free radical agent known as TEMPO
(2,2,6,6-tetramethyl-1-piperidinyloxy). See U.S. Pat. No.
5,728,747.
[0098] More recently, methods for producing a (meth)acrylic polymer
which is hydroxyl-terminated at both ends have been reported. See
U.S. Pat. No. 5,852,129. The method involves preparation of a
(meth)acrylic polymer by polymerizing a (meth)acrylic monomer using
an organic halide or a halogenated sulfonyl compound as an
initiator and, a metal complex with a central metal selected from
the elements belonging to the groups 8, 9, 10 and 11 in the
periodic table as a catalyst. This (meth)acrylate polymer contains
a terminal structure of the general formula:
--CH.sub.2--C(R.sub.1)(CO.sub.2R.sub.2)(X). This terminal halogen
is converted into a hydroxyl-containing substituent by reacting
with a polymerizable alkenyl group and a hydroxyl group. The '129
patent also describes a method to introduce a hydroxyl group at
each end, for example, by polymerizing a (meth)acrylic monomer
using a hydroxyl-containing halide as an initiator in the
above-described scheme.
[0099] b) Polymeric Brushes of the Present Invention
[0100] While the general methodology of making polymeric brushes
has been known, the methods for making polymeric brushes with
multiple functional groups on the brushes and the use of such
multifunctionalized polymeric brushes in preparing macromolecular
arrays remains to be explored.
[0101] Any support for the preparation of macromolecular arrays
must provide optimal spacing of initiation sites, wettability of
the surface by both organic solvents and aqueous solutions, and
minimize non-specific binding of ligands to the surface. The
spacing of synthesis initiation sites on a solid support can affect
not only the synthesis of the array but also the binding events
between an immobilized macromolecule and its 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.
[0102] 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.
[0103] In view of the above considerations, the present invention
provides polymeric brushes that allow control of functional site
density, wettability and porosity.
[0104] As will become clearer from the disclosure herein and in the
accompanying examples, this invention provides novel polymeric
brush compositions comprise individual polymer chains, wherein the
individual polymer chains include multiple functional groups, such
as hydroxyl groups, for example 2, 3, 4 or more. See, for example,
FIGS. 1A-C, and 6. The invention also provides methods for forming
such polymeric brushes on substrates such as glass or silica.
[0105] The polymeric brush support can be tailored to provide
optimal properties for synthesis and for biological assays. For
example, the final concentration of functional groups (amine or
hydroxyl) in the polymeric brush can be controlled by varying the
relative amounts of nonfunctionalized and functionalized monomers
used in forming the polymer. Additionally, the porosity and
solubility of the polymer films can be controlled by varying the
concentrations of monomers and crosslinking agents in the
composition. Thus, a high degree of crosslinking gives a rigid
insoluble polymer with low pore size, whereas omitting the
crosslinking agent altogether will result in soluble linear polymer
chains (with functional groups) extending off the surface of the
substrate from the attachment sites.
[0106] The polymeric brushes on substrates such as glass or silica
are useful for synthesizing arrays of macromolecules such as
polypeptides, polynucleotides and polysaccharides or other
macromolecules of interest. The polymeric brushes provide a porous
three-dimensional matrix functionalized with reactive groups that
serve as starting points for macromolecular array synthesis. These
arrays can also be used for assays involving macromolecules such as
hybridization-based nucleic acid sequence analysis or
ligand-peptide or ligand-enzymatic interactions.
[0107] One of the chief advantages of these brushes is that they
provide a much larger number of synthesis sites per unit area of
substrate than is offered by the current generation of
monofunctional silane-derivatized glass surfaces, while maintaining
a similar or greater spacing between sites. The extent of binding
of "target" molecules to the immobilized macromolecules is
substantially increased, which enhances detection, and the
multiplicity of binding sites within the polymer support may
provide additional kinetic enhancement.
[0108] The lateral surface density of polymer chains can be, for
example, 0.1-1000 pmoles/cm.sup.2 substrate surface area, or e.g.,
1-100. The lateral surface density of attachment sites on the
polymer chain, wherein individual polymer chains have multiple
attachment sites, can be, for example, 0.1 to 1,000,000
pmoles/cm.sup.2 substrate surface area, e.g., 1-1,000.
