U.S. patent application number 10/840841 was filed with the patent office on 2004-10-14 for chemical amplification for the synthesis of patterned arrays.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Beecher, Jody E., Goldberg, Martin J., McGall, Glenn H..
Application Number | 20040203056 10/840841 |
Document ID | / |
Family ID | 21856245 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040203056 |
Kind Code |
A1 |
Beecher, Jody E. ; et
al. |
October 14, 2004 |
Chemical amplification for the synthesis of patterned arrays
Abstract
Radiation-activated catalysts (RACs), autocatalytic reactions,
and protective groups are employed to achieve a. highly sensitive,
high resolution, radiation directed combinatorial synthesis of
pattern arrays of diverse polymers. When irradiated, RACs produce
catalysts that can react with enhancers, such as those involved in
autocatalytic reactions. The autocatalytic reactions produce at
least one product that removes protecting groups from synthesis
intermediates. This invention has a wide variety of applications
and is particularly useful for the solid phase combinatorial
synthesis of polymers.
Inventors: |
Beecher, Jody E.; (Mountain
View, CA) ; Goldberg, Martin J.; (San Jose, CA)
; McGall, Glenn H.; (Mountain View, CA) |
Correspondence
Address: |
BANNER & WITCOFF LTD.,
ATTORNEYS FOR AFFYMETRIX
1001 G STREET , N.W.
ELEVENTH FLOOR
WASHINGTON
DC
20001-4597
US
|
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
21856245 |
Appl. No.: |
10/840841 |
Filed: |
May 7, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10840841 |
May 7, 2004 |
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09578282 |
May 25, 2000 |
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6770436 |
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09578282 |
May 25, 2000 |
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08969227 |
Nov 13, 1997 |
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6083697 |
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60030826 |
Nov 14, 1996 |
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Current U.S.
Class: |
435/6.11 ;
536/25.32 |
Current CPC
Class: |
B01J 2219/00675
20130101; C07H 21/04 20130101; C07K 1/04 20130101; C40B 50/14
20130101; B01J 2219/00617 20130101; B01J 2219/00711 20130101; B01J
2219/00432 20130101; B01J 2219/00722 20130101; C07H 21/00 20130101;
C07K 1/045 20130101; B01J 2219/00576 20130101; C07B 61/00 20130101;
B01J 2219/00637 20130101; C12Q 1/6837 20130101; B01J 2219/00659
20130101; B01J 2219/00605 20130101; B82Y 30/00 20130101; B01J
2219/00497 20130101; B01J 2219/00596 20130101; B01J 2219/00612
20130101; B01J 2219/00427 20130101; B01J 2219/00608 20130101; B01J
2219/00626 20130101; C40B 40/06 20130101; C40B 60/14 20130101; B01J
19/0046 20130101; Y02P 20/55 20151101; B01J 2219/00585 20130101;
B01J 2219/00527 20130101; B01J 2219/00529 20130101; B01J 2219/0061
20130101; C07B 2200/11 20130101 |
Class at
Publication: |
435/006 ;
536/025.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1-16. (canceled)
17. A method for synthesizing polymers of diverse sequences
comprising the steps of a) forming a surface comprising i) a
photosensitive compound or group, said photosensitive compound or
group producing a catalyst when irradiated, and ii) an
autocatalytic compound or group, said autocatalytic compound or
group generating a protecting group removing product when said
autocatalytic compound is activated by said catalyst; b)
irradiating at least a part of said surface to remove said
protecting group; c) contacting the surface with a first monomer;
d) irradiating at least another part of said surface to remove said
protecting group; and e) contacting the surface with a second
monomer.
18. The method recited in claim 17 wherein said photosensitive
compound is a PAC.
19. The method recited in claim 17 wherein said autocatalytic
compound is a masked acid.
20. The method recited in claim 17 wherein said synthesis
intermediate is a linker molecule.
21. The method recited in claim 17 wherein said synthesis
intermediate is a nucleotide.
22. The method re cited in claim 17 wherein said synthesis
intermediate is a [[an]] polynucleotide.
23. The method recited in claim 17 wherein said synthesis
intermediate is an amino acid.
24. The method recited in claim 17 wherein said synthesis
intermediate is a polypeptide.
25. The method recited in claim 17 wherein said removable
protecting group is an acid removable group.
26. The method recited in claim 17 wherein said photosensitive
compound is a toluenesulfonic acid.
27. The method recited in claim 17 wherein said autocatalytic
compound is a pentafluorobenoic acid.
28. The method recited in claim 17 wherein said acid removable
protecting group is 5' demethoxytrityl.
29-40. (canceled)
41. A method for hybridizing nucleic acid comprising the steps of:
a) forming a surface comprising i) a photosensitive compound or
group, said photosensitive ii) compound or group producing a
catalyst when irradiated, and ii) an autocatalytic compound or
group, said autocatalytic compound or group generating a protecting
group removing product when said autocatalytic compound is
activated by said catalyst; b) irradiating at least a part of said
surface to remove said protecting group; c) contacting the surface
with a first monomer; d) irradiating at least another part of said
surface to remove said protecting group; e) contacting the surface
with a second monomer; f) repeating steps a to e to synthesize
polynucleotide arrays of desired length; and g) hybridizing a
target nucleic acid to said polynucleotide arrays.
42. The method recited in claim 41 wherein said synthesis
intermediate is a linker molecule.
43. The method recited in claim 41 wherein said synthesis
intermediate is a nucleotide.
44. The method recited in claim 41 wherein said synthesis
intermediate is a [[an]] polynucleotide.
45. The method recited in claim 41 wherein said removable
protecting group is an acid removable group.
46. The method recited in claim 41 wherein said photosensitive
compound is a toluenesulfonic acid.
47. The method recited in claim 41 wherein said autocatalytic
compound is apentafluorobenzoic acid.
48. The method recited in claim 41 wherein said acid removable
protecting group is 5' dimethoxytrityl.
49. A method for synthesizing a polymer array on a substrate
comprising the steps of: a) providing a surface of the substrate
having one or more synthesis intermediates bound thereon and a
catalyst system, the synthesis intermediates having a reactive
group protected from reaction by a protective group; b) irradiating
the surface of the substrate with radiation of certain wavelength;
c) initiating a catalytic reaction to remove the protective group
from the synthesis intermediates to produce unprotected reactive
groups; d) reacting a subsequently added synthesis intermediate
with the unprotected reactive group, the subsequently added
synthesis intermediate having a reactive group protected from
reaction by a protective group; e) repeating steps b) through d),
or not, until a desired polymer sequence is obtained.
50-51. (canceled)
Description
STATEMENT OF RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/030,826 filed Nov. 14, 1996, hereby
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Embodiments of the present invention relate to spatially
defined chemical synthesis involving lithographic processes. In
particular, embodiments of the present invention are directed to
novel methods and compositions for synthesizing arrays of diverse
polymer sequences, such as polypeptides and polynucleotides.
According to a specific aspect of the invention, a method of
synthesizing diverse polymer sequences, such as peptides or
polynucleotides, is provided. The diverse polymer sequences are
useful for example, in nucleic acid analysis, gene expression
monitoring, receptor and nucleic acid binding studies, surface
based DNA computation, and integrated electronic circuits and other
miniature device fabrication.
[0003] Methods of synthesizing polymer sequences such as nucleotide
and peptide sequences are known. Methods of synthesizing
oligonucleotides are found in, for example, Oligonucleotide
Synthesis: A Practical Approach, Gait, ed., IRL Press, Oxford
(1984), incorporated herein by reference in its entirety for all
purposes. The so-called "Merrifield" solid phase peptide synthesis
has been in common use for several years and is discussed in
Merrifield, J. Am. Chem. Soc. (1963) 85:2149-2154, incorporated
herein by reference for all purposes. Solid-phase synthesis
techniques have been provided for the synthesis of several peptide
sequences on, for example, a number of "pins." See e.g., Geysen et
al., J. Immun. Meth. (1987) 102:259-274, incorporated herein by
reference for all purposes. Other solid-phase techniques involve,
for example, synthesis of various peptide sequences on different
cellulose disks supported in a column. See Frank and Doring,
Tetrahedron (1988) 44:6031-6040, incorporated herein by reference
for all purposes. Still other solid-phase techniques are discussed
in U.S. Pat. No. 4,728,502 (issued to Hamill) and PCT Publication
No. WO 90/00626 (Beattie, inventor).
