U.S. patent application number 13/563932 was filed with the patent office on 2012-11-22 for photoacid generators for the synthesis of oligo-dna in a polymer matrix.
This patent application is currently assigned to AFFYMETRIX, INC.. Invention is credited to Andrea Cuppoletti, Glenn H. McGall.
Application Number | 20120296107 13/563932 |
Document ID | / |
Family ID | 38685960 |
Filed Date | 2012-11-22 |
United States Patent
Application |
20120296107 |
Kind Code |
A1 |
McGall; Glenn H. ; et
al. |
November 22, 2012 |
Photoacid Generators for the Synthesis of Oligo-DNA in a Polymer
Matrix
Abstract
Compounds represented by the following structural formulas can
be used as photoacid generators: ##STR00001## Such compounds are
useful, for example, in fabricating arrays of polymers.
Inventors: |
McGall; Glenn H.; (Palo
Alto, CA) ; Cuppoletti; Andrea; (Livermore,
CA) |
Assignee: |
AFFYMETRIX, INC.
Santa Clara
CA
|
Family ID: |
38685960 |
Appl. No.: |
13/563932 |
Filed: |
August 1, 2012 |
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Application
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13103621 |
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8258199 |
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13563932 |
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12882064 |
Sep 14, 2010 |
7964654 |
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12263623 |
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12882064 |
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Current U.S.
Class: |
558/32 ; 558/37;
560/110; 560/129; 560/19; 560/81 |
Current CPC
Class: |
Y10S 430/12 20130101;
C07C 219/32 20130101; C07C 205/42 20130101; Y10S 430/106 20130101;
C07C 309/64 20130101; C07C 205/59 20130101; Y10S 430/127 20130101;
Y10S 430/114 20130101 |
Class at
Publication: |
558/32 ; 558/37;
560/19; 560/81; 560/110; 560/129 |
International
Class: |
C07C 305/18 20060101
C07C305/18; C07C 69/00 20060101 C07C069/00; C07C 69/78 20060101
C07C069/78; C07C 229/42 20060101 C07C229/42; C07C 69/76 20060101
C07C069/76 |
Claims
1. A compound represented by Structural Formula (I) ##STR00020## or
a salt thereof, wherein: R.sub.1 is --H, --COOR, a substituted
alkyl group, or an alkenyl or aryl group; R.sub.2 is a sulfonate,
substituted acetate or benzoate group; R.sub.3 is --NRR', --COOR,
an alkyl or alkenyl group or a substituted alkoxy or aryl group;
and R and R' are independently --H or an alkyl, alkenyl or aryl
group.
2-21. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Methods of synthesizing polymer sequences such as nucleotide
and peptide sequences are known. Synthesis of individual
oligonucleotides is described in Oligonucleotide Synthesis: A
Practical Approach, Gait, ed., IRL Press, Oxford (1984),
incorporated herein by reference in its entirety for all purposes.
Similarly, the "Merrifield" solid phase peptide synthesis has been
in common use for many years and is discussed in Merrifield, J. Am.
Chem. Soc. (1963) 85:2149-2154, incorporated herein by reference
for all purposes.
[0002] The in situ fabrication of a plurality of polymers or
"catamers," including peptides and oligonucleotides, on a single
solid support (a plurality of pins attached to a support, each pin
having a unique polymer) to subsequently be used for analytical
purposes was described in WO86/06487, published Nov. 6, 1986,
entitled "Method for determining mimotopes," by Hendrik M. Geysen,
incorporated herein by reference for all purposes.
[0003] The combination of solid phase synthetic chemistry and
photolithographic technology from the semiconductor industry
allowed for the first time for the fabrication of high density
arrays of polymers. See Fodor, S. P. A., Read, L. J., Pirrung, M.
C., Stryer, L., Lu, A. T. and Solas, D., Light-Directed, Spatially
Addressable Parallel Chemical Synthesis, (1991) Science 251,
767-773, incorporated herein by reference for all purposes.
[0004] These techniques disclosed in Fodor et al. provide for total
independent access to sites on the substrate at each synthetic
step, allowing for massive parallel synthesis of the desired
polymer (e.g., peptide, oligonucleotide) on the array. In turn,
combinatorial masking strategies allow for the fabrication of a
large number of chemical entities in a relatively small number of
steps. In addition, light-directed synthesis allows for a high
degree of miniaturization because the density of synthesis sites is
bounded only by physical limitations on spatial addressability,
here the diffraction of light.
[0005] These photolithograph techniques have been employed
commercially to produce high density oligonucleotide arrays which
may be used, for example, to simultaneously monitor the expression
of the entire set of human genes or to finely map the genome of a
human subject. This technology has in turn led to diagnostic
applications of the high density arrays for human disease. See
www.affymetrix.com.
SUMMARY OF THE INVENTION
[0006] The present invention provides compounds represented by
Structural Formula (I):
##STR00002##
or a salt thereof, wherein:
[0007] R.sub.1 is --H, --COOR, a substituted alkyl group, or an
alkenyl or aryl group;
[0008] R.sub.2 is a sulfonate, substituted acetate or benzoate
group;
[0009] R.sub.3 is --NRR', --COOR, an alkyl or alkenyl group or a
substituted alkoxy or aryl group; and
[0010] R and R' are independently --H or an alkyl, alkenyl or aryl
group.
[0011] The invention also provides compounds represented by
Structural Formula (II):
##STR00003##
or a salt thereof, wherein:
[0012] R.sub.4 and R.sub.5 are independently --H, --COOR, a
substituted alkyl group or an alkenyl or aryl group;
[0013] R.sub.6 is a sulfonate, substituted acetate or benzoate
group;
[0014] R.sub.7 is --NRR', --COOR, an alkyl or alkenyl group or a
substituted alkoxy or aryl group; and
[0015] R and R' are independently --H or an alkyl, alkenyl or aryl
group.
[0016] The invention additionally provides compounds represented by
Structural Formula (III):
##STR00004##
or a salt thereof, wherein:
[0017] R.sub.8 is a sulfonate, substituted acetate or benzoate
group;
[0018] R.sub.9 is --H, --COOR, a substituted alkyl group or an
alkenyl or aryl group;
[0019] R.sub.10 is --NRR', --COOR, an alkyl or alkenyl group or a
substituted alkoxy or aryl group; and
[0020] R and R' are independently --H or an alkyl, alkenyl or aryl
group.
[0021] In one aspect, the present invention discloses methods of
generating acid by exposing a compound represented by one of
Structural Formulae (I)-(III) to light of an appropriate
wavelength.
[0022] In another aspect, the present invention discloses methods
for fabricating arrays of polymers. One disclosed method includes
the steps of: providing a solid substrate comprising a reactive
group protected by an acid labile protective group; coating said
solid substrate with a film, said film comprising a photoacid
generator represented by Structural Formula (I), (II) or (III) and
optionally an acid scavenger; activating said photo acid generator
in selected regions of said substrate by selective application of
light having a predetermined wavelength to provide an acid;
exposing said reactive group having said protective group to said
acid in the presence of said scavenger, when present, such that
said protective group is removed to provide an exposed reactive
group; reacting said exposed reactive group with a monomer, wherein
the monomer is coupled to said exposed reactive group; and
repeating the steps of coating, activating, exposing and reacting
to produce the array of polymers.