[0109] The polymer brushes can be used to form arrays of nucleic
acids. Arrays of nucleic acids immobilized on a surface are
described in detail, for example, in U.S. Pat. No. 5,744,305. On a
substrate, nucleic acids for example with different sequences are
immobilized each in a predefined area on a surface. For example,
10, 50, 60, 100, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
or 10.sup.8 different monomer sequences may be provided on the
substrate. The nucleic acids of a particular sequence are provided
within a predefined region of a substrate, having a surface area,
for example, of about 1 cm.sup.2 to 10.sup.-10 cm.sup.2. In some
embodiments, the regions have areas of less than about 10.sup.-1,
10.sup.-2, 10.sup.-3, 10.sup.-4, 10.sup.-5, 10.sup.-6, 10.sup.-7,
10.sup.-8, 10.sup.-9, or 10.sup.-10 cm.sup.2. For example, in one
embodiment, there is provided a planar, non-porous support having
at least a first surface, and a plurality of different nucleic
acids attached to the first surface at a density exceeding about
400 different nucleic acids/cm.sup.2, wherein each of the different
nucleic acids is attached to the surface of the solid support in a
different predefined region, has a different determinable sequence,
and is, for example, at least 4 nucleotides in length. The nucleic
acids may be, for example, about 4 to 20 nucleotides in length. The
number of different nucleic acids may be, for example, 1000 or
more.
[0110] The polymer brushes also may be provided on porous silica
substrates having a high porosity, for example, a primarily
inorganic porous substrate including a support region, and a porous
region in contact with the support region, wherein the porous
region includes pores with a pore size of 1-500 nm, the porous
region having a porosity of, e.g., 10-90%, and a porous surface
thickness of 0.01-20 .mu.m, as described in PCT/US00/09206, the
disclosure of which is incorporated herein.
[0111] c) Methods for Forming Polymeric Brushes
[0112] In one aspect, a method is provided for preparing
covalently-anchored polymer brushes of amine- or
hydroxy-functionalized polymers on glass or silica substrates. The
polymer film is "grafted" onto the substrate covalently through a
surface polymerization scheme as shown in FIGS. 1A-C and 6. An
initiator is provided and one end of the initiator is covalently
bound to the substrate surface, while the initiator has a radical
generation site, distally from the substrate, to participate in the
polymerization. Under appropriate conditions that promote free
radical polymerization, monomers are contacted with the substrate.
The polymeric chain is propagated to the desired length and the
polymerization can be terminated when desired.
[0113] In one aspect, a glass substrate is pre-silanized with an
azo type initiator, such as 4,4' azobis(pentanamide propyl
triethoxysilane) (AIBN-APS) (I). See FIG. 2. The silanized
substrate with the bound initiator is shown in FIG. 4. In this
example, upon activation, such as by heating, N.sub.2 is extruded,
leaving two carbon radicals.
-alkyl-(Me)(CN)C--NN--C(Me)(CN)-alkyl.fwdarw.2
[-alkyl-(Me)(CN)C.]+N2
[0114] Mixtures of suitably functionalized monomers that can
function as initiating points for macromolecular synthesis as well
as those monomers that can function as "inert" diluents (or capping
agents) are then reacted with the carbon radicals and
polymerization begins. See FIG. 6. Using such a mixture of
functionalized and diluent monomers, the average spacing of
macromolecular synthesis initiation sites on the substrate is
altered. This method provides effective control of not only
functional site density but also other surface properties such as
surface wettability and nonspecific binding of macromolecules.
[0115] AIBN-APS can be readily prepared by art-known methods. One
exemplary method of preparation is shown in FIG. 2. Another example
of an azo-type initiator (II) and its synthesis are shown in FIG.
3. See also, Chang and Frank, Langmuir, 12:5824-29 (1996); Chang
and Frank, Langmuir, 14:326-334 (1998); Prucker and Ruhe, supra;
Japanese Patent H1-234479; and Japanese Patent H3-99702.
[0116] Azo type initiators are described for example in Pruker and
Ruhe, Macromol., 31:592-601 (1998). It should be understood that
the present invention is not limited to azo-type initiators. In
fact, any known initiator can be used, so long as the initiator can
be covalently linked to the substrate on one end while it carries a
radical generation site distally to initiate polymerization.