[0004] Each of the above techniques produces only a relatively low
density array of polymers. For example, the technique discussed in
Geysen et al. is limited to producing 96 different polymers on pins
spaced in the dimensions of a standard microliter plate.
SUMMARY OF THE INVETION
[0005] Improved methods of forming high density arrays of peptides,
polynucleotides, and other polymer sequences in a short period of
time have been devised using combinatorial solid phase synthesis.
Very Large Scale Immobilized Polymer Synthesis (VLSIPS) technology
has greatly advanced combinatorial solid phase polymer synthesis
and paved the way to clinical application of deoxyribonucleic acid
(DNA) array chips such as those sold under the trademark GENECHIP.
See Kozal et al., Nature Medicine, Vol. 2, pp. 753-759 (1996),
incorporated herein by reference in its entirety for all purposes.
VLSIPS technology is disclosed in Pirrung et al., U.S. Pat. No.
5,143,854 (see also PCT Publication No. WO 90/15070), Fodor et al.,
PCT Publication No. WO 92/10092, and PCT Publication No. WO
95/11995; Fodor et al., Science (1991) 251:767-777, all
incorporated herein by reference in their entirety for all
purposes. Known embodiments of VLSIPS technology employ
radiation-labile protecting groups and photolithographic masks to
achieve spatially defined combinatorial polymer synthesis on a
substrate surface. In those embodiments, masks are used to control
the selective exposure to radiation in specific locations of a
surface provided.with linker molecules containing radiation-labile
protecting groups. In the exposed locations, the radiation-labile
protecting groups are removed. The surface is then contacted with a
solution containing a desired monomer. The monomer has at least one
site that is reactive with the newly exposed reactive moiety on the
linker and at least a second reactive site protected by one or more
radiation-labile protecting groups. The desired monomer is then
coupled to the unprotected linker molecules. The process can be
repeated to synthesize a large number of polymer sequences in
specific locations.
[0006] Other methods for synthesizing high density polymer arrays
employ blocks containing channels for reagent delivery at
preselected sites on the substrate. See PCT Publication No. WO
93/09668, incorporated herein by reference for all purposes. In
certain embodiments, a block is contacted with the substrate and
the reagents necessary to form a portion of the immobilized polymer
are permitted to access the substrate via the channel(s). The block
or substrate can be rotated and the process repeated to form arrays
of polymers on the substrate. The block channel method can be
combined with light-directed methodologies.
[0007] Certain embodiments of the present invention provide novel
methods, compositions, and devices useful in synthesizing novel
high density arrays of diverse polymer sequences. The polymer
sequences are fashioned from individual synthesis intermediates and
include diverse naturally or non-naturally occurring peptides,
nucleotides, polypeptides or polynucleotides. The methods of the
present invention utilize a novel chemical amplification process
using a catalyst system which is initiated by radiation to assist
in the synthesis the polymer sequences. Methods of the present
invention include the use of photosensitive compounds which act as
catalysts to chemically alter the synthesis intermediates in a
manner to promote formation of polymer sequences. Such
photosensitive compounds include what are generally refered to as
radiation-activated catalysts (RACs), and more specifically photo
activated catalysts (PACs). The RACs can by themselves chemically
alter the synthesis intermediate or they can activate an
autocatalyfic compound which chemically alters the synthesis
intermediate in a manner to allow the synthesis intermediate to
chemically combine with a later added synthesis intermediate or
other compound.
[0008] According to one embodiment of the present invention, one or
more linker molecules are bound to or otherwise provided on the
surface of a substrate, such as a glass plate. The unbound portion
of the linker molecule, also referred to as the terminal or free
end of the linker molecule, has a reactive functional group which
is blocked, protected or otherwise made unavailable for reaction by
a removable protective group. Once the protective group is removed,
the functional group is made available for reaction, i.e. the
reactive functional group is unblocked. A photo activated catalyst
(PAC) is also located or otherwise provided on the surface of the
substrate in the vacinity of the linker molecules. An autocatalytic
compound may also be present on the surface of the substrate. The
photo activated catalyst by itself or in combination with
additional catalytic components is referred to herein as a catalyst
system.
[0009] Using lithographic methods and techniques well known to
those of skill in the art, a set of first selected regions on the
surface of the substrate is exposed to radiation of certain
wavelengths. The radiation activates the PAC which then either
directly or through an autocatalytic compound catalytically removes
the protecting group from the linker molecule making it available
for reaction with a subsequently added synthesis intermediate.
[0010] According to one embodiment of the present invention, the
radiation causes the structure of the PAC to change and to produce
a catalyst capable of initiating the autocatalytic compound, also
referred to herein as an enhancer, to undergo a reaction producing
at least one product that removes the protective groups from the
linker molecules in the first selected regions; The use ofPACs and
autocatalytic compounds advantageously amplifies through catalysis
the number of linker molecules having their protective groups
removed. Stated differently, the radiation initiates a chemical
reaction which catalyzes the removal of a large number of
protective groups. With the protective groups removed, the reactive
functional groups of the linker molecules are made available for
reaction with a subsequently added synthesis intermediate or other
compound.
[0011] The substrate is then washed or otherwise contacted with an
additional synthesis intermediate that reacts with the exposed
functional groups on the linker molecules to form a sequence. In
some preferred embodiments, the enhancers are autocatalytic
compounds or groups that undergo autocatalysis when initiated by a
RAC such as a PAC. The synthesis intermediate also has a reactive
functional group which is blocked or otherwise made unavailable for
reaction by a removable protective group. In this manner, a
sequence of monomers of any desired length can be created by
stepwise irradiating the surface of the substrate to initiate a
catalytic reaction to remove a protective group from a reactive
functional group on a already present synthesis intermediate and
then introducing a monomer, i.e. a synthesis intermediate, that
will react with the reactive functional group, and that will have a
protective group for later removal by a subsequent irradiation of
the substrate surface.
[0012] Accordingly, a second set of selected regions on the
substrate which may be the same or different from the first set of
selected regions on the substrate is, thereafter, exposed to
radiation and the removable protective groups on the synthesis
intermediates or linker molecules are removed. The substrate is
then contacted with an additional subsequently added synthesis
intermediate for reaction with exposed functional groups. This
process is repeated to selectively apply synthesis intermediates
until polymers of a desired length and desired chemical sequence
are obtained. Protective groups on the last added synthesis
intermediate in the polymer sequence are then optionally removed
and the. sequence is, thereafter, optionally capped. Side chain
protective groups, if present in the polymer sequence, are also
removed. The technique, when it employs photon radiation, is
referred to as "photochemical amplification for the synthesis of
patterned arrays" or "PASPA".
[0013] According to one embodiment of the present invention, the
RAC produces an acid when exposed to radiation; the enhancer is an
ester labile to acid catalyzed thermolytic cleavage which itself
produces an acid; the protecting group is an acid removable
protecting group, and the monomer is a nucleotide containing an
acid removable protecting group at its C-5' hydroxyl group, for
eaxmple when synthesis is carried out in the 3' to 5' direction. It
is to be understood that the teachings of the present invention are
equally useful in carrying out synthesis of polynucleotides in the
5' to 3' direction. In that instance, the protective group is
present at the 3' hydroxyl group. In an alternate embodiment of the
present invention, the monomer is an amino acid containing an acid
removable protecting group at its amino or carboxy terminus and the
linker molecule terminates in an amino or carboxy acid group
bearing an acid removable protective group.
[0014] Using the techniques disclosed herein, it is possible to
advantageously irradiate relatively small. and precisely known
locations on the surface of the substrate. The radiation does not
directly cause the removal of the protective groups, such as
through a photochemical reaction upon absorption of the radiation
by the synthesis intermediate or linker molecule itself, but rather
the radiation acts as a signal to initiate a chemical catalytic
reaction which removes the protective group in an amplified manner.
Therefore, the radiation intensity as used in the practice of the
present invention to initiate the catalytic removal by a catalyst
system of protecting groups can be much lower than, for example,
direct photo removal, which can result in better resolution when
compared to many non-amplified techniques.