[0023] In one aspect of the invention, the monomers are nucleotides
and the polymer is an oligonucleotide. In another aspect of the
invention, the monomers are amino acids and the polymer is a
polypeptide.
[0024] Another method of the invention includes the steps of:
providing a substrate comprising a hydroxyl group protected by an
acid labile protective group; coating said substrate with a film,
said film comprising a photo acid generator represented by one of
Structural Formulae (I)-(III) and optionally an acid scavenger;
activating said photo acid generator in selected regions of said
substrate by selective application of light having a predetermined
wavelength to provide an acid; exposing said hydroxyl group
protected by said protective group to said acid in the presence of
said scavenger, when present, such that said protective group is
removed to produce a deprotected hydroxyl group; reacting said
deprotected hydroxyl group with a nucleotide monomer, wherein the
nucleotide monomer is coupled to said deprotected hydroxyl group;
and repeating the steps of coating, activating, exposing and
reacting to produce the array of oligonucleotides.
[0025] In an aspect of the invention, the method also includes the
steps of: stripping the film from the substrate with an appropriate
solvent after removal of the protective group to provide a
partially completed substrate comprising an exposed reactive
hydroxyl group; reacting said hydroxyl group with a deoxynucleotide
with a reactive group at its 5' or 3' hydroxyl group and an acid
labile protective group at the other 5' or 3' hydroxyl group; and
repeating the steps of coating, activating, exposing, stripping,
and reacting to provide the array of oligonucleotides. The above
methods can also be applied to fabricate arrays of other polymers
such as carbohydrates and nucleic acid peptides.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0026] The following definitions are applicable to the terms set
forth below unless otherwise indicated. Halogen or "halo" is
fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, aralkyl, alkylaryl,
and the like denote both straight and branched groups; but
reference to an individual radical such as "propyl" embraces only
the straight chain alkyl group, a branched chain isomer such as
"isopropyl" being specifically referred to. Aryl includes a phenyl
radical or an ortho-fused bicyclic carbocyclic radical having about
nine to ten ring atoms in which at least one ring is aromatic.
Heteroaryl encompasses a radical attached via a ring carbon of a
monocyclic aromatic ring containing five or six ring atoms
consisting of carbon and one to four heteroatoms each selected from
the group consisting of non-peroxide oxygen, sulfur, and N(X)
wherein X is absent or is H, O, (C.sub.1-C.sub.4)alkyl, phenyl or
benzyl, as well as a radical of an ortho-fused bicyclic heterocycle
of about eight to ten ring atoms derived therefrom, particularly a
benz-derivative or one derived by fusing a propylene, trimethylene,
or tetramethylene diradical thereto.
[0027] "Alkyl" refers to a straight chain, branched or cyclic
saturated chemical group containing only carbon and hydrogen. Alkyl
groups include, without limitation, ethyl, propyl, pentyl,
cyclopentyl and 2-methylbutyl. Alkyl groups are unsubstituted or
substituted with 1 or more substituents (e.g., halogen, alkoxy,
amino).
[0028] "Alkenyl" refers to a straight chain, branched or cyclic
chemical group containing only carbon and hydrogen and having at
least one double bond. Alkylene groups include, without limitation,
ethenyl, propenyl, butenyl, pentenyl, and 2-methylbutenyl. Alkenyl
groups are unsubstituted or substituted with 1 or more substituents
(e.g., halogen, alkoxy, amino).
[0029] "Alkynyl" refers to a straight chain, branched or cyclic
chemical group containing only carbon and hydrogen and having at
least one triple bond. Alkynyl groups include, without limitation,
ethylyne, propylene, butylyne, pentylyne and hexylyne. Alkynyl
groups are unsubstituted or substituted with 1 or more substituents
(e.g., halogen, alkoxy, amino).
[0030] "Aryl" refers to a monovalent, unsaturated aromatic
carbocyclic group. Aryl groups include, without limitation, phenyl,
naphthyl, anthryl and biphenyl. Aryl groups are unsubstituted or
substituted with 1 or more substituents (e.g. halogen, alkoxy,
amino). "Arylene" refers to a divalent aryl group.
[0031] A "photoacid generator" is a compound or substance which
produces acid (H.sup.+ or H.sub.3O.sup.+) upon exposure to light
having a predetermined wavelength.
[0032] An "acid scavenger" is a compound or substance which acts to
neutralize, adsorb and/or buffer acids, e.g., a base or alkaline
compound. Acid scavengers act to reduce the amount or concentration
of protons or protonated water, i.e., H.sup.+ or H.sub.3O.sup.+. In
the context of the present invention, an acid scavenger acts to
neutralize, diminish, or buffer acid produced by a photoacid
generator. Preferably, an acid scavenger exhibits little or no
stratification within a film over time or following exposure to
heat.
[0033] In accordance with an aspect of the present invention, acid
scavengers may be further subdivided into "organic bases" and
"polymeric bases." A polymeric base is an acid scavenger (e.g.,
basic unit) attached to a longer polymeric unit. A polymer is
typically composed of a number of coupled or linked monomers. The
monomers can be the same (to form a homopolymer) or different (to
form a copolymer). In a polymeric base, at least some of the
monomers act as acid scavengers.
[0034] An organic base is defined as a base which is joined to or
part of a non-polymeric unit. Non-limiting examples of organic
bases include, without limitation, amine compounds (e.g., primary,
secondary and tertiary amines). Generally any type of acid
scavenger, defined here as a traditional Lewis Base, an electron
pair donor, can be used in accordance with the present
invention.
[0035] In one aspect of the invention, amine compounds are
represented by the following structure:
##STR00005##
wherein R.sub.1, R.sub.2 and R.sub.3 are independently H, an alkyl
group, an alkenyl group, an alkynyl group or an aryl group, or one
or more of R.sub.1, R.sub.2 and R.sub.3 taken together with the
nitrogen atom form a carbocyclic or heterocyclic ring. In a
particular aspect, two or three of R.sub.1, R.sub.2 and R.sub.3 are
alkyl groups.
[0036] In a particular aspect, one or more of R.sub.1, R.sub.2 and
R.sub.3 taken together with the nitrogen atom form a carbocyclic
(excluding the pictured nitrogen atom) or heterocyclic ring. For
example, R.sub.1 and R.sub.2 taken together with the nitrogen atom
form a ring. In another example, R.sub.1 and R.sub.2 taken together
with the nitrogen atom form a ring and R.sub.2 and R.sub.3 taken
together with the nitrogen atom form a ring. In a further example,
R.sub.1 and R.sub.2 taken together with the nitrogen atom form a
ring, R.sub.2 and R.sub.3 taken together with the nitrogen atom
form a ring and R.sub.1 and R.sub.3 taken together with the
nitrogen atom form a ring. Such rings are typically carbocyclic or
include only carbon and nitrogen atoms, such as 5-, 6-, 7- and
8-membered rings.