[0117] Surface initiating sites include silane compounds, such as
(X).sub.a(Y).sub.bSi-(Z)-Q, where b=3 minus a; X is Cl, OMe, or
OEt; Y is C1-4 alkyl; Z is C2-C20 alkyl, alkylaryl or
polyoxyalkylidine; and Q is a radical forming precursor group. Q is
H or alkyl when a diluent silane is used.
[0118] Other initiators include nitroxyl (Husseman et al.,
Macromol., 32:1421-31 (1999)), halo (Huang and Wirth, Anal. Chem.,
69:4577-80 (1997)) and thiocarbamate (Kobayashi et al., J. Appl.
Poly. Sci., 49:447423 (1999)). Examples of initiator moieties
include:
[0119] --C(CN)(R.sup.1)--N.dbd.N--C(CN)(R.sup.2)R.sup.3;
[0120] --CR.sup.1(R.sup.2)--S--C(.dbd.S)--N(R.sup.3).sub.2;
[0121] --CR.sup.1(R.sup.2)--O--N(R.sup.3)R.sup.4; and
[0122] --C(R.sup.1)(R.sup.2)X;
[0123] where R.sup.1-4 are independently alkyl and X is I, Cl or
Br.
[0124] The monomers are those that are capable of undergoing free
radical polymerization. In one aspect, the monomer is 2-hydroxy
ethyl methacrylate (HEMA), which is polymerized to provide a
hydroxy functionalized vinyl polymer network. A variety of monomers
that provide the desired functional groups can be used. Some
monomers that meet these criteria can be represented by the generic
structures shown below: ##STR4##
[0125] wherein R.sub.1 is hydrogen or lower alkyl; R.sub.2 and
R.sub.3 are independently hydrogen, or --Y-Z, wherein Y is lower
alkyl, and Z is hydroxyl, amino, or C(O)--R, where R is hydrogen,
lower alkoxy or aryloxy.
[0126] Some specific examples of vinyl monomers that can be used in
the methods of this invention are shown in FIG. 5. It is
appreciated that while the specific monomers disclosed herein
provide functional groups such as hydroxyl, amino, carboxyl groups,
by selecting appropriately functionalized monomers, one can prepare
polymeric brushes that offer additional functional groups such as
thiol, cyano, isocyanate, thiocyanate or isothiocyanate. Selection
of such appropriate monomers is within the ordinary skill in the
art.
[0127] The resulting films exhibit excellent stability against a
variety of conditions on the macromolecular arrays. Such conditions
include synthesis, as well as assay conditions.
[0128] As discussed above, the polymeric brush and the silane layer
can be tailored to provide optimal properties such as suitable
functional group spacing, improved wettability, and minimized
non-specific binding of macromolecules. For example, the thickness
of the polymeric brush can be controlled by varying the polymer
chain length and the number of surface initiators. The final
density of functional groups (e.g. amine or hydroxyl) on the brush
can be controlled simply by varying the relative amounts of
non-functionalized and functionalized monomers.
[0129] In addition, the spacing between adjacent films on the brush
can be controlled by interspersing polymers comprising diluent
monomers. Thus, it is possible, and may be desirable in some cases,
to reduce the density of polymeric films as well as the functional
sites on the brush. A functional site, as used herein, refers to an
attachment site on the polymer brush comprising a functional group
that permits attachment of a molecule to the polymer. Surface
density of initiator sites can also be varied by diluting the
initiator-silane reagent with a non-functional silane such as
alkyl-SiX.sub.3, wherein X is a halogen or alkoxy. The porosity and
solubility of the polymeric brushes can be controlled by varying
the concentrations of vinyl monomers, crosslinking agents, and
surface initiators in the composition.
[0130] The free radical polymerization is typically conducted for a
sufficient amount of time, such that the desired conversion is
achieved. The amount of time needed may depend upon the temperature
of the polymerization. In some aspects, the lower the temperature,
the longer the amount of time needed to achieve a desired
conversion. Typically, the polymerization is conducted from about 1
to about 20 hours, preferably from about 1.5 to about 10 hours,
more preferably from about 2 to about 8 hours and most preferably
from about 2.5 to about 6 hours.