[0015] The present invention is advantageous because it makes
possible the synthesis of polymers of any desired chemical sequence
at known locations on a substrate with high synthesis fidelity,
small synthesis feature, and improved manufacturability.
Embodiments of the present invention are useful in fabricating high
density nucleic acid probe arrays or immobilizing nucleic acid
sequences on a surface of a substrate. High density nucleic acid
probe arrays provide an efficient means to analyze nucleic acids,
to monitor gene expression and to perform computation.
[0016] It is therefore an object of the present invention to
provide methods of manufactuing high density polymer arrays using
chemical amplification techniques. It is a further object of the
present invention to provide methods of manufacturing polymer
arrays using less time and lower radiation intensities to improve
polymer purity, to improve the spatial resolution and contrast
between polymer and substrate and to decrease the area on the
substrate where polymer sequences can be synthesized allowing many
and different polymer sequences on the same substrate. It is a
still further object of the present invention to provide methods of
removing protecting groups from synthesis intermediates in the
formation of polymer sequences using photosensitive compounds to
initiate catalytic reactions. It is an even still further object of
the present invention to improve precision, contrast, and ease of
manufacture in the production of polymer arrays.
[0017] These and other objects, features and advantages of the
present invention will become apparent by reference to the
remaining portions of the specification and the attached
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In the course of the detailed description of certain
preferred embodiments to follow, reference will be made to the
attached drawings, in which,
[0019] FIG. 1 is a graph of concentration of PAC versus irradiation
time in seconds.
[0020] FIG. 2 is an image showing 5 .mu.m and 2 .mu.m features
obtained by the process of the present invention.
[0021] FIG. 3 is an array produced according to the method of the
present invention.
[0022] FIG. 4 is a graph showing the nonlinear behavior of the
response as a function of the irradiation does.
[0023] FIG. 5 is a graph of the photokinetic response as a function
of trioctylamine concentration.
[0024] FIG. 6 is a chromatogram of a labeled T.sub.6 polymer
synthesized with the chemically amplified photo process.
[0025] FIG. 7 is a chromatogram of a labeled T.sub.6 polymer
synthesized with TCA/DCM.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0026] The principles of the present invention may be applied with
particular advantage to provide a method of preparing selected
polymer sequences in a precise manner in a polymer array by using
radiation to initiate the catalytic removal of protective groups to
allow polymer chain formation in a stepwise method.
[0027] As used herein, the following terms are intended to have the
following general meanings:
[0028] 1. Ligand: A ligand is a molecule that is recognized by a
receptor. Examples of ligand that can be investigated by this
invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones, opiates, steroids, peptides, enzyme substrates,
cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids,
oligosaccharides, and proteins.
[0029] 2. 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 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, each set of which is readily known to those
of skill in the art. The particular ordering of monomers within a
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 and is not limited to a single "mer". For
example, dimers of the 20 naturally occurring L-amino acids form a
basis set of 400 monomers for synthesis of polypeptides. Monomers
can also include trimers, oligomers, polymers and so forth.
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 Ser. No.
796,727, filed Nov. 22, 1991, entitled "POLYMER REVERSAL ON SOLID
SURFACES," incorporated herein by reference for all purposes.
[0030] 3. Peptide: A peptide is a polymer in which the monomers are
a -amino acids and are joined together through amide bonds,
alternatively referred to as a polypeptide. Amino acids may be the
L-optical isomer or the D-optical isomer. The term "polypeptide" as
used herein refers to two or more amino acid monomers in length or
greater and often includes more than 20 amino acid monomers or
monomers on the order of hundreds. Standard abbreviations for amino
acids are used (e.g., P for proline). Identification of amino acids
and their abbreviations are well-known and are included in Stryer,
Biochemistry, Third Ed., 1988, which is incorporated herein by
reference for all purposes.
[0031] 4. Receptor: A receptor is a molecule that has an affinity
for a ligand. Receptors may be naturally-occurring or man-made
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
antiligands. 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. Specific examples of receptors which can be investigated
by this invention include but are not restricted to:
[0032] a.) Microorganism receptors: The determination of ligands
that bind to microorganism receptors such as specific transport
proteins or enzymes essential to survival of microorganisms would
be a useful tool for discovering new classes of antibiotics. Of
particular value would be antibiotics against opportunistic fuingi,
protozoa, and bacteria resistant to antibiotics in current use.
[0033] b.) Enzymes: For instance, a receptor can comprise a binding
site of an enzyme such as an enzyme responsible for cleaving a
neurotransmitter; determination of ligands for this type of
receptor to modulate the action of an enzyme that cleaves a
neurotransmitter is useful in developing drugs that can be used in
the treatment of disorders of neurotransmission.
[0034] c.) Antibodies: For instance, the invention may be useful in
investigating a receptor that comprises a ligand-binding site on an
antibody molecule which combines with an epitope of an antigen of
interest; analyzing a sequence that mimics an antigenic epitope may
lead to the development of vaccines in which the immunogen is based
on one or more of such sequences or lead to the development of
related diagnostic agents or compounds useful in therapeutic
treatments such as for autoimmune diseases (e.g., by blocking the
binding of the "self" antibodies).
[0035] d.) Nucleic Acids: Sequences of nucleic acids may be
synthesized to establish sequences recognized by various receptor
molecules, such as protein or other DNA or RNA molecules. Nucleic
acids within the scope of the present invention include naturally
occuring or synthetic nucleic acids, nucleic acid analogs, modified
nucleic acids, nucleic acids containing modified nucleotides,
modified nucleic acid analogs, peptide nucleic acids and the like
or mixtures thereof.
[0036] e.) Catalytic Polypeptides: Polymers, preferably
polypeptides, which are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products. Such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, which
functionality is capable of chemically modifying the bound
reactant. Catalytic polypeptides and others are discussed in, for
example, PCT Publication No. WO 90/05746, WO 90/05749, and WO
90/05785, which are incorporated herein by reference for all
purposes.
[0037] f.) Hormone receptors: Determination of the ligand which
binds with high affinity to a receptor such as the receptors for
insulin and growth hormone is useful in the development of, for
example, an oral replacement of the daily injections which
diabetics must take to relieve the symptoms of diabetes or a
replacement for growth hormone. Other examples of hormone receptors
include the vaso-constrictive hormone receptors; determination of
ligands for these receptors may lead to the development of drugs to
control blood pressure.
[0038] g.) Opiate receptors: Determination of ligands which bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0039] 5. Substrate: A material having a rigid or semi-rigid
surface usually made from glass or suitable polymer materials. 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 optionally may be released upon completion of the
synthesis. Substrates are well known in the art and are readily
commercially available through vendors such as USPG, PPG
Industries, AFG Industries and others.
[0040] 6. Protective Group: A material which may be selectively
removed to expose an active site such as, in the specific example
of an amino acid, an amine group. By way of illustration,
protecting groups include but are not limited to those that are
photolabile (see Fodor et al., PCT Publication No. WO 92/10092.
(previously incorporated by reference), U.S. Ser. No. 07/971,181,
filed Nov. 2, 1992, and U.S. Ser. No. 08/310,817, filed Sep. 22,
1994 (all of which are incorporated herein by reference in their
entirety for all purposes)), acid labile, and base labile. For an
extensive listing of protective groups useful in the practice of
the present invention, see also Greene, T. W. and Wuts, P. G. M.,
Protective Groups in Organic Synthesis, (1991), incorporated herein
by reference in its entirety for all purposes. Useful
representative acid sensitive protective groups include
dimethoxytrityl (DMT), tert-butylcarbamate (tBoc) and
trifluoroacetyl (Tfa). Useful representative base sensitive
protective groups include 9-fluorenylmethoxycarbonyl (Fmoc),
isobutyrl (iBu), benzoyl (Bz) and phenoxyacetyl (pac). Other
protective groups include acetamidomethyl, acetyl,
tert-amyloxycarbonyl, benzyl, benzyloxycarbonyl,
2-(4-biphenylyl)-2-propyloxycarbonyl; 2-bromobenzyloxycarbonyl,
tert-butyl, tert-butyloxycarbonyl,
1-carbobenzoxamido-2,2,2-trifluoroethyl, 2,6-dichlorobenzyl,
2-(3,5-dimethoxyphenyl)-2-propyloxycarbonyl, 2,4-dinitrophenyl,
dithiasuccinyl, formyl, 4-methoxybenzenesulfonyl, 4-methoxybenzyl,
4-methylbenzyl, o-nitrophenylsulfenyl,
2-phenyl-2-propyloxycarbonyl,
.alpha.-2,4,5-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl,
xanthenyl, benzyl ester, N-hydroxysuccinimide. ester, p-nitrobenzyl
ester, p-nitrophenyl ester, phenyl ester, p-nitrocarbonate,
p-nitrobenzylcarbonate, trimethylsilyl and pentachlorophenyl ester
and the like.