[0037] R.sub.1, R.sub.2 and R.sub.3 are often unsubstituted groups,
however, substitution is permitted. When R.sub.1, R.sub.2 and
R.sub.3 are substituted, substituents can be selected to enhance
the solubility of the base.
[0038] Exemplary groups of organic base additives include (A) mono,
di and tri-alkylamines, (B) anilines and substituted anilines, (C)
substituted pyridines, (D) substituted guanidines, (E) bicyclic
mono and di-azo compounds, and (F) bifunctional bases containing
amino and hydroxyl functionalities. Specific examples of organic
base additives are shown below:
TABLE-US-00001 ORGANIC BASE ACID SCAVENGER AND REPORTED BL (MP)
CH.sub.3--(CH.sub.2).sub.7--NH.sub.2 175.degree. C.
CH.sub.3--(CH.sub.2).sub.7--NH--(CH.sub.2).sub.6--CH.sub.3
297.degree. C. ##STR00006## 162.degree. C. @ 15 mm
CH.sub.3--(CH.sub.2).sub.7--N--((CH.sub.2).sub.6--CH.sub.3).sub.2
365.degree. C. CH.sub.3--(CH.sub.2).sub.7--N--(CH.sub.3).sub.2
195.degree. C. ##STR00007## 88.degree. C. @ 43 mm ##STR00008##
104.degree. C. ##STR00009## 115.degree. C. at 20 mm ##STR00010##
233.degree. C. ##STR00011## 115.degree. C. @ 11 mm ##STR00012##
159.degree. C. ##STR00013## 223.degree. C. ##STR00014## 160.degree.
C.
[0039] In accordance with an aspect of the present invention,
boiling points and melting points are of great importance in
determining whether the compound in question will act as an
effective acid scavenger. For example, in some systems a prebake
step is employed. If an acid scavenger, particularly an organic
base, has a low boiling point, it could tend to evaporate,
diminishing the effective amount of scavenger. Thus, if the
synthesis methodology employed in the context of the present
invention includes exposure of the acid scavenger to a high
temperature, a person of skill in the art will compensate by
choosing an acid scavenger with a high boiling point or that is a
solid at the temperature in question. Alternatively, if a low
boiling point acid scavenger is used, a person of skill in the art
can compensate for exposure to a high temperature by starting off
with a higher concentration of the low boiling point acid scavenger
or supplementing the mixture with more acid scavenger to compensate
for evaporation.
[0040] Organic base concentrations typically range from 0.1 to 4.0
molar equivalents, relative to the PAG. More preferably, organic
base concentrations range from 0.1 to 3.0 molar equivalents, such
as 0.5 to 2.0 molar equivalents. In accordance with an aspect of
the present invention, organic bases advantageously have one or
more of the following physical properties: (1) a boiling point
above 150.degree. C. and preferably above 200.degree. C., (2) a pKa
greater than 7 and less than 14 and more preferably between 8 and
10, (3) a sterically hindered nitrogen, and (4) solubility both in
a PAG formulation and in a thin film.
[0041] Suitable polymeric bases include basic homopolymers and
copolymers, including those formed from amine-containing monomers.
Examples of such polymeric bases are polyvinylpyridone,
polyvinylpyridine and polyvinylimidizaole. Additional suitable
polymeric bases include polymers containing base functionalities
(e.g., amines, preferably sterically hindered amines). Further
suitable polymeric bases are polymer backbones (e.g., alkylene
backbones such as ethylene) to which one or more of the organic
bases described above are directly or indirectly attached. In some
examples, the nitrogen atom is attached directly or indirectly
(e.g., via an alkylene group) to a polymer backbone and R.sub.3 is
absent. Typically, molecular weights of the polymeric basess range
between 2 K and 150 K, more preferably 10 K and 150 K, such as 10 K
and 50 K. Polymeric base concentrations generally range from 0.05%
to 5% by weight of a film.
[0042] A "film" as used herein refers to a layer or coating having
one or more constituents, applied in a generally uniform manner
over the entire surface of a substrate, for example, by spin
coating. For example, in accordance with an aspect of the present
invention, a film is a solution, suspension, dispersion, emulsion,
or other acceptable form of a chosen polymer. For example, a film
can include a photoacid generator and optionally a base and a
sensitizer, generally in combination with a film-forming polymer.
Film-forming polymers are polymers, which after melting or
dissolution in a compatible solvent, can foil a uniform film on a
substrate.
[0043] A "sensitizer" is a compound which aids in the use of
certain photoacid generators ("PAGs"). While the instant invention
is not limited by any particular mechanism of action or proposed
mechanism of action, the sensitizer is understood to extend the
photosensitivity of the PAG, i.e., to shift the photo sensitivity
to a longer wavelength of electromagnetic radiation. The
sensitizer, also called a photosensitizer, is capable of activating
the FAG at, for example, a longer wavelength of light in accordance
with an aspect of the present invention. Preferably, the
concentration of the sensitizer is greater than that of the PAG,
such as 1.1 times to 5 times greater, for example, 1.1 times to 3
times greater the concentration of PAG. Exemplary sensitizers
suitable for use in the invention include isopropylthioxanthone
(ITX) and 10H-phenoxazine (PhX).
[0044] A "substrate" is a material having a rigid, semi-rigid or
gelatinous surface. Typical examples include glass or suitable
polymer materials. In some embodiments of the present invention, 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.
[0045] A "labile protective group" is a moiety which may be
selectively removed to expose an active site such as an amino
functionality in peptide or amino acid or a hydroxyl group in a
nucleic acid or nucleotide. In accordance with one aspect of the
present invention, protective groups may be removed under a variety
of condition. For example, an "acid labile protective group" is
removed by exposure to acid. For an extensive listing of labile
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. 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.
[0046] 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, simply a
"region" or a "feature". The predefined region may have any
convenient shape, e.g., circular, rectangular, elliptical,
wedge-shaped, etc. In accordance with the present invention, the
arrays of the present invention have features on the order of
10-100 .mu.m, i.e. 10.times.10 .mu.m.sup.2 to 100.times.100
.mu.m.sup.2 for approximately square features. More preferably the
features will be on the order of 1-10 .mu.m. It is also an object
of the present invention to provide features having sub-micron
dimensions. Such features are preferably on the order of 100-1000
nm. Within these regions, the polymer synthesized therein is
preferably synthesized in a substantially pure form. However, in
other embodiments of the invention, predefined regions may
substantially overlap. In such embodiments, hybridization results
may be resolved by software for example.