[0131] The polymer produced by the process of the present invention
can have a variety of molecular weights. In some aspects, the
molecular weight may depend on the amount of initiator used,
because the amount of initiator used may determine how many chains
are initiated.
[0132] The polymerization reactions may be conducted in a variety
of media, for example suspension, emulsion, bulk, that is neat or
without solvent, or in aqueous or nonaqueous solution. When used,
suitable solvents include aromatic hydrocarbons, such as benzene,
toluene, xylenes, pyridines or other solvents that have comparably
small chain transfer constants with the particular monomer(s) used
in the polymerization. The polymerization can be carried out at
temperatures ranging from about -80.degree. C. to about 80.degree.
C. The preferred reaction temperature range may be from about
25.degree. C. to about 70.degree. C. Any of the known class of
polymerization initiators is suitable provided it has requisite
solubility in the solvent or monomer mixture chosen and has an
appropriate half-life at the temperature of polymerization. In some
aspects, the initiator has a half-life that is short when compared
to the total time required for the polymerization process. The
process of the invention is carried out preferably as a batch
process, but when needed can be carried out in any of the standard
polymerization processes, for example semi-batch, starved feed, or
continuous processes.
[0133] The polymerization reactions of the present invention can in
some aspects be supplemented with a solvent or cosolvent to help
ensure that the reaction mixture remains a homogeneous single phase
throughout the monomer conversion. Any solvent or cosolvent may be
selected providing that the solvent media is effective in
permitting a solvent system which avoids precipitation or phase
separation of the reactants or polymer products until after all
polymerization reactions have been completed.
[0134] Exemplary solvents or cosolvents include polymer product
compatible aliphatic alcohols, glycols, ethers, glycol ethers,
pyrrolidines, N-alkyl pyrrolidinones, N-alkyl pyrrolidones,
polyethylene glycols, polypropylene glycols, amides, carboxylic
acids and salts thereof, esters, organosulfides, sulfoxides,
sulfones, alcohol derivatives, hydroxyether derivatives, such as
butyl CARBITOL.TM. or CELLOSOLVE.TM., amino alcohols, ketones, and
the like, derivatives thereof, and mixtures thereof. When mixtures
of water and water soluble or miscible organic liquids are selected
as the reaction media, the water to cosolvent weight ratio
typically ranges from about 100:0 to about 10:90, and preferably
from about 97:3 to about 25:75.
[0135] The polymerization reaction rate of the monomers may be
accelerated and the reaction time reduced by the addition of a
catalytic amount of a protic acid that will not also initiate
cationic polymerization. The protic acid may be selected from the
group consisting of organic acids such as sulfonic, phosphoric,
carboxylic acids, camphor sulfonic acid and nitroxides containing
acid functional groups, such as 3-carboxyl-proxyl. Suitable amounts
can be easily determined with ordinary skill.
[0136] The polymerization process of the present invention may be
repeated a number of times within the same reaction vessel by the
delayed and stepwise addition of more monomer or monomers with
varying amounts of initiators and terminating agents.
[0137] The processes of the present invention can be selected to
form a wide variety of polymers. Further, the process of the
present invention can be selected to polymerize a mixture of two or
more different polymerizable monomers to form copolymers
therefrom.
[0138] Optionally, known additives may be selected in the
polymerization reactions, which additives may provide performance
enhancements to the resulting product. Such additives may include
colorants, lubricants, release or transfer agents, surfactants,
stabilizers, antifoams, and the like.
[0139] II. Methods of Preparing Macromolecular Arrays on Polymeric
Brush Substrates
[0140] a) General
[0141] Examples of macromolecules that can be prepared on polymeric
brush substrates of this invention include nucleic acids and
polynucleotides comprising both linear and cyclic nucleotides,
peptides, polysaccharides, phospholipids, heteromacromolecules 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 macromolecules which will be
apparent upon review of this disclosure. Such macromolecules are
"diverse" when different (i.e., non-identical) monomers are used at
different predefined regions of a substrate.
[0142] It should be understood that any multifunctionalized
polymeric brush substrate, can be used to prepare macromolecular
arrays of this invention. Thus, the polymeric brush substrates
contemplated for the purposes of preparing macromolecular arrays
are not limited to the above-described polyhydroxy functionalized
or polyamino acrylate polymeric brush substrates.