[0041] 7. 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 mm.sup.2, more preferably less than 1 cm.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..sup.2m. Within
these regions, the polymer synthesized therein is preferably
synthesized in a substantially pure form.
[0042] 8. Substantially Pure: A polymer is considered to be
"substantially pure" within a predefined region of a substrate when
it exhibits characteristics that distinguish it from other
predefined regions. Typically, purity will be measured in terms of
biological activity or function as a result of uniform sequence.
Such characteristics will typically be measured by way of binding
with a selected ligand or receptor. Preferably the region is
sufficiently pure such that the predominant species in the
predefined region is the desired sequence. According to preferred
aspects of the invention, the polymer is at least 5% pure, more
preferably more than 10% to 20% pure, more preferably more than 95%
pure, where purity for this purpose refers to the ratio of the
number of polymer molecules formed in a predefined region having a
desired sequence to the total number of Molecules formed in the
redefined region.
[0043] 9. Catalyst: A catalyst is any material that is not consumed
in a chemical reaction and that affects the rate of the reaction.
Reactions that are affected by catalysts are termed catalytic
reactions. Autocatalytic reactions are reactions in which at least
one of the products is also a catalyst for the reaction. An
autocatalyst is a material that undergoes a reaction that produces
a product that is also a catalyst for that same reaction. Some
autocatalytic reactions have a relatively slow rate of reaction at
the initial stage but the reaction is accelerated as it proceeds as
more catalytic product is accumulated. Where a substance or a
combination of substances undergoes two or more simultaneous
reactions that yield different products, the distribution of
products could be influenced by the use of a catalyst that
selectively accelerates one reaction relative to the other(s).
[0044] 10. Radiation-Activated Catalyst (RAC): A radiation
activated catalyst (RAC) is a compound or group which produces at
least one catalyst when exposed to radiation. RACs include but are
not limited to radicals, acids, bases, ions, and metals.
[0045] 11. Enhancer: An enhancer is any material that amplifies a
radiation-initiated chemical signal so as to increase the effective
quantum yield of the radiation. Enhancers include but are not
limited to catalytic materials. The use of an enhancer in
radiation- assisted chemical processes is termed chemical
amplification. Chemical amplification has many benefits. Non
limiting examples of the benefits of chemical amplification include
the ability to decrease the time and intensity of iradiation
required to cause a desired chemical reaction. Chemical
amplification also improves the spatial resolution and contrast in
patterned arrays formed using this technique.
[0046] 12. Radiation sensitizer: A radiation sensitizer is any
material that shifts the wavelengths of radiation required to
initiate a desired reaction.
[0047] The present invention provides methods, devices, and
compositions for the formation of arrays of large numbers of
different polymer sequences. The methods and compositions provided
herein involve the conversion of radiation signals into chemical
products in an amplified manner that are particularly useful in
polymer synthesis. The invention also includes the arrays formed
using the methods and compositions disclosed herein. One aspect of
the invention includes methods, compositions, and devices for the
synthesis of an array of different polymers in selected and
predefined regions of a substrate. Another aspect of the invention
includes those arrays and various methods of using them.
[0048] Such arrays are used in, for example, nucleic acid analysis.
Polynucleotide or nucleic acid arrays are especially suitable for
checking the accuracy of previously elucidated sequences and for
detecting mutations and polymorphisms. Such arrays are also used in
screening studies to evaluate their interaction with receptors such
as antibodies and nucleic acids. For example, certain embodiments
of the invention provide for the screening of peptides to determine
which if any of a diverse set of peptides has strong binding
affinity with a receptor.
[0049] In some embodiments, the arrays formed by the present
invention are used in competitive assays or other well-known
techniques. to screen for compounds having certain activities. For
example, vast collections of synthetic or natural compounds are
immobilized on predefined regions of a substrate. The reaction of
the immobilized compounds (or compound) with various test
compositions such as the members of a chemical library or a
biological extract are tested by dispensing small aliquots of each
member of the library or extract to a different region. In one
embodiment, a large collection of human receptors is deposited on a
substrate, one in each region to form an array. A plant or animal
extract is then screened for binding to various receptors of the
array.
[0050] Nucleic acid sequences can also be immobilized in specific
locations or predefined regions of a substrate using the current
invention. In some embodiments, such immobilized nucleic acid
arrays are used in hybridization assays for gene expression
monitoring, nucleic acid amplifications, nucleic acid computation,
and nucleic acid analysis in general.
[0051] The present invention has certain features in common with
the radiation directed methods discussed in U.S. Pat. No.
5,143,854, previously incorporated herein by reference. The
radiation-directed methods discussed in that patent involve
activating predefined regions of the substrate and then contacting
the substrate with a preselected monomer solution. The predefined
regions can be activated with, for example, a light source shown
through a mask (much in the manner of photolithographic techniques
used in integrated circuit fabrication). Other regions of the
substrate remain inactive because they are blocked by the mask from
illumination. Thus, a light pattern defines which regions of the
substrate react with a given monomer. By repeatedly activating
different sets of predefined regions and providing different
monomer compositions thereto, a diverse array of polymers is
produced on or near the substrate.
[0052] In some preferred embodiments of the present invention, a
substrate with a linker having a protective group is provided with
a radiation-activated catalyst and an enhancer. The RAC is
selectively irradiated to generate a catalyst in preselected
regions. The catalyst and the enhancer assist the removal of the
protective groups on the linker. The linker, having a newly exposed
reactive group, is contacted with a monomer capable of reacting
with the linker. The monomer also has a protective group which can
be removed in a subsequent reaction step. In this step wise manner,
diverse arrays of polymers are synthesized at preselected regions
of a substrate.
Photochemical Ainplification for the Synthesis of Patterned
Arravs
[0053] One embodiment of the present invention includes a
photochemical amplification method wherein photon radiation signals
are converted into chemical signals in a manner that increases the
effective quantum yield of the photon in the desired reaction. The
use of photochemical amplification in methods of synthesizing
patterned arrays (PASPA) is particularly advantageous since the
time and the intensity of irradiation required to remove protective
groups is decreased relative to known ph6tochernical methods. The
methods of the present invention advantageously produce patterned
arrays having improved spatial resolution and contrast.
[0054] In general, radiation signals are detected by a catalyst
system including, for example, a photo activated catalyst (PAC).
The catalyst activates an enhancer, which increases the effective
quantum yield of the photons in subsequent chemical reactions. Such
subsequent reactions include the removal of protective groups in
the synthesis of patterned arrays.
[0055] In certain embodiments, a photo activated acid catalyst
(PAAC) is irradiated. The resulting acid produced from the PAAC
activates an enhancer to undergo an acid- catalyzed reaction to
itself produce an acid that removes acid labile protecting groups
from a linker molecule or synthesis intermediate. The combination
of PACs and enhancers converts and amplifies the photon signal
irradiated on the surface of the substrate. Because of the
amplification, the effective quantum yield of the radiation
directed at the surface of the substrate is much larger than one,
resulting in high sensitivity.
[0056] According to one embodiment of the present invention, linker
molecules having reactive functional groups protected by protecting
groups are provided on the surface of a substrate. A catalyst
system including a PAC and an enhancer are also provided on the
surface. A set of selected regions on the surface of the substrate
is exposed to radiation using well-known lithographic methods
discussed, for example, in Thompson, L. F.; Wilson, C. G.; and
Bowden, M. J., Introduction to Microlithography; American Chemical
Society, 1994, pp. 212-232, incorporated herein by reference in its
entirety for all purposes.
[0057] The PAC catalyst activated by the region-selective
irradiation discussed above acts to initiate a reaction of the
enhancer. The enhancer produces at least one product that removes
the protecting groups from the linker molecules in the first
selected regions. Preferably, the enhancer is capable of removing
protective groups in a catalytic manner. The substrate is then
washed or otherwise contacted with a first monomer that reacts with
exposed finctional groups on the linker molecules. Those bound
monomers are termed first-bound monomers.
[0058] A second set of selected regions is, thereafter, exposed to
radiation. The radiation-initiated reactions remove the protecting
groups on molecules in the second set of selected regions, i.e. the
linker molecules and the first-bound monomers. The substrate is
then contacted with a second monomer containing a removable
protective group for reaction with exposed functional groups. This
process is repeated to selectively apply monomers until polymers of
a desired length and desired chemical sequence are obtained.
Protective groups are then optionally removed and the sequence is,
thereafter, optionally capped. Side chain protective groups, if
present, are also optionally removed.
[0059] In one preferred embodiment, the monomer is a
2'-deoxynucleoside phosphoramidite containing an acid removable
protecting group at its 5' hydroxyl group. As stated previously, in
an alternate embodiment, the protecting group is present at the 3'
hydroxyl group if synthesis of the polynucleotide is from the 5' to
3' direction. The nucleoside phosphoroamidite is represented by the
following formula: 1
[0060] wherein the base is adenine, guanine. thymine, or cytosine,
R.sub.1 is a protecting group which makes the 5' hydroxyl group
unavailable for reaction and includes dimethoxytrityl,
tert-butyloxycarbonyl or any of the protecting groups previously
identified; R.sub.2 is cyanoethyl, methyl, t-butyl, trimethylsilyl
and the like and R.sub.3 and R.sub.4 are isopropyl, cyclohexone and
the like. Exocyclic amines present on the bases can also be
protected with acyl protecting groups such as benzoyl, isobutyryl,
phenoxyacetyl and the like. The linker molecule contains an acid-
or base- removable protecting group. Useful linker molecules are
well known to those skilled in the art and representative examples
include oligQ ethers such as hexaethylene glycol, oligomers of
nucleotides; esters, carbonates, amides and the like. Useful
protecting groups include those previously listed and others known
to those skilled in the art.
[0061] In another preferred embodiment, the monomer is an amino
acid containing an acid- or base- removable protecting group at its
amino or carboxy terminus and the linker molecule teminates in an
amino or carboxy acid group bearing an acid- or base removable
protecting group. Protecting groups include tert-butyloxycarbonyl,
9-fluorophenylmethoxycar- bonyl, and any of the protective groups
previously mentioned and others known to those skilled in the
art.
[0062] It is apparent to those skilled in the art that
photochemically amplified radiation- based activation is not
limited to photo activated enhancers or catalysts or to acid or
base production cascades. Various compounds or groups can produce
catalysts or enhancers in response to radiation exposure.
Non-limiting examples include photogeneration of radicals using
diphenylsulfide, benzoylperoxide, 2,2'-azobis(butyronitril- e),
benzoin and the like; cations such as triarylsulfonium salts,
diaryl iodonium salts and the like; and anions.
Radiation-Activated Catalvsts (RACs)
[0063] Useful RACs within the scope of the present invention
include those that are capable of directly or indirectly catalyzing
the removal of a protective group from a linker moleculer or
polymer chain and are chosen based upon their sensitivity to
radiation at certain wavelengths. Useful wavelengths include those
within the infrared, visible, ultraviolet and X-ray ranges. In one
embodiment, the RACs produce acids or bases upon exposure to
radiation of certain wavelengths for use in activating enhancers or
other catalysts in the chemically amplified removal of protecting
groups.
[0064] Preferably, the RAC chosen for a particular synthesis
strategy does not unduly interfere with subsequent or previous
synthesis steps in the formation of the polymer. Surprisingly, the
method of the present invention advantageously allows the use of
photocatalysts or products of photocatalysts that can be
detrimental in known methods of synthesizing polymer arrays. For
example, some PAACs produce strong acids that cause significant
depurination and thus could not be. used directly for
polynucleotide synthesis. However, the method of the present
invention allows the use of these types of PAACs that produce
strong acids since only small amounts of the PAACs are needed and
accordingly only a small amount of strong acid is produced. Another
important consideration is the radiation sensitivity of the various
compounds employed.
[0065] One preferred class of RACs include PAACs such as
naphthoquinone diazide sulfonic acids such as those disclosed by
Kosar, Light Sensitive Systems, John Wiley & Sons, 1965, pp.
343 to 352, incorporated herein by reference in its entirety for
all purposes. These PAACs form an acid in response to radiation of
different wavelengths ranging from visible to X-ray. Preferred
PAACs include the 2, 1, 4 diazonaphthoquinone sulfonic acid esters
and the 2, 1, 5-diazonaphthoquinone sulfonic acid esters. Other
useful PACs include the family of nitrobenzyl esters, and the
s-triazine derivatives. Suitable s-triazine acid generators are
disclosed, for example, in U.S. Pat. No. 4,189,323, incorporated
herein by reference. Non-ionic PAACs including halogenated
non-ionic, photoacid generating compounds such as, for example, 1,
1-bis [pchorophenyl] -2, 2, 2-trichloroethane (DDT); 1, 1- bis
[p-methoxyphenyl]-2, 2, 2-trichloroethane; 1, 2, 5, 6, 9,
10-hexabromocyclododecane; 1, 10-dibromodecane; 1,
1-bis[p-chlorophenyl] -2, 2-dichloroethane; 4, 4 dichloro-2-
(trichloromethyl) benzhydrol (Kelthane);
[0066] hexachlorodimethyl sulfone; 2-chloro-6-(trichloromethyl)
pyridine; o, o-diethyl -o- (3, 5, 6-trichloro-2-pyridyl)
phosphorothionate; 1, 2, 3, 4, 5, 6 hexachlorocyclohexane;
N(1,1-bis[p-chlorophenyl]-2, 2, 2 trichloroethyl)acetamide; tris
[2, 3-dibromopropyl] 20 isocyanurate; 2, 2-bis [p-chlorophenyl]-1,
1 dichloroethylene; tris [trichloromethyl] striazine; and their
isomers, analogs, homologs, and residual compounds are also
suitable for some applications. Suitable PAACs are also disclosed
in European Patent Application Nos. 0164248 and 0232972, both
incorporated by reference for all purposes. PAACs that are
particularly preferred for deep UV exposure include 1,1-bis
(p-chlorophenyl)-2,2,2-tri- chloroethane (DDT); 1,1 -bis
(p-methoxyphenol)-2,2,2,-trichloroethane;
1,1-bis(chlorophenyl)-2,2,2 trichloroethanol; tris
(1,2,3-methanesulfonyl) benzene; and tris (trichloromethyl)
triazine.
[0067] Onium salts are preferred for some embodiments as PACs. When
synth esizing polynucleotide arrays, a radiation sensitizer is
employed to shift the radiation sensitivity of the onium salts away
from wavelengths damaging to the starting materials. Suitable
radiation sensitizers for use with onium salts or other RACs are
well known in the art and include benzophenone, thiophene,
fluorene, anthraquinone, quinoline, phenanthracene, flavone,
micheler's ketone, chrysene, anthracene, eosin and the like. It is
to be understood that additional sensitizers are well known to
those skilled in the art and are readily identifiable based upon
the present disclosure.
[0068] Examples of onium salts useful in the present invention
include those having halogen (i.e. I, Br, Cl etc.) complex anions
of divalent to heptavalent metals or non- metals, for example, Sb,
Sn, Fe, Bi, Al, Ga, In, Ti, Zr, Sc, Cl, Cr, Hf, and Cu as well as
B, P, and As. Examples of suable onium salts are diaryl-diazonium
salts and onium salts of group VI and VII of the Periodic Table,
for example, halonium salts, quaternary ammonium, phosphonium and
arsonium salts, aromatic sulfonium salts and sulfoxonium salts or
seleonium salts. Examples of suitable preferred onium salts can be
found in U.S. Pat. Nos. 4,442,197; 4,603,101; and 4,624,912, all
incorporated herein by reference. Sulfonium analogs can be prepared
using Group VI elements such as O, S, Se, Te. Onium analogs can be
prepared by using Group VII elements such as I, Br, and Cl. For a
review on onium salts as photoacid generators, see Pappas, J.
Imaging Technology (1985), 11,146, incorporated herein by
reference. Another group of suitable acid generators is the family
of sulfonated esters including sulfonyloxy ketones. Suitable
sulfonated esters have been reported in J. of Photopolymer Science
and Technology (991), 4, 3, 337-340, incorporated herein by
reference, including benzoin tosylate, t-butylphenyl
alpha-(p-toluenesulfonyloxy)-acetate, and t-butyl
alpha-(p-toluenesulfonyloxy)-acetate. Both ionic, including
di-tert-butylphenyl iodonium triflate (TBI-T),
di-tertbutylphenliodonium camphorsulfonate (TBI-CAM) and
di-tert-butylphenyl iodonium dichloracetate (TBI-DCA), and
nonionic, including napthalimidotriftete and phthalimidotosylate or
mixture of those photoacids are useful in the present invention.
Usefil PACs within the scope of the present invention include 2
[0069] Alkyl refers to saturated or unsaturated, straight chain or
branched, carbon atoms having from 1 to 50 carbons, preferrably
from 1 to 30 carbon atoms and more preferrably from 1 to 10 carbon
atoms. Aromatic groups include straight chain or cyclic aromatics,
substituted or unsubstituted having from 1 to 50 carbons,
preferrably from 1 to 30 carbon atoms and more preferrably from 1
to 10 carbon atoms. One preferred PAC for polynucleotide synthesis
is the o-nitrobenzyl ester of toluenesulfonic acid, such as the
2-nitro-3,4-dimethoxybenzyl tosylate having the structure: 3
[0070] When irradiated, the ester produces catalytic amounts of
p-toluenesulfonic acid. Other PACs useful in the practice of the
present invention include the following: 4
[0071] wherein R is sulfonate, tosylate, mesolate, PF.sub.6.sup.-
or BF.sub.4.sup.- with or without the presence of a sensitizer of
the formula: 5
Catalvtic Enhancers
[0072] In some preferred embodiments of polynucleotide synthesis,
masked acids including esters, anhydrides, and nitrites are used as
autocatalysts. In one preferred specific embodiment, the RAC is a
PAAC which generates an acid upon exposure to radiation of suitable
wavelength. The catalytic enhancer is an ester labile to
acid-catalyzed thermolytic cleavage by the acid produced by the
PAAC. The enhancer, itself, produces an acid which is used to
removed an acid labile protective group. Post-exposure baking of
the substrate is required, in some embodiments, because the acid
autocatalysis occurs only when heated. A preferred catalytic masked
acid for polynucleotide synthesis is the ester of the
pentafluorobenzoic acid such as the 1,4-cyclohex-2-enediylbi-
s(pentafluorobenzoate) illustrated below: 6
[0073] The acid catalyzes the cleavage of the ester to produce
pentafluorobenzoic acid, benzene, and regenerates the catalytic
acid. The acid produced effectively removes acid- labile groups,
yet does not cause the degradation or depurination of
polynucleotides. 15 Other useful catalytic enhancers within the
scope of the present invention include those identified in
Ichimura, Mol. Cryst. Liq. Cryst. (1996) vol. 280 pp. 307-312 and
Ichimura, Chem. Lett. (1995) pp. 551-552 each of which are hereby
incorporated by reference in their entireties and those of the
following general formulas where R is any suitable group: 7
[0074] The selection of temperature is also dependent upon the
subsequent synthesis steps. A too high temperature may damage
synthesis intermediates. A too low temperature may not be
sufficient to activate thernolysis. A suitable range of
temperatures to induce acidcatalyzed thermolysis of 1,4-cyclohex-2-
enediylbis (pentafluorobenzoate) is 70-100.degree. C.
[0075] Using the guidance provided herein, suitable reaction
conditions (including temperature) can be determined for a variety
of embodiments by one having skill in the art. For example, the
chemical and thermal stability of various compounds is known or can
be determined readily. A series of experiments showing the
efficiency of synthesis as a function of temperature, irradiation
intensity, or exposure time is within the skill of those in the
art.
[0076] If an acid autocatalysis system is used, the protecting
group could, but not necessarily, be an acid removable protecting
group, and the. monomer could be a nucleotide containing an acid
removable protecting group at its C-5' or C-3' hydroxyl group.
Radiation Sensitizers and Substrates
[0077] The selection of radiation sources is based upon the
sensitivity spectrum of the RAC. Potential damage to synthesis
substrates, intermediates, or products is also considered. In some
preferred embodiments, the radiation could be ultraviolet (UV),
infrared (IR), or visible light. In a specific embodiment, the
radiation source is a light beam with a wavelength in the range of
from 190-500 nm, preferably from 250-450 nm, more preferably from
365-400 nm. Specific radiation wavelengths include 193 nm, 254 nm,
313 nm, 340 nm, 365 nm, 396 nm, 413 nm, 436 nm, and 500 nm.
Suitable light sources include high presure mercury arc lamps and
are readily commercially available from Oriel, OAI, Cannon, A,B
Manufacturing. The sensitivity spectrum of the RAC can be altered
by providing radiation sensitizers. The energy of the sensitizer
must be matched to the PAC and include
2-ethyl-9,10-dimethoxy-anthracene, perylene, phenothiazine,
xanthone and the like. Many radiation sensitizers are known to
those skilled in the art and include those previously mentioned. It
is to be understood that one of ordinary skill in the art will be
able to readily identify additional radiation sensitizers based
upon the present disclosure.
[0078] In preferred embodiments, the substrate is conventional
glass, pyrex, quartz, any one of a variety of polymeric materials,
or the like. Of course, the substrate may be made from any one of a
variety of materials such as silicon, polystyrene, polycarbonate,
or the like. In operation, the surface of the substrate is
appropriately treated by cleaning with, for example, organic
solvents, methylene chloride, DMF, ethyl alcohol, or the like.
Optionally, the substrate may be provided with appropriate linker
molecules on the surface thereof. The linker molecules may be, for
example, aryl acetylene, ethylene glycol oligomers containing from
2-10 monomers or more, diamines, diacids, amino acids, or
combinations thereof. Thereafter, the surface is provided with
protected surface active groups such as tertbutyloxycarbonyl (TBOC)
or fluorenylmethoxycarbonyl (FMOC) protected amino acids. Such
techniques are well known to those of skill in the art.
[0079] In light-directed methods, the light shown through the mask
is diffracted to varying degrees around the edges of the dark
regions of the mask. Thus, some undesired removal of photosensitive
protecting groups at the edges of "dark" regions occurs. This
effect is exacerbated by the repeated mask translations and
subsequent exposures, ultimately leading to inhomogeneous synthesis
sites at the edges of the predefined regions. Since in one
embodiment of the present invention, the RAC catalyzes cleavage of
the enhancer to produce an acid used to remove an acid-labile
protective group, the effective quantum yield of the radiation is
much larger than one, resulting in a high sensitivity.
Additionally, the sensitivity of the process can be tuned by
controlling the concentrations of the RAC or photocatalyst and the
enhancer on the polymer matrix. Higher concentration results in a
higher sensitivity. Other advantages will be apparent to those
skilled in the art.
Application of Chemicsl Amplification Techniques
[0080] The techniques of the present invention are useful in many
fields, particularly in nucleic acid analysis, gene expression
monitoring, amplification of nucleic acids, -drug discovery,
fabrication of miniature electronic, mechanic or other devices, and
DNA based computation.
A. Nucleic Acid Analysis
[0081] The present invention provides an efficient means for
fabricating high density polynucleotide arrays, which have been
successfully employed in a variety of nucleic acid analysis
applications. Polynucleotide arrays are usefull in a variety of
applications including but not limited to detecting specific
mutations or polymorphisms and checking the accuracy and resolving
ambiguity of previously elucidated sequences.
B. Gene Expression Monitoring
[0082] Polynucleotide arrays can be used for simultaneously
monitoring the expression of multiple genes and eventually all
genes as transcript sequences become available. Gene expression
monitoring at the MRNA level can be carried out by extracting MRNA
or total RNA from tissue or cell samples; fragmenting and labeling
the RNA samples; hybridizing the fragmented RNA samples to
polynucleotide arrays and detecting the hybridization pattern to
determine quantitatively the level of specific mRNAs. Various
levels of transcript processing, such as RNA splicing, can also be
monitored using polynucleotide arrays. Specific embodiments for
gene expression monitoring are disclosed in U.S. patent application
Ser. No. 08/529,115, filed Sep. 15, 1995, and PCT Application
No.
[0083] PCT/US96/14839, filed Sep. 13, 1996, incorporated by
reference herein in their entirety for all purposes.
[0084] The present invention is also used to immobilize nucleic
acid sequences on a substrate. Immobilized nucleic acid sequences
are used for various hybridization assays. Hybridization of such
immobilized nucleic acids with niRNA samples (or immobilized MRNA
samples) is detected to monitor gene expression in some
embodiments.
C. Drug Discovery
[0085] The significantly enhanced resolution made possible by the
present invention permits the synthesis of more polymers on a given
surface area. Therefore, the invention can be used for building
chemical library and screening for biological activities of a large
number of compounds in drug discovery using combinatorial
chemistry.
D. DNA Computation
[0086] Polynucleotides have been used in DNA based computation.
Spatially defined polynucleotide arrays are useful for certain DNA
computation tasks. DNA computation employs the ligation, enzymatic
cleavage and hybridization of polynucleotides. In some embodiments,
polynucleotide arrays are used for accessing the result of DNA
computation by detecting the presence of specific polynucleotides
by specific hybridization. In some other embodiments, DNA
computation is accomplished by manipulating polynucleotide arrays
fabricated with chemical amplification.
Alternative Embodiments
[0087] According to other embodiments, spatially defined polymer
synthesis will be performed by depositing a photoresist such as
those used extensively in the semiconductor industry, more fully
discussed in Ghandi, "VLSI Fabrication Principles," Wiley (1983)
Chapter 10, incorporated herein by reference in its entirety for
all purposes. According to these embodiments, a resist is
deposited, selectively exposed, and etched, leaving a portion of
the substrate exposed for coupling. These steps of depositing
resist, selectively removing resist and monomer coupling are
repeated to form polymers of desired sequence at desired
locations.
[0088] In some specific embodiments, a positive-tone resist
comprised of diazonapthoquinone-novolac (DNQ/N) is incorporated in
a cresol-formaldehyde polymer matrix. This resist and its variants
are used routinely in the microelectronics industry for submicron
resolution lithography, as more filly discussed in Reiser,
"Photoreactive Polymers: the Science and Technology of Resists",
Wiley (1989), incorporated herein by reference in its entirety for
all purposes. High contrast detritylation at a resolution of <4
microns has been demonstrated in simple contact printing
experiments with this resist. Unfortunately, the alkaline
conditions needed to develop the DNQ/N resists (aqueous
[OH.sup.-]>0.1 M ) complicates its direct use in a multi-step
polymer synthesis, such as the polynucleotide array fabrication
process, because of the hydrolysis of akali-labile nucleobase
protecting groups that are used to prevent side reactions during
synthesis with standard phosphoramidite monomers using
dimethoxytrityl (DMT) as a protecting group. A preferred embodiment
uses alkali-resistant acid labile nucleobase protecting groups,
such as monomethoxytrityl (MMT), and akali-labile 5' hydroxyl group
to avoid this difficulty. MMT is completely resistant to the
aqueous alkali developer, and readily removed with dilute acid
post-synthesis. Alkali labile protection is used for the 5'
hydroxyl group so that it will be susceptible to cleavage in the
same alkaline solutions used for resist development, so that the
two processes occur simultaneously. One preferred embodiment uses
benzyol group as alkali-labile protection group because the benzyol
group is sufficiently selective for the 5' hydroxyl group in
preparing the monomer. More sterically hindered acyl protecting
moieties, such as isobutyrl or pivaloyl, can also be used to
enhance selectivity in monomer preparation.
EXAMPLE I
Removal of Protecting Groups bv Acid Amplification
[0089] Efficient removal of protective groups as taught by the
present invention is demonstrated in the following experiment.
[0090] A system using an ester of toluenesulfonic acid as a PAAC
and an autocatalytic ester of pentafluorobenzoic acid
(1,4-cyclohex-2-enediylbis- -(pentafluorobenzoate)) as an enhancer
was employed. An experiment was conducted to determine time and
intensity required to achieve efficient deprotection.
[0091] The synthesis of
1,4-cyclohex-2-enediylbis-(pentafluorobenzoate) and
2-nitro-3,4-dimethoxybenzyl tosylate were carried out according to
Houlihan et al., Chemistry of Mat. 3:462-471, 1991. The yields were
54% and 62%, respectively.
[0092] Solutions containing poly (methyl methacrylate) (PMMA,
average molecular weight of 15,000 dalton) (14.0 wt %),
1,4-cyclohex-2-enediylbis- -(pentafluorobenzoate) (7.0 wt %), and
2-nitro-3,4-dimethoxybenzyl tosylate (0.5, 0.8, 1.2, 1.6, or 2.3 wt
%) in cyclohexanone were spin coated as ca. 1 .mu.m thick films
onto glass substrates bearing 5'-dimethoxytrityl (DMT) protected
foundation molecules. In this case the surface of the glass
substrate was reacted with DMT-hexaethyloxy-glycol-C-
E-phosphoramidite.
[0093] The resulting films were dried (prebaked) at 85.degree. C.
for 1 min. and then exposed with increasing doses of light (365400
nm) from a collimated source (Oriel, Straford, Conn.) through a
chrome on quartz mask in contact with the substrate. After
exposure, the films were postbaked at 85.degree. C. for 1 min. and
stripped by rinsing with acetone (2 min.).
[0094] The free hydroxyl group was then reacted with a solution of
Fluoreprime(c) fluorescein amidite in acetonitrile, using a
modified Applied Biosystems Inc. (ABI) DNA synthesizer. The
fluorescein amidite was coupled with the free hydroxyl groups, but
not the DMT protected hydroxyl groups. The fluorescent output of
the surface of the substrate was measured using a scanning
fluorescence microscope. The coupling efficiency as measured by
fluorescence intensity was used as a measurement of deprotection
efficiency.
[0095] Another glass slide was deprotected with
ethanolamine-ethanol (1:1 v/v, 30 min.) as complete deprotection
control, the fluorescent output of the surface of the substrate was
also measured using a scanning fluorescence microscope. The
efficiency of deprotection was expressed in percentage of
deprotection using the control slide as 100% deprotected.
[0096] Complete coupling occurred at low doses, ranging from 660
mJ/cm.sup.2 to less than 33 mJ/cm.sup.2. As shown in FIG. 1 the
required exposure time was dependent on the amount of PAC in the
substrate. When a formulation containing 0.02 g of PAC and 0.09 g
of ester (enhancer) per 1 g of PMMA stock solution were used, the
required exposure dose was 0.1 J/cm.sup.2 corresponding to an
exposure time of 3 seconds.
EXAMPLE II
High Resolution Synthesis of Polynucleotide and Hybridization with
an Polynucleotide Probe
[0097] Another important consideration for applying the techniques
disclosed herein is whether the deprotection procedure interferes
with the subsequent synthesis and functioning of the desired
polymer arrays. The following experiment shows that functional
polynucleotide arrays were synthesized by the method of the current
invention.
[0098] A combination of a PAC and an enhancer in the form of a
masked acid was used to synthesize a standard checkerboard pattern
of an polynucleotide on a glass slide. The resulting glass slide
containing polynucleotide arrays was hybridized to a complementary
polynucleotide probe to test resolution and integrity of the
arrays.
[0099] Solutions containing poly (methyl methacrylate) (PMMA,
average molecular weight 15,000) (14.0 wt %), 1,4
cyclohex-2-enediyl-bis(pentaflu- orobenzoate) (7.0 wt %), and
2-nitro-3,diethoxybenzyl tosylate (1, 2 wt %) in cyclohexanone were
spin coated as approximately 1 .mu.m thick films onto glass
substrates bearing 5' dimethoxytrityl (DMT) protected foundation
molecules.
[0100] The resulting films were dried (prebaking) at 85.degree. C.
for 1 min. and then exposed to light (0.2 J/cm2, 365-400 nm) from a
collimated source (oriel) through a chrome on quartz mask in
contact with the substrate.
[0101] After exposure, the films were postbaked at 85.degree. C.
for 1 min. and stripped by rinsing with acetone, ethanol, and
acetone again (each rinse 2 min.). The free hydroxyl group was then
reacted with a DMT protected nucleotide phosphoramidite in
acetonitrile, using a modified Applied Biosystems Inc. (ABI) DNA
synthesizer. This coat/expose/strip process was repeatedly used to
build an pblynucleotide of the sequence 5'-CATTTACAGC-3'.
[0102] The resulting polynucleotide was deprotected with
ethanolamine--ethanol (1:1 v/v, 18 h) and then hybridized to a
fluorescent labeled target containing the complementary sequence
5'-GCTGTAAATG-3'.
[0103] The high fluorescence intensity achieved, as observed with a
scanning fluorescence microscope is a measurement of the combined
efficiency of polynucleotide synthesis and hybridization. Yield of
the polymer prepared using the method of invention was comparable
to that of the standard MeNPoc VLSIPS method. Data showed a
checkerboard pattern with a feature size of 10 .mu.m. The high
intensity of fluorescence also indicated a good fidelity of the
synthesized polynucleotides, as demonstrated by the efficient
hybridization of complementary probes to the arrays.
[0104] As shown in FIG. 2, resolution showing 5 .mu.m and 2.mu.m
lines were printed with the process of the present invention. A
poly(ethylene glycol) linker molecule containing a DMT protected
hydroxyl group was covalently bound to a substrate. The surface of
the substrate was then coated with polymer containing a PAC and an
enhancer, irradiated and heated as described above. The polymer
film was then removed followed by reaction of the free hydroxyl
groups with a biotin phosphoramidite. The image of FIG. 2 was
obtained by incubating the substrate with a collodial gold label
conjugated to strepavidin and detected using a Zeiss microscope
with a CCD camera.
[0105] As can be seen in FIG. 3, afluoroscent image of a probe
array was made according to the teachings of the present invention.
Probes vary from 10 to 20 bases in length and were prepared by
repeating the synthesis steps, i.e. coating the substrate with e
polymer containing a catalyst system, exposing the substrate to
radiation to initiate a catalytic reaction to remove protective
groups from reactive functional groups, stripping away the polymer
layer and then adding a monomer to react with the free reactive
group, on the order of thirty times. Feature sizes in FIG. 3 vary
from 100 to 20 microns.
EXAMPLE III
Lithographic Evaluation
[0106] As shown in FIG. 4, the high contrast observed in photo
processes reflects the nonlinearity of the response as a function
of the irradiation dose. In traditional photo resists, this
nonlinearity stems from the solubility behavior of the polymer.
Although the catalytic photo process described in this application
does not involve a development step, nonlinear behavior was
observed. This probably results from a titration effect: a quantity
of acid must accumulate before the DMT group is removed.
[0107] The lithographic behavior of the process was evaluated by
spin coating a 0.5 .mu.m thick film of poly (methyl methacrylate)
(PMMA) containing the nitrobenzyl ester PAC (0.5 wt %) and the
enhancer (8 wt %) having the following structures: 8
[0108] onto a glass substrate bearing covalently bound
polynucleotides whose terminal 5' hydroxyl groups were DMT
protected. The coated substrate was prebaked at 85.degree. C. for 2
min, irradiated with varying doses at 365 nm, and postbaked at
85.degree. C. for 2 min. The polymer coating was then removed with
an acetone wash and the surface treated with a fluorescent coupling
reagent. As shown by the sensitivity curve in FIG. 4, the
lithographic process generated a direct image with a sensitivity of
600 mJ/cm.sup.2 at 365 nm and a contrast of 3.0. By increasing the
concentration of the PAC, the sensitivity of the system can be
significantly improved. However, this may result in a decrease in
the contrast. The contrast was calculated using the contrast
equation as defined in Reiser, Arnost, Photoreactive Polymers: the
Science and Technology of Resists, pp. 226-228 (1989), incorporated
in its entirety herein by reference for all purposes.
[0109] In addition to tuning the sensitivity and the contrast by
altering the concentration of the PAC and the enhancer, it is also
possible to affect these two properties by adding an amine to the
formulation to improve environmental stability and resolution of
the resist. Photokinetic response was measured as a function of the
concentration of trioctylamine. As shown in FIG. 5, the dose
required to reach complete detritylation increased with increasing
concentrations of trioctylamine (increasing from 130, 240, 400, and
650 nJ/cm.sup.2 for added base of 0.0, 0.08, 0.24 and 0.56 wt%
respectively.
EXAMPLE IV
Coupling Efficiency in Polynucleotide Array Fabrication
[0110] We have used the chemically amplified photo process in
conjunction with nucleoside phosphoramidite coupling chemistry to
fabricate polynucleotides with mixed and unmixed sequences. By
employing a cleavable linker and a fluorescent label (FL*label) at
the 3' end of the.sequence, the polynucleotide can be removed from
the glass substrate and analyzed by HPLC. A typical probe sequence
has the following construction (where B represents a nucleotide
base):
SUBSTRATE--Linker--FL*Iabel--3'-BBBBBB5'--OH
[0111] After synthesis, the sequence was simultaneously cleaved
from the surface and deprotected by soaking in
ethanol/ethylenediamine (1:1 v/v) for 15 h at 25.degree. C. The
sequence was then directly analyzed using HPLC with an anion
exchange column and a fluorescence detector. To compare the
chemically amplified photo process to traditional polynucleotide
chemistry, each probe sequence was synthesized twice: once using
the chemically amplified photo process for the deprotection step
and once using the traditional deprotecting reagent, 3%
trichloroacetic acid in dichloromethane (TCA/DCM).
[0112] FIGS. 6 and 7 show the chromatograms of a labeled T.sub.6
polymer synthesized with the chemically amplified photo process and
TCA/DCM, respectively. The predominant peak at 21.7 min corresponds
to the full length polymer, while the small peaks eluting earlier
represent the shorter truncated polymers. The integration data
showed that the yields for the full length polymer are 63% using
the photo process and 80% using TCA/DCM, corresponding to a step
wise efficiency of 93% and 96%, respectively. Further analyses of
other sequences indicated that the step wise coupling efficiency
for the photo process ranges from 90-96%, approaching the
efficiencies achieved using TCA/DCM as the deprotecting
reagent.
[0113] The present invention provides methods, compositions, and
apparatus involving synthesis of polymers on substrates. It is to
be understood that the embodiments of the present invention which
have been described are merely illustrative of some applications of
the principles of the invention. Numerous modifications may be made
by those skilled in the art without departing from the true spirit
and scope of the invention. By way of example, the invention has
been described primarily with reference to the use of PAACs,
catalytic compounds labile to acid-cleavage, such as acid
thermolytic cleavage, and acid removable protective groups, but it
will be readily recognized by those of skill in the art that
photobases, base labile protective groups, and other systems
involving chemical amplification can be used. It should be apparent
to those of skill in the art that protecting groups can be the
photocatalyst generator and can undergo autocatalytic reactions. It
should also be readily recognized by those of skin in the art that
sources of radiation other than light could be used. For example,
in some embodiments, it may be desirable to use initial compounds
for generating catalysts or acids which are sensitive to electron
beam irradiation, x-ray irradiation, in combination with electron
beam lithography, or x-ray lithography techniques. The scope of the
invention should, therefore, be determined not with reference to
the above description, but should instead be determined with
reference to the appended claims, along with the full scope of
equivalents to which such claims are entitled.
[0114] All of the references cited in this application are
incorporated herein by reference in their entirety for all purposes
even if not listed as such anywhere else in this application.
Sequence CWU 1
1
2 1 10 DNA Artificial Sequence polynucleotide synthesized on a
glass slide 1 catttacagc 10 2 10 DNA Artificial Sequence
polynucleotide probe 2 gctgtaaatg 10
* * * * *