[0047] "Damage to the polymer" means degradation or harm to a
polymeric sequence such as deletions or substitutions of one
monomer sequence for another, damage to the monomer itself, a
linker or substrate. It is an object of the present invention to
maintain the integrity of the synthesized polymer in all facets of
synthesis and/or use for detection of hybridization or binding. It
is an object of one aspect of the present invention that the
reagents and conditions used to deprotect the monomer (e.g.,
exposure of acid labile protective group to acid), whether attached
to a linker or growing polymer chain, do not substantially degrade
or harm the polymer, monomer, linker or substrate. Preferably, the
reagents and conditions used to deprotect will not damage the
polymer at all or will do so only minimally such that the polymer
can still be specifically recognized by its counterpart (e.g.
ligand-receptor). For example, if the polymer is a nucleic acid, it
can only sustain damage, e.g., depurination, to the extent that it
can still undergo specific Watson-Crick base pairing with a
complementary nucleic acid such that specific hybridization is
detectable over non-specific hybridizations. Similarly, if a
peptide or its amino acids are chemically damaged, the damage must
not be to such an extent that a ligand, e.g., an antibody, fails to
recognize the peptide. Acceptable levels of damage will be readily
appreciated by those of skill in the art. In constructing an array
of polymers in accordance with the present invention, it is
acceptable that some polymers of a group are extensively damaged as
long as there are sufficient other members of the group that are
either undamaged or minimally damaged to allow specific recognition
of the polymer.
[0048] The present invention has many preferred embodiments and
relies on many patents, applications and other references for
details known to those of the art. Therefore, when a patent,
application, or other reference is cited or repeated below, it
should be understood that it is incorporated by reference in its
entirety for all purposes as well as for the proposition that is
recited.
[0049] As used in this application, the singular form "a," "an,"
and "the" include the corresponding plural references unless the
context dictates otherwise. Likewise, plural references include the
singular unless the context indicates otherwise.
[0050] Throughout this disclosure, various aspects of this
invention can be presented in a range format. It should be
understood that such description is merely for convenience and
brevity and should not be construed as an unwarranted 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.
[0051] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques of organic chemistry,
polymer technology, molecular biology (including recombinant
nucleic acid techniques), cell biology, biochemistry, and
immunology as would be understood by one of the ordinary skill.
Such conventional techniques include polymer array synthesis,
hybridization, ligation, and detection of hybridization using a
label. Specific illustrations of suitable techniques can be had by
reference to the examples herein below. However, other equivalent
conventional procedures can, of course, also be used. Such
conventional techniques and descriptions 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), Stryer, L. (1995) Biochemistry (4th Ed.)
Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical
Approach" 1984, IRL Press, London, Nelson and Cox (2000),
Lehninger, Principles of Biochemistry 3.sup.rd Ed., W.H. Freeman
Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5.sup.th
Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein
incorporated by reference in their entirety.
[0052] The present invention can employ solid substrates, including
arrays in some preferred embodiments. Methods and techniques
applicable to polymer (including protein) array synthesis have been
described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.
5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,
5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215,
5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734,
5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324,
5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860,
6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT
Applications Nos. PCT/US99/00730 (International Publication Number
WO 99/36760) and PCT/US01/04285 (International Publication Number
WO 01/58593), which are all incorporated herein by reference in
their entirety.
[0053] Patents that describe synthesis techniques in specific
embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216,
6,310,189, 5,889,165, and 5,959,098, which are all incorporated by
reference in their entirety. Nucleic acid arrays are described in
many of the above patents, but the same general methodologies are
applicable to polypeptide arrays.
[0054] The present invention also contemplates many uses for
polymers attached to substrates. These uses include gene expression
monitoring, profiling, library screening, genotyping and
diagnostics. Gene expression monitoring, and profiling methods can
be shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135,
6,033,860, 6,040,138, 6,177,248 and 6,309,822, which are all
incorporated by reference in their entirety. Genotyping and uses
therefore are shown in U.S. Ser. Nos. 60/319,253, 10/013,598 (U.S.
Patent Application Publication 20030036069), and U.S. Pat. Nos.
5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799
and 6,333,179, which are incorporated by reference in their
entirety. Other uses are embodied in U.S. Pat. Nos. 5,871,928,
5,902,723, 6,045,996, 5,541,061, and 6,197,506, which are
incorporated by reference in their entirety.
[0055] The present invention also contemplates sample preparation
methods in certain preferred embodiments. Prior to or concurrent
with genotyping, the genomic sample may be amplified by a variety
of mechanisms, some of which may employ PCR. See, e.g., PCR
Technology: Principles and Applications for DNA Amplification (Ed.
H.A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide
to Methods and Applications (Eds. Innis, et al., Academic Press,
San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19,
4967 (1991); Eckert et al., PCR Methods and Applications 1, 17
(1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S.
Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675,
and each of which is incorporated herein by reference in their
entirety. The sample may be amplified on the array. See, for
example, U.S. Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300,
which are incorporated herein by reference in their entirety.
[0056] Other suitable amplification methods include the ligase
chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989),
Landegren et al., Science 241, 1077 (1988) and Barringer et al.
Gene 89:117 (1990)), transcription amplification (Kwoh et al.,
Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO 88/10315),
self-sustained sequence replication (Guatelli et al., Proc. Nat.
Acad. Sci. USA, 87, 1874 (1990) and WO 90/06995), selective
amplification of target polynucleotide sequences (U.S. Pat. No.
6,410,276), consensus sequence primed polymerase chain reaction
(CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase
chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and
nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.
Nos. 5,409,518, 5,554,517, and 6,063,603, each of which is
incorporated herein by reference). Other amplification methods that
may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810,
4,988,617 and in U.S. Ser. No. 09/854,317. Each of the above
references is incorporated herein by reference in its entirety.
[0057] Additional methods of sample preparation and techniques for
reducing the complexity of a nucleic sample are described in Dong
et al., Genome Research 11, 1418 (2001), in U.S. Pat. Nos.
6,361,947, 6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491
(U.S. Patent Application Publication 20030096235), 09/910,292 (U.S.
Patent Application Publication 20030082543), and 10/013,598, each
of which is incorporated herein by reference in its entirety.
[0058] Numerous methods for conducting polynucleotide hybridization
assays have been well developed. 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 (2.sup.nd 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 Davism, P.N.A.S, 80: 1194 (1983). Methods
and apparatus for carrying out repeated and controlled
hybridization reactions have been described in U.S. Pat. Nos.
5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of
which is hereby incorporated by reference in its entirety.
[0059] The present invention contemplates detection of
hybridization between a ligand and its corresponding receptor by
generation of specific signals. See U.S. Pat. Nos. 5,143,854,
5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601;
6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S.
Ser. No. 60/364,731 and in PCT Application PCT/US99/06097
(published as WO99/47964), each of which also is hereby
incorporated by reference in its entirety. Each of these references
is incorporated herein by reference in its entirety.
[0060] Methods and apparatus for signal detection and processing of
intensity data are disclosed in, for example, U.S. Pat. Nos.
5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758;
5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555,
6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S.
Ser. No. 60/364,731 and in PCT Application PCT/US99/06097
(published as WO 99/47964), each of which also is hereby
incorporated by reference in its entirety.
[0061] The practice of the present invention may also employ
conventional biology methods, software and systems. Computer
software products of the invention typically include computer
readable medium having computer-executable instructions for
performing the logic steps of the method of the invention. Suitable
computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM,
hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The
computer executable instructions may be written in a suitable
computer language or combination of several languages. Basic
computational biology methods are described in, e.g. Setubal and
Meidanis et al., Introduction to Computational Biology Methods (PWS
Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.),
Computational Methods in Molecular Biology, (Elsevier, Amsterdam,
1998); Rashidi and Buehler, Bioinformatics Basics: Application in
Biological Science and Medicine (CRC Press, London, 2000) and
Ouelette and Bzevanis Bioinformatics: A Practical Guide for
Analysis of Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd
ed., 2001). See U.S. Pat. No. 6,420,108. Each of these references
is incorporated herein by reference in its entirety.
[0062] The present invention may also make use of various computer
program products and software for a variety of purposes, such as
probe design, management of data, analysis, and instrument
operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729,
5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127,
6,229,911 and 6,308,170. Each of these references is incorporated
herein by reference in its entirety.
[0063] Light patterns can also be generated using Digital
Micromirrors, Light Crystal on Silicon (LCOS), light valve arrays,
laser beam patterns and other devices suitable for direct-write
photolithography. See, e.g., U.S. Pat. Nos. 6,271,957 and
6,480,324, incorporated herein by reference.
[0064] Additionally, the present invention may have preferred
embodiments that include methods for providing genetic information
over networks such as the Internet as shown in U.S. Ser. Nos.
10/063,559 (United States Publication No. 20020183936) and U.S.
Provisional Applications 60/349,546, 60/376,003, 60/394,574 and
60/403,381). Each of these references is incorporated herein by
reference in its entirety.
[0065] The present invention provides methods, devices, and
compositions for the formation of arrays of large numbers of
different polymer sequences. In one aspect of the present
invention, the methods and compositions provided herein involve the
conversion of radiation signals into chemical products 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.
[0066] Such arrays are used in, for example, in 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. Polymer
arrays are also used in screening studies to evaluate their
interaction with, for example, receptors such as antibodies in the
case of peptide arrays or with nucleic acids in the case, for
example of oligonucleotide arrays. 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.
[0067] In some embodiments of the present invention, 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.
[0068] 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.
[0069] The present invention has certain features in common with
the radiation directed methods discussed in U.S. Pat. No.
5,143,854, 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.
[0070] According to another aspect of the present invention, there
is no requirement for the use of masks. Predefined regions of the
array may be activated by light without the use of photomasks, for
example without limitation, by spatial light modulation as
discussed in U.S. Pat. No. 6,271,957 and related applications
(parent and progeny patents).
[0071] According to one aspect of the present invention, linker
molecules having reactive functional groups protected by acid
labile protecting groups are provided on the surface of a
substrate. In one preferred embodiment of the present invention, a
photoacid generator ("PAG") is provided on the surface, preferably
in a film with an acid scavenger. This is also called a "resist
mixture."
[0072] In another aspect of the present invention, the resist
mixture additionally contains a sensitizer. 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.; Willson, C. G.; and Bowden, M. J., Introduction to
Microlithography; American Chemical Society, 1994, pp. 212-232,
incorporated herein by reference in its entirety.
[0073] According to an aspect of the present invention, acid is
generated in the selected regions from the PAG by exposure of the
PAG to light of a predetermined wavelength. The generated acid
contacts the protected group(s) for long enough and under
appropriate conditions to remove the protective group. In
accordance with an aspect of the present invention, the protective
group is preferably a DMT group and it protects a hydroxyl group.
The hydroxyl group can be, for example, part of a substrate, part
of a linker, a 5'-hydroxyl group of a nucleotide or deoxynucleotide
or a 3'-hydroxyl group of a nucleotide or deoxynucleotide. After
sufficient exposure of the protective groups to the acid such that
the protective group is removed, but no or substantially no damage
is done to any polymer, the surface of the array is stripped,
preferably in an appropriate solvent leaving protected and
unprotected groups. In one aspect of the invention, the protective
groups are exposed to the acid for up to 3 hours, such as up to 1
hour, and typically from 2-30 or 5-15 minutes.
[0074] Monomers having an acid labile protective group are allowed
to react with the exposed groups from the acid treatment. The
surface is again coated with one of the resist mixtures described
above.
[0075] In a particular embodiment of the invention,
deoxynucleotides having one hydroxyl group with an acid labile
protective group and the other with a reactive group, preferably a
phosphoramidite group, are allowed to react with the exposed
hydroxyl groups from the acid treatment, allowing coupling of the
nucleotide to the hydroxyl group. The surface is again coated with
one of the resist mixtures described above.
[0076] 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.
According to one aspect of the present invention, the monomers are
preferably nucleotides. In accordance with an aspect of the present
invention, growing chains of nucleic acid are preferably capped in
between synthesis rounds. By terminating chain growth where a
monomer should have been added but was not, capping limits the
production of incorrect nucleotide sequences. Side chain protective
groups for exocyclic amines for example are also preferably
protected by techniques well known in the art during synthesis and
deprotected at the conclusion of synthesis of the nucleotide
array.
[0077] In one preferred embodiment, the monomer is a
2'-deoxynucleoside phosphoramidite containing an acid labile
protecting group at its 5'-hydroxyl group. Accordingly, a "monomer"
is understood to include both the individual units of a finished
polymer (e.g., oligonucleotide, polypeptide) and compounds that
become individual units of a finished polymer upon attaching to a
substrate and optionally further reaction (e.g., to remove
protecting groups, to oxidize phosphite esters to phosphate
esters). 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 in accordance with one
aspect of the present invention by the following formula:
##STR00015##
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 known to those of skill in the art;
R.sub.2 is cyanoethyl, methyl, t-butyl, trimethylsilyl or the like;
and R.sub.3 and R.sub.4 are isopropyl, cyclohexyl 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 oligo 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.
[0078] 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 terminates in an
amino or carboxy acid group bearing an acid- or base removable
protecting group. Protecting groups include tert-butyloxycarbonyl,
9-fluorophenylmethoxycarbonyl, and any of the protective groups
previously mentioned and others known to those skilled in the
art.