[0143] The above-described macro molecular arrays can be prepared
on the polymeric brush substrates above using a number of art-known
methods, including light-directed methods, flow channel and
spotting methods, pin-based methods and bead-based methods.
[0144] b) Light-Directed Methods
[0145] "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, 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,
comprising monomers that are protected with photolabile protecting
groups. 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.
[0146] Using photolithographic techniques described above, the
photolabile protecting groups can be removed in one preselected
area and a monomer bearing a chemically-removable protecting group
is attached. Standard, chemically-removable protecting groups
include those groups which are commercially available and which are
known to be removable under typical chemical conditions. Examples
of such protecting groups include FMOC, DMT, BOC, t-butyl esters
and t-butyl ethers. See also copending U.S. Provisional Application
No. 60/146,574, filed Jul. 30, 1999, and copending application Ser.
No. 08/630,148, filed Apr. 10, 1996 for additional disclosure of
suitable protecting groups.
[0147] Following the attachment of such a protected monomer, the
protecting group is removed. Conditions for the removal are known
in the art. See, for example, Greene, et al., Protective Groups In
Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y.,
1991, incorporated herein by reference. The reactive functionality
which was previously protected with the chemically-removable
protecting group is then re-protected with a photolabile protecting
group, using, for example, a derivative of the formula:
R--O--C(O)--X in which R is a photo-cleavable moiety (e.g.,
o-nitrobenzyls, pyrenylmethyl, Ddz, various benzoin groups,
bromonitroindole) and X is a suitable leaving group (e.g., Cl, F,
pentafluorophenoxy, p-nitrophenoxy, N-succinimidyloxy,
adamantanecarboxy, or tetrazolyl).
[0148] The re-protection of surface functional groups with such
reagents is typically carried out in an organic solvent containing
a non-nucleophilic base (e.g., 2,6-lutidine, pyridine,
triethylamine or diisopropylethylamine). In some embodiments, a
nucleophilic catalyst (e.g., N-methylimidazole,
hydroxybenzotriazole or 4-(N,N-dimethylamino) pyridine) is also
included to provide further enhancement of the rate and efficiency
of the re-protection step. Following the addition of the
photolabile protecting groups, the VLSIPS.TM. cycles can be
continued using photolithographic deprotection, followed by
coupling of an additional monomer, protecting group replacement,
etc., until the desired macromolecular array is completed.
[0149] In one aspect, the macromolecule produced is a
polynucleotide. Standard phosphoramidite chemistry or H-phosphonate
methods or other coupling methods known to those of skill in the
art can be used for monomer coupling monomers. Additionally, the
photolabile protecting group which is illustrated (MeNPOC) can be
replaced with another photolabile protecting group such as NVOC, or
those photolabile protecting groups described in co-pending
applications as referred to above. Once the chemically-removable
protecting group has been removed, a photolabile protecting group
can be added using a mixed anhydride of the protecting group.
[0150] In another aspect, the macromolecule is a peptide. For
peptide synthesis, commercially-available amino acids having
chemically-removable protecting groups can be used, for example
FMOC-amino acids. After exchange of the protecting groups, the
coupling steps can be carried out using BOP/HOBt activation and
coupling methods. Those of skill in the art will understand that
other coupling methods as well as other amino acid monomers having
chemically-removable protecting groups can be used in the present
invention.
[0151] In still another aspect, all preselected areas are
derivatized with a first monomer, each of the monomers having a
chemically-removable protecting group. Following the addition of
the first monomer to each of the preselected regions, the
protecting groups are all removed in a single step using chemical
deprotection in the form of a wash across the solid support.
Alternatively, vapor-phase deprotection can also be used. See the
U.S. Pat. Nos. 5,599,695, and 5,831,070. Reprotection of each of
the growing macromolecule with a photolabile protecting group is
then carried out in the form of another wash across the entire
solid support. Following this reprotection, photolithographic
techniques of macromolecule synthesis can be continued using
monomers having chemically-removable protecting groups.
[0152] c) Flow Channel or Spotting Methods
[0153] Additional methods applicable to array synthesis on a single
substrate are described in U.S. Pat. Nos. 5,677,195 and 5,384,261,
incorporated herein by reference. 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.
[0154] A typical "flow channel" method applied to the present
invention can generally be described as follows. Diverse
macromolecules 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] d) Pin-Based Methods
[0160] Pin-based methods for the preparation of macromolecular
arrays are described in detail in U.S. Pat. No. 5,288,514. 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.
[0161] 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 polynucleotides, 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.
[0162] 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 aspects, the spacer may provide for a
cleavable function by way of, for example, exposure to acid or
base.
[0163] e) Bead Based Methods
[0164] A general approach for bead based synthesis is described in
the U.S. Pat. No. 5,384,261. For the synthesis of molecules such as
polynucleotides 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.
[0165] 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).
[0166] 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.
[0167] 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.
[0168] 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 the U.S. Pat. No. 5,639,603.
[0169] Applications
[0170] The advent of methods for the synthesis of diverse molecules
on solid supports has resulted in the genesis of a multitude of
diagnostic applications for such arrays. A number of these
diagnostic applications involve contacting a sample with a solid
support, or chip, having multiple attached biological
macromolecules such as peptides and polynucleotides, 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.
[0171] Methods for making arrays of polynucleotide 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
polynucleotide sequence have been described. U.S. Pat. No.
5,556,752 describes methods of making arrays of unimolecular,
double-stranded polynucleotides 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
polynucleotides can also be used to screen for new drugs having
particular binding affinities. More recently, complete n-mer array
probes with a wide scope of general applicability have been
described. See U.S. Provisional Application No. 60/100,393, filed
Sep. 15, 1998; and U.S. Ser. No. 09/394,230, filed Sep. 13,
1999.
[0172] 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 arrays of biological polymers and small molecules as
well as in any of the above-noted assay methods.
EXAMPLES
[0173] The following examples are offered solely for the purposes
of illustration, and are intended neither to limit nor to define
the invention.
Example 1
Preparation of Polymeric Brushes
[0174] Soda lime glass or silicon (100) substrates are cleaned with
piranha solution (30% of hydrogen peroxide and 70% of sulfuric
acid) at 90.degree. C. for 30 minutes, washed with copious amount
of deionized water and dried with a stream of N.sub.2. The cleaned
substrate is then silanized with azobis(pentanamide propyl
triethoxysilane), known as AIBN-APS, structure and preparation of
which are shown in FIG. 2. See Japanese Patent H1-234479; and
Japanese Patent H3-99702. The method consists of immersing the
glass or silica substrate in a 1% of AIBN-APS solution in toluene
for several hours. After reaction, the substrate is washed with
fresh toluene and dried with a stream of N.sub.2. The reaction
progress of silanation on silica can be monitored by ellipsometric
thickness measurements.
[0175] The AIBN-APS-silanized substrate is subjected to radical
polymerization. The substrate is immersed in a 25-50% solution of
2-hydroxy ethylmethacrylate (HEMA) in degassed DMF for various
reaction times and temperatures. At a reaction temperature of
70.degree. C., the surface AIBN molecule dissociated into two
radicals, initiating polymerization to form hydroxyl-functionalized
methacrylate polymer. The substrates were then washed thoroughly
with DMF and water, and thoroughly dried. The resulting film
thickness on silicon is monitored by ellipsometry or AFM (atomic
force microscopy). For example, a range of 5-30 nm thick pHEMA film
is obtained after a 24-hour polymerization.
[0176] Additional details for preparing polymeric brushes are known
in the art. See for example, U.S. Pat. Nos. 5,852,129, 5,728,747,
5,807,937, 5,708,102, and 5,677,388. See also, Chang and Frank,
Langmuir, 12: 5824-29 (1996); Chang and Frank, Langmuir, 14:
326-334 (1998); Prucker and Ruhe, supra; Japanese Patent H1-234479;
and Japanese Patent H3-99702.
Example 2
Preparation of Polynucleotide Array on Polymeric Brushes
[0177] A 5 nm thick pHEMA film on a glass substrate was used.
Fluorescein molecules are attached to the surface by standard
procedures as described for example in PCT/JUS00/09206. A
representative control silanated substrate, flat soda lime glass,
silanated with bis (2-hydroxyethyl)-3-aminopropyltriethoxysilane,
and the pHEMA modified glass were compared.
[0178] Fluoreprime Stain Assay
[0179] Quantitative studies of the synthesis, density and
uniformity of silica substrates was conducted using methods based
on surface fluorescence as described in McGall et al; J. Am. Chem.
Soc.; 119: 5081-5090 (1997). Fluorescent "staining" of the surface
was performed as described, with the exception that a fluorescein
concentration of 0.5 mM in a solution containing 50 mM DMT-T-CEP in
acetonitrile was used. The fluorescein phosphoramidite is coupled
to the free hydroxyl groups with the standard protocol. Substrates
are then deprotected for a minimum of one hour in a 1:1 solution of
ethylenediamine/ethanol, rinsed with deionized water, and blown dry
with dry nitrogen. The substrate is then scanned using confocal
microscopy. The signal obtained is a function of the number of
available hydroxyl groups on the surface. In this case, the
relative values as compared to other types of similarly treated
glass is an indication of the relative density and capacity of the
surface. This technique also provides a visual picture of the
surface with respect to quality and uniformity of the surface. The
technique is not limited to hydroxyl groups but may be modified to
measure other groups of interest for support of polymer of interest
on the surface by using the appropriately functionalized molecule
for detection.
[0180] The pHEMA film had a much higher hydroxyl content/unit area
of the substrate as evidenced by fluorescent staining analysis. As
shown in FIG. 7, the average fluorescent intensity from the
fluorescein-stained stripe on the pHEMA-modified glass was at least
60-times higher than the BIS-silane control initially, and this
increases to a >300-fold higher ratio after a 40-hour period in
6.times.SSPE buffer at 25.degree. C. This increase is primarily due
to a loss of the fluorescence intensity on the control substrate
due to the known hydrolysis of the "bis" silane bonded phase in
aqueous phosphate buffers. It appears, then that the pHEMA film is
more stable towards hydrolytic degradation than the silanated
layer.
[0181] HPLC Quantitation Assay
[0182] The HPLC quantitaion assay is performed substantially as
described in U.S. Pat. No. 5,843,655. HPLC (high performance liquid
chromatography) analyses are performed on a Beckman System Gold ion
exchange column using fluorescence detection at 520 nm. Elution is
performed with a linear gradient of 0.4M NaClO.sub.4 in 20 mM Tris
pH 8, at a flow rate of 1 mL/min, or other suitable buffer system.
The HPLC quantitation assay is used to measure the site density
available for generating polymers, and the coupling efficiency of
each subsequent addition of monomer to the growing chain.
[0183] In this technique, attached to the surface were a cleavable
sulfone linker (5'-phosphate-ON reagent, ChemGenes Corporation), a
spacer molecule ("C3," a three carbon spacer phosphoramidite from
Glen Research), and a fluorophore (5-carboxyfluorescein-CX CED
phosphoramidite from BioGenex). The purpose of the spacer molecule
is to discriminate between fluorescent molecules that have attached
to the intended synthesis sites vs. those that have remained on the
surface without chemical attachment. Synthesis was also
accomplished on the surfaces using traditional acid-based
polynucleotide chemistry (trityl chemistry). Similar chemistries
can be applied for the synthesis of polynucleotide, peptide,
oligosaccarides, peptide nucleic acids, and other polymers. The
description relating to the peptide nucleic acids can be found in
the PCT publication WO92/20702, published Nov. 26, 1992.
[0184] After synthesis the surface is treated with a known solution
volume of reagent necessary to cleave the linker to release
3'-C.sub.3-fluorescein-5', and this is typically cleaved in
solution overnight (1:1 by volume ethylenediamine/water). The
resulting solution is diluted and coinjected with an internal
standard onto and analyzed by HPLC. The internal standard is a
3'-C.sub.3-C.sub.3-fluorescein-5' chain prepared separately on an
ABI synthesizer. Concentration is determined by UV-Vis spectra on a
Varian Cary 3E spectrophotometer (Varian). Integration of HPLC peak
areas can be used to determine total site density and cleanliness
of coupling.
[0185] The control silanized substrates had a density of hydroxyl
groups of 110 pmoies/cm.sup.2, whereas the pHEMA modified glass had
a density of hydroxyl groups of 11,800. Thus the pHEMA modified
glass included 107 times more hydroxyls per unit area.
Example 3
Hybridization Assay on Polymeric Brush Arrays of
Polynucleotides
[0186] The polynucleotide polymeric brush array included
polynucleotides attached to a pHEMA coated glass substrate made as
described in Example 1.
[0187] Full length probes capable of hybridization, typically
20-mer probes, were synthesized using Affymetrix synthesizers as
described in U.S. Pat. No. 5,405,783, using nucleoside
phosphoramidites equipped with 5'-photolabile MeNPOC protecting
groups. The sequence used was a 20 mer probe such as (3')-AGG TCT
TCT GGT CTC CTT TA (5') (SEQ ID NO:1), with the 3' end attached to
the surface. The non-photolabile protecting groups were removed
post synthesis in 1:1 ethylenediamine/ethanol (v/v) for a minimum
of 4 hours.
[0188] Hybridization assays were performed on glass slides without
further processing. Each slide was placed in about 10-15 mls of
10-50 nM target oligonucleotide in hybridization buffer with gentle
stirring. The two hybridization buffers used is 6.times.SSPE. The
target sequence is the exact complement of the probe sequence, such
as: (5') Fluorophore-TCC AGA AGA CCA GAG GAA AT (SEQ ID NO:2).
[0189] The pattern and intensity of surface fluorescence was imaged
with a specially constructed scanning laser confocual fluorescence
microscope. Where necessary, the photon multiplier tube gain was
adjusted to keep signals within rage for the detector.
[0190] The polynucleotide polymeric brush array was hybridized from
one hour to several hours with control oligonucleotide (10 nM in
6.times.SSPE) at various temperatures. Standard hybridization
protocols were used, as described, for example, in PCT/US00/09206.
Any equivalent hybridization protocol known in the art can also be
employed.
[0191] With respect to hybridization characteristics, synthesis on
the pHEMA film also gives a much higher surface concentration of
probes, and this results in substantially higher hybridization
signals. FIG. 8 shows the hybridization signal ratio developed in
40 hours. The intensity ratio, initially .about.5.times. higher
than the control, increases to .about.9 times higher after 40-hour
period of hybridization. Hybridization signal continues to increase
on the film with time, whereas the silanated substrate is already
saturated at the 1 hour time point. Apparently, while the pHEMA
film clearly has a much higher surface hydroxyl content, and
capacity for binding target molecules, the kinetics of binding may
be somewhat slower. This would be the expected result from crowding
of the probes on the fully hydroxylated polymer "brush", leading to
substantial inaccessibility of the probes.
[0192] A lower hydroxyl concentration is likely to be more optimal,
as greater probe spacing improves their accessibility to target
molecules in solution. Dilution of the probe concentration in the
film can be achieved by carrying out the polymerization with a
mixture of functional and non-functional monomers to form a
copolymer with functional groups spaced further apart along the
chains. Such "diluted" functionalized polymeric brush substrates
are envisioned to provide optimal probe density (which is still
comparable to or greater than that obtainable on traditional
substrates), while providing comparable or greater signal.
[0193] After approximately 40 hours of hybridization, the
temperature was raised to 45.degree. C., which resulted in rapid
decrease in signals for both the control and pHEMA modified
substrates. This is due to the dissociation of the bound target
molecules from the surface probes as the duplex is destabilizied
with increasing temperature. However, there is no observable
decrease in the signal ratio of the pHEMA sample to the control
substrate, suggesting that hybridization affinities of the
oligonucleotide probes are equivalent on both substrates.
[0194] In another experimental run, in a hybridization on a
substrate made as in Example 1, the hybridization in SSPE (sodium
chloride, sodium phosphate, EDTA) buffer was 2.3.times. higher than
the control at 25.degree. C. at 1 hr. After 45.degree. C. for 16
hours, the intensity increased 135 fold higher than the
standard.
[0195] All publications, patents and patent applications referred
to herein are incorporated herein by reference in their
entirety.
[0196] The above description is illustrative and not restrictive.
Many variations of the invention will become apparent to those of
skill in the art upon review of this disclosure. Merely by way of
example a variety of substrates, polymers, initiators, synthesis
initiation sites, and other materials may be used without departing
from the scope of the invention.
Sequence CWU 1
1
2 1 20 DNA Artificial Sequence hybridization probe 1 atttcctctg
gtcttctgga 20 2 20 DNA Artificial Sequence hybridization probe 2
tccagaagac cagaggaaat 20
* * * * *