U.S. patent application number 11/369628 was filed with the patent office on 2006-07-06 for methods and compositions for monitoring polymer array synthesis.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Anthony D. Barone, Evelyn Chai, Glenn H. McGall, Nam Quoc Ngo.
Application Number | 20060147985 11/369628 |
Document ID | / |
Family ID | 36127671 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060147985 |
Kind Code |
A1 |
Barone; Anthony D. ; et
al. |
July 6, 2006 |
Methods and compositions for monitoring polymer array synthesis
Abstract
VLSIPS.TM. manufacturing processes are of increasing commercial
importance. The present invention provides methods and compositions
for monitoring the efficiency and quality of polymer synthesis in
VLSIPS.TM. arrays. Methods for monitoring polymer synthesis in an
array on a substrate are provided. Mono-isomeric labels for the
labeling of synthetic polymer arrays are provided. Methods and
compositions for post-synthetically labeling polymers in polymer
arrays are also provided.
Inventors: |
Barone; Anthony D.; (San
Jose, CA) ; McGall; Glenn H.; (Palo Alto, CA)
; Chai; Evelyn; (Foster City, CA) ; Ngo; Nam
Quoc; (Campbell, CA) |
Correspondence
Address: |
BANNER & WITCOFF LTD.,;COUNSEL FOR AFFYMETRIX
1001 G STREET , N.W.
ELEVENTH FLOOR
WASHINGTON
DC
20001-4597
US
|
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
36127671 |
Appl. No.: |
11/369628 |
Filed: |
March 7, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
08574461 |
Nov 30, 1995 |
7026114 |
|
|
11369628 |
Mar 7, 2006 |
|
|
|
60003726 |
Sep 13, 1995 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
536/25.32 |
Current CPC
Class: |
C12Q 1/6837 20130101;
G01N 33/54366 20130101; C12Q 2565/137 20130101; C12Q 2523/107
20130101; C12Q 2545/114 20130101; C07H 21/04 20130101; C12Q 1/6837
20130101 |
Class at
Publication: |
435/006 ;
536/025.32 |
International
Class: |
C40B 40/08 20060101
C40B040/08; C12Q 1/68 20060101 C12Q001/68; C07H 21/04 20060101
C07H021/04 |
Claims
1. A detectable monomeric polymer synthesis reagent with the
structure A-B, wherein A comprises a detectable chromogenic moiety
and B comprises a polymer integration element, said integration
element comprising a polymer joining agent selected from the group
consisting of an amine, a carboxyl, an oxygen, and a phosphate, and
wherein A-B is a single isomer.
2. The polymer synthesis reagent of claim 1, wherein the
chromogenic moiety is a fluorophore.
3. The polymer synthesis reagent of claim 1, wherein the polymer
synthesis reagent is a nucleic acid synthesis reagent, wherein B
comprises the structure ##STR24## and wherein L is a linking chain
selected from the group of linking chains consisting of an alkyl
linking chain from 1 to 30 carbons in length, wherein one or more
carbon is optionally substituted with a heteroatom selected from
the group consisting of N, S, O and P, and wherein the alkyl
linking group optionally includes one or more sites of
unsaturation, an alkyl linking chain from 1 to 30 carbons in
length, wherein one or more carbon is optionally replaced with a
heteroatom selected from the group consisting of N, S, O and P, and
wherein the alkyl linking group optionally includes one or more
sites of unsaturation; R.sub.1 is selected from the group
consisting of hydrogen, alkyl, and aryl; R.sub.2 is selected from
the group consisting of hydrogen, alkyl, and aryl; X is a nucleic
acid integration element comprising a phosphorous atom, Y is
selected from the group consisting of hydrogen, alkyl, and aryl;
Y.sub.2 is an alkyl chain; and Z comprises a protecting group.
4. The polymer synthesis reagent of claim 1, wherein the polymer
synthesis reagent is a nucleic acid synthesis reagent, wherein B
comprises the structure ##STR25## and wherein L is selected from
the group of alkyl linking chains consisting of an alkyl linking
chain from 1 to 30 carbons in length, wherein one or more carbon is
optionally substituted with a heteroatom selected from the group
consisting of N, S, O and P, and wherein the alkyl linking group
optionally includes one or more sites of unsaturation, and an alkyl
linking chain from 1 to 30 carbons in length, wherein one or more
carbon is optionally replaced with a heteroatom selected from the
group consisting of N, S, O and P, and wherein the alkyl linking
group optionally includes one or more sites of unsaturation; and
R.sub.1 is selected from the group consisting of hydrogen, alkyl,
and aryl.
5. A labeled oligonucleotide array attached to a solid substrate,
wherein the oligonucleotides of the array comprise a single isomer
of a detectable label.
6. A labeled oligonucleotide array attached to a solid substrate,
wherein the label is a mono-isomeric label comprising the structure
##STR26## wherein F comprises a fluorescent group. L is selected
from the group of alkyl linking chains consisting of an alkyl
linking chain from 1 to 30 carbons in length, wherein one or more
carbon is optionally substituted with a heteroatom selected from
the group consisting of N, S, O and P, and wherein the alkyl
linking group optionally includes one or more sites of
unsaturation, and an alkyl linking chain from 1 to 30 carbons in
length, wherein one or more carbon is optionally replaced with a
heteroatom selected from the group consisting of N, S, O and P, and
wherein the alkyl linking group optionally includes one or more
sites of unsaturation; R.sub.1 is selected from the group
consisting of hydrogen, alkyl, and aryl; R.sub.2 is selected from
the group consisting of hydrogen, alkyl, and aryl; X is a
nucleotide or a cleavable linker; Y is selected from the group
consisting of hydrogen, alkyl, and aryl; Y.sub.2 is selected from
the group consisting of a hydrocarbon chain and a substituted
hydrocarbon chain; and, Z is selected from the group consisting of
a nucleotide and a nucleic acid.
7. The nucleic acid synthesis reagent of claim 6, wherein the
nucleic acid synthesis reagent has the structure ##STR27## wherein
R.sub.1 is selected from the group consisting of alkyl, aryl, and
hydrogen; R.sub.2 is selected from the group consisting of alkyl,
and aryl; and FL is a fluorescent moiety.
8. The isomeric nucleic acid synthesis reagent of claim 6, wherein
the compound has the structure ##STR28##
9. The array of claim 6, wherein the composition further comprises
a cleavable linker.
10. A method of post-synthetically labeling an oligonucleotide
array, comprising: (i) providing a polymer array which comprises a
plurality of polymers, wherein each polymer comprises a labeling
site; and (ii) attaching a detectable label to the labeling
site.
11. The method of claim 10, wherein the detectable label comprises
a fluorophore.
12. The method of claim 10, wherein step (i) of said method
comprises synthesizing a polymer array, which polymer array
comprises polymers attached to a substrate, said polymers
comprising a labeling linker, which labeling linker comprises an
attachment site for the detectable label.
13. The method of claim 10, wherein step (i) of said method
comprises synthesizing a polymer array, which polymer array
comprises polymers attached to a substrate, said polymers
comprising a cleavable linker and a labeling linker, which labeling
linker comprises an attachment site for the detectable label, a
site for attachment to the cleavable linker and a protected site
for the attachment of the detectable label.
14. The method of claim 10, wherein step (i) of said method
comprises synthesizing a polymer array, which polymer array
comprises polymers attached to a substrate, said polymers
comprising a cleavable linker and a labeling linker, which labeling
linker comprises a protected attachment site for the detectable
label, and wherein step (ii) of the method comprises deprotecting
the labeling linker, thereby making the protected attachment site
into an unprotected attachment site, and incubating the polymer
array with a detectable labeling reagent, which detectable labeling
reagent comprises a site which is reactive with the unprotected
attachment site, and which labeling reagent comprises the
detectable label.
15. The method of claim 14, wherein said protected attachment site
comprises a DMT protective group.
16. The method of claim 10, wherein said labeling site is located
proximal to a cleavage site in the polymers of the polymer
array.
17. A post-synthetic labeling linker which comprises a site for
polymer elongation, a site for attaching a polymer to a substrate
and an attachment site for attaching a detectable label.
18. The labeling linker of claim 17, wherein the linker has the
structure ##STR29## wherein: R.sub.1 is selected from the group
consisting of hydrogen, alkyl and aryl; R.sub.2 is selected from
the group consisting of hydrogen, alkyl and aryl; R.sub.3