[0079] According to one aspect of the present invention, spatially
defined polymer synthesis will be performed by depositing a
photoresist such as Ghand's "VLSI Fabrication Principles," Wiley
(1983), incorporated herein by reference in its entirety. According
to these embodiments, a resist is deposited, selectively exposed,
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 defined sequences at
desired locations. In some specific embodiments, a positive tone
resist comprised of diazonapthoquinone-novolac (DQN/N) is
incorporated in a creasole-formaldehyde polymer matrix. This resist
and its variants are used routinely in the microelectronics
industry for submicron resolution lithography, as more fully
discussed in Reiser, "Photoreactive Polymers: The Science and
Technology of Resist," Riley (1989), incorporated herein by
reference in its entirety. However, it has been discovered in
accordance with an aspect of the present invention that substantial
and non-obvious refinements to the procedures developed for the
microelectronics industry are necessary to allow similar procedures
to work with certain polymers of the present invention, e.g.,
nucleic acids. It is also known to those of skill in the art that
other polymers such as peptides are not stable at all conditions
employed in the microelectronics industry.
[0080] High contrast detritylation of less than 4 microns has been
demonstrated with simple contact printing with a resist.
Unfortunately, the alkaline conditions needed (aqueous [OH] of 0.1
M) complicates its direct use in a multistep polymer synthesis,
such as polynucleotide array fabrication because of the hydrolysis
of nucleobase exocyclic amine protecting groups that are used to
prevent side reactions during synthesis with standard
phosphoramidite monomers.
[0081] As various well known methods for chemical removal of
protecting groups involving application of alkali conditions
resulted in undesired side reactions such as removal of exocyclic
amino protecting groups, reagents and methods were developed for
light-directed synthesis of DNA probes, utilizing phosphoramidite
monomers having photolabile protecting groups. These methods and
reagents are described in the various references incorporated by
reference above.
[0082] Under some circumstances, photodeprotection yields truncated
probe sequences due to incomplete removal of the photoprotecting
group following application of light. Incomplete removal of a
photodeprotecting group may impose limitations on probe length. For
example, if one imagines a stepwise yield of photolysis of 85% and
25 successive steps are carried out to provide 25-mer
oligonucleotides, less than 2% of the probes will reach the desired
length of 25.
[0083] In addition, relative to conventional DMT-protected
phosphoramidite monomers, photolabile-protected phosphoramidite
monomers are costly to obtain. A manufacturing process that uses
DMT-protected phosphoramidite monomers should therefore be cheaper,
and by analogy to well-established efficiencies of acid-mediated
DMT removal, should also be higher-yielding, perhaps even
approaching a 99% stepwise yield. A high-yielding synthesis method
would substantially decrease the number of truncated probes and
enable the ability to produce long-mer probes (e.g., 50-mer,
60-mer, 70-mer etc.) with relative ease. Shorter probes could also
be constructed by the same method if desired.
[0084] In accordance with one aspect of the present invention,
methods and compositions to generate localized photo-generation of
appropriate acid species to effect protecting group (e.g., DMT)
removal from growing strands of polynucleotides were developed. The
traditional semiconductor field employs photoacid generator
compounds (i.e., PAGs) in conjunction with "sensitizer"
compounds.
[0085] Sensitizer compounds work to allow some PAGs to produce acid
at an acceptable wavelength of light. Many PAGs are known from the
computer chip industry. Many of them require activation energies,
i.e., wavelengths of light, which can cause damage to the DNA being
synthesized on the substrate. For example wave lengths of light
which are perfectly tolerable for computer chips (<300 nm) would
cause severe damage to DNA oligonucleotides, rendering these PAGs
useless for oligonucleotide array synthesis. However, an
appropriate sensitizer can render the same PAG activatable by
longer wavelengths of light.
[0086] In the computer chip industry, after exposure of PAGs to
light a baking step is traditionally employed to maximize the
effect of the liberated acid. However, a traditional baking step is
likely to lead to substantial damage in the way of depurination of
the probes. Probes which have undergone depurination, i.e., the
loss of the base structure on A and G nucleotides, will not
hybridize as well, or possibly at all, to corresponding homologous
DNA or RNA.
[0087] Solutions to acid induced depurination are known in the art.
Analogues of standard DNA, for example 2'-O-methyl (2'-OMe)
nucleoside modifications, are known to be more resistant to such
degradation. However, utilization of such analogues is
substantially more expensive than the corresponding underivatized
analog. Moreover, analogues such as 2'-OMe nucleosides alter the
hybridization properties of the probes, which would require changes
to probe/array design.
[0088] In accordance with the present invention, probes may be
prepared using standard DMT-containing monomers and detritylation
with a photoacid generator used under appropriate conditions, i.e.,
conditions described in accordance with an aspect of the present
invention that substantially reduce or eliminate acid induced
depurination. In accordance with an aspect of the present
invention, the exposure time of the polymer to the acid is an
important consideration. Another key aspect of an aspect of the
invention is the photolysis time, which must be of sufficient
duration to generate a suitable quantity of acid and achieve
essentially quantitative detritylation, but not so long that
depurination becomes a factor. It has been discovered in accordance
with an aspect of the present invention that a heating step
following photoactivation of the PAG, which is routinely employed
and taught in the semiconductor industry, should not be used in
conjunction with certain polymers contemplated by the present
invention, including especially polynucleotides, e.g. DNA
oligonucleotides. If growing polynucleotide chains are baked after
activation of the photoacid generator, it appears that the
resulting heat in conjunction with a localized low pH causes
depurination. Thus, post-UV light exposure baking is to be avoided
in accordance with an aspect of the present invention.
[0089] In accordance with this aspect of the present invention, the
photoacid causes minimal or insubstantial damage to the polymers
making up the array. What damage may be endured by the polymer in
question will be determined by the nature of the polymer and the
assay or experiment to be conducted with the array. This will be
apparent to the person of skill in the art. For example, if an
array of oligonucleotides is fabricated, a certain amount of
depurination may be tolerated if the probes on the array can still
be used to reliably and specifically detect sequences in a sample.
Preferably, depurination occurs in less than 25%, less than 20%,
less than 15%, less than 10%, less than 5%, less than 3%, less than
2% or less than 1% of nucleotides susceptible to depurination.
[0090] In accordance with another aspect of the present invention,
the PAG must be chosen (in conjunction with a sensitizer as
necessary) such that that wavelength of light of activation does
not fall below about 310 nm. For example, many PAGs used in the
semiconductor industry require UV light having a wavelength of less
than 300 nm. Indeed, literature references speak of using "short
UV" PAGs wherein wavelengths of light of 220 to 260 nm are used. In
accordance with an aspect of the present invention, such short LTV
wavelengths are unacceptable with respect to nucleic acids. For
nucleic acids, UV light of wavelength greater than 310 nm, such as
330 to 365 nm is typically used. More preferably, UV light of
around 365 nm is used.
[0091] According to one aspect of the present invention a process
is provided for fabricating an array of polymers, the process
having the steps of providing a substrate having a reactive group
protected by a protective group; coating the substrate with a film
having an activatable deprotecting agent; activating the
deprotecting agent in selected regions by selective application of
an activator to provide an activated deprotecting agent; and
exposing the monomer having the protective group to the activated
deprotecting group under appropriate conditions such that the
protecting group is removed to provide an exposed reactive group
wherein the step of exposing does not result in substantial damage
to the polymer. In accordance with the present invention, the
reactive group may be located on a linker having one end bound to a
substrate with the reactive group at the opposite end or other
exposed site of the linker, a monomer attached to a linker or a
polymer (here, two or more monomers) attached to a linker.
[0092] Typically, the array of polymers is an array of nucleic
acids. More typically, the array of nucleic acids is an array of
oligonucleotides. The monomers for such arrays are preferably
naturally or non-naturally occurring nucleotides. More preferably,
the nucleotides employed in the present invention are selected from
the group consisting of G, A, T, and C. Preferably, a nucleotide is
protected at its 5' hydroxyl end by a dimethoxytrityl ("DMT")
protective group. In the most preferred embodiments, the nucleotide
is selected from the group G, A, T, and C and is protected at its
5' hydroxyl group by a DMT protective group. In another aspect of
the present invention, the nucleotide is protected at its 3'
hydroxyl group with a DMT protective group. Thus, in accordance
with the present invention, nucleotides may be synthesized in the
5' to 3' direction or a 3' to 5' direction.
[0093] In still another preferred embodiment of the present
invention, the array of polymers is an array of peptides, where the
monomers are amino acids. Suitable amino acids include naturally
occurring amino acid and non-naturally occurring amino acids.
Preferably, the amino acid is selected from the group consisting of
the L form of alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, praline, serine,
threonine, tryptophan, tyrosine and valine. Preferably, an amino
acid is protected at its amino terminus functionality by a
tert-butyloxycarbonyl ("tBOC") protective group during
synthesis.
[0094] According to another aspect of the present invention,
suitable amino acids include peptide nucleic acids (PNAs). PNAs
include a peptide backbone with nitrogenous bases attached to this
backbone, such that they can serve as mimics of nucleic acids
(including oligomers). Preferably, PNAs have a greater affinity for
a complementary nucleic acid sequence than the analogous native
nucleic acid. Suitable PNA repeat units are shown by the following
structural formulae:
##STR00016##
where B represents a base, typically adenine, cytosine, guanine or
thymine. Other backbones are suitable, provided that the resulting
PNAs are capable of hybridizing with nucleic acids.
[0095] Syntheses of PNAs are described in Hyrup and Nielsen,
Bioorg. Med. Chem. (1996) 4:5-23; and Vilaivan and Lowe, J. Am.
Chem. Soc. (2002) 124:9326-9327, the contents of which are
incorporated herein by reference.
[0096] In an aspect of the invention, density of PNAs in an array
and any linker groups are selected such that a 2:1 complex of PNA
to a hybridized DNA or RNA sample can be formed. In another aspect
of the invention, a chimeric polymer of PNA and a nucleic acid is
prepared.
[0097] In another aspect of the instant invention, the process
described above has an additional step of reacting the monomer with
an exposed reactive group with a second monomer having a reactive
group protected by a protective group. In another preferred
embodiment of the instant invention, the process further includes
repeating all the steps to obtain the desired polymer array.
[0098] Originally the term lithography referred to a method of
printing using a nonpolar ink applied to a hydrophilic master plate
patterned with a hydrophobic image. As used at the present date,
the term is generally used to describe a number of methods for
replicating a predetermined master pattern on a substrate. Common
applications of this technology involve replication effected by
first coating the substrate with a radiation-sensitive polymer film
(a resist) and then exposing the film to actinic radiation in a
predefined pattern. The radiation-induced chemical changes that
result, alter the chemical properties of the exposed regions of the
coated substrate such that they can be differentiated in subsequent
developmental steps.
[0099] In yet another preferred embodiment of the instant
invention, the step of coating is performed by applying to the
substrate a film of a polymer solution containing the activatable
deprotecting agent. Preferably, the polymer solution is a
composition of a certain percentage of poly(methyl methacrylate).
Preferably, the activatable deprotecting agent is a photoacid
generator.
[0100] One group of photoacid generators of the invention is
represented by Structural Formula (I):
##STR00017##
or a salt thereof, wherein:
[0101] R.sub.1 is --H, --COOR, a substituted alkyl group, or an
alkenyl or aryl group;
[0102] R.sub.2 is a sulfonate, substituted acetate or benzoate
group;
[0103] R.sub.3 is --NRR', --COOR, an alkyl or alkenyl group or a
substituted alkoxy or aryl group; and
[0104] R and R' are independently --H or an alkyl, alkenyl or aryl
group.
[0105] A second group of photoacid generators of the invention is
represented by Structural Formula (II):
##STR00018##
or a salt thereof, wherein:
[0106] R.sub.4 and R.sub.5 are independently --H, --COOR, a
substituted alkyl group or an alkenyl or aryl group;
[0107] R.sub.6 is a sulfonate, substituted acetate or benzoate
group;
[0108] R.sub.7 is --NRR', --COOR, an alkyl or alkenyl group or a
substituted alkoxy or aryl group; and
[0109] R and R' are independently --H or an alkyl, alkenyl or aryl
group.
[0110] A third group of photoacid generators of the invention is
represented by Structural Formula (III):
##STR00019##
or a salt thereof, wherein:
[0111] R.sub.8 is a sulfonate, substituted acetate or benzoate
group;
[0112] R.sub.9 is --H, --COOR, a substituted alkyl group or an
alkenyl or aryl group;
[0113] R.sub.10 is --NRR', --COOR, an alkyl or alkenyl group or a
substituted alkoxy or aryl group; and
[0114] R and R' are independently --H or an alkyl, alkenyl or aryl
group.
[0115] Where the activatable deprotecting agent is a photoacid
generator, it is particularly preferred that the monomer is a
nucleotide and the protecting group is DMT. It is also preferred in
this situation that the monomer is an amino acid and the protecting
group is tBOC.
[0116] In other embodiments of the instant invention, the array of
polymers comprises a polymer at least 25 monomers in length. In
another preferred embodiment, the polymer is at least 50 monomers
in length. In further embodiments, the polymer may range up to 200
monomers in length. In other preferred embodiments, the polymers
are at least 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150
monomers in length. More preferably, the polymers referred to above
are nucleic acids or oligonucleotides.
[0117] Still other photoacid generators ("PAGs") are known and
suitable for use in the present invention, such as combination with
the PAGs represented by Structural Formulae (I)-(III) or separately
in certain steps of a synthetic process. Common commercial ionic
PAGs include onium and organometallic salts such as diaryliodonium
and triarylsulfonium salts and (cyclopentadienyl)(arene)iron.sup.+
salts of the anions PF.sub.6.sup.-, SbF.sub.6.sup.-,
CF.sub.3SO.sub.3.sup.-, C.sub.4F.sub.9SO.sub.3.sup.- and
C.sub.8F.sub.17SO.sub.3.sup.-. Also known are sulfonium salts
(e.g., triphenylsulfonium hexafluorophosphate, triflate, toslyate,
and camphorsulfonate). PAGs previously used in the synthesis of
biological polymers include 2,6-dinitrobenzyl tosylate and
Bis(4-t-butyl phenyl) iodonium PF.sub.6.sup.-.
[0118] The photochemical reaction of many onium salts generates a
strong Bronsted acid. In this regard, numerous PAGs are known from
the semiconductor industry. However, in the semi-conductor
industry, the wafer is subjected to a baking step after generation
of the acid by photolysis, where the exposed wafers are subjected
to elevated temperatures. In accordance with the present invention,
it has been discovered that baking has a deleterious effect on some
polymers, in particular nucleic acids. Thus, while onium salts and
other PAGs used in the semiconductor industry are of interest to
the present invention, protocols for the usage of these compounds
must be varied significantly as described in accordance with one
aspect of the present invention.
[0119] Onium salts are known to have high quantum yields of acid
production, good absorption properties and good solubility in many
resist films. However, it is also known in accordance with the
present invention that the wavelengths of light commonly used to
activate onium salts for semi-conductors can not be used with some
polymers, particularly nucleic acids. In this regard, it is common
in the semi-conductor industry to use low wavelength UV light (e.g.
less than 300 nm) to activate onium salts. See, e.g., Wallraff, G.
M. and Hinsberg, W. D., Lithographic Imaging Techniques for the
formation of Nanoscopic Features, Chem. Rev. 1999, 99, 1801-1821,
which is incorporated herein be reference for all purposes.
[0120] In accordance with the present invention, it is known that
such wavelengths of light are unacceptable for the synthesis of
nucleic acids. Such wavelengths of UV light cause numerous forms of
damage to a nucleic acid chain, including cross-linking of bases.
Nucleic acids synthesized under these conditions would be unable to
effectively hybridize to their homologous counterparts. To use PAGs
in accordance with the present invention, they must be capable of
being directly or indirectly activated by light in the range of 330
nm to about 365 nm and generate acid at an acceptable level and
rate (photospeed) at those longer wavelengths.
[0121] In accordance with an aspect of the present invention, both
ionic and non-ionic photoacid generators are contemplated to be
used in combination with the photoacid generators represented by
Structural Formulae (I)-(III). Ionic PAGs are thermally stable and
have a wide range of spectral absorption. However, ionic PAGs have
a limited solubility in organic solvents. Non-ionic PAGs have
better solubility in organic solvents, but have less thermal
stability than ionic PAGs. However, as discussed above, the thermal
stability is a less important consideration here than in the
computer industry.
[0122] In accordance with an aspect of the present invention, the
polymers synthesized by the techniques of the present invention do
not undergo undue or substantial damage during the synthesis. In
this regard, it is known that exposure of nucleic acid polymers to
acids can result in damage, including for example depurination. In
the context of nucleic acid arrays, which are used to detect the
hybridization of homologous species of nucleotides, the nucleic
acid attached to the substrate can undergo some depurination and
still act to satisfactorily hybridize homologous nucleic acids.
However, if the damage is too great, the hybridization will not
occur at all or will not occur reliably. A substantial number of
damaged probes in a feature could result in a false negative. Thus,
in certain embodiments of the instant invention, the acid from a
photoacid generator is not allowed to substantially damage the
nucleic acids being synthesized. In accordance with the present
invention, substantial damage means that the polymer or nucleic
acid is unable to be used for the intended use for the array. Thus,
in the context of a nucleic acid array, substantial damage would
mean that the array could not be used to reliably detect nucleic
acids. For a protein array, substantial damage would mean that the
peptide was damaged to the extent that it could not be recognized
by an antibody or protein receptor.
[0123] In one aspect of the present invention, the polymer is a
nucleic acid and the monomer is a nucleotide and substantial damage
is determined by determining the level of false negatives (e.g.,
the loss of signal from hybridization) generated by hybridizing the
array with a known sample having known complementary nucleic acids
to said array. In accordance with this aspect, the array can be
tested by hybridizing it with a test or control sample having
nucleic acids which should give a positive signal on the array if
the oligonucleotides, for example, on the array have been
synthesized without substantial damage. After hybridization of the
control sequence, the array can be scanned and the features
analyzed with the corresponding control probes. If the control
probes have suffered no damage during fabrication, a high intensity
result should be observed. However, if minimal damage occurred the
signal might still be present, but diminished, for example by 50%.
If the array were intended to detect rare species such a diminution
would probably be unacceptable. The batch of arrays containing such
defects would likely be disposed of. If no signal were seen or if
the signal was diminished by 90% or more, the batch of such arrays
would probably be disposed of regardless of the proposed end use of
such arrays.
[0124] In accordance with another aspect of the present invention,
the polymer arrays generated by the teachings of instant invention
are subjected to quality control. In preferred embodiments,
oligonucleotide or nucleic acid arrays of the present invention are
subject to quality control to determine fidelity of the synthesis
and the ability of the probes to bind to homologous nucleic acids.
In a preferred embodiment of the present invention, quality control
is performed by synthesizing nucleic acid arrays containing probes
of known sequence. These arrays are then hybridized to control
sequences corresponding to the known sequence. It is then
determined whether sufficient signal is generated by hybridization
to the control sequences.
[0125] In preferred embodiments, the arrays are synthesized on
wafers, containing scores of joined individual arrays. Quality
control is performed on a wafer by sawing off a few arrays for the
control testing described above. According to an aspect of the
instant invention, if the few arrays pass the control hybridization
test, the wafer passes the test and it then may be segregated into
individual arrays. If the control experiments fail, the wafer fails
and is discarded.
[0126] In accordance with another aspect of the present invention,
an array of oligonucleotides is produced using a PAG and DMT
protected nucleotides to produce features preferably on the order
of 10-100 .mu.m. More preferably, features are on the order of 1-10
.mu.m. In another preferred embodiment, features are on the order
of 100-1000 nm.
[0127] In accordance with an aspect of the present invention, one
purpose of adding an acid scavenger to the resist mixture is to
modulate contrast/sensitivity and decrease background (e.g.,
spontaneous detritylation).
[0128] In accordance with an aspect of the present invention,
standard DMT-protected phosphoramidite nucleotide monomers are used
in conjunction with one or more acid scavengers. Generally, an
activated DMT-protected phosphoramidite monomer is coupled to a
support-bound hydroxyl functionality and oxidized in the typical
manner. The support (i.e., wafer or chip) is subsequently coated
with a PAG formulation that contains a photo acid generator, a
polymeric matrix, solvent and an acid scavanger. Preferably, an
acid scavenger is either an organic base or a polymeric base
described herein.
[0129] In a preferred embodiment of the present invention, arrays
of the instant invention are synthesized on commercially available
silicon wafers.
[0130] The foregoing invention has been described in some detail by
way of illustration and examples, for purposes of clarity and
understanding. It will be obvious to one of skill in the art that
changes and modifications may be practiced within the scope of the
appended claims. Therefore, it is to be understood that the above
description is intended to be illustrative and not restrictive. 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 following appended claims, along
with the full scope of equivalents to which such claims are
entitled.
* * * * *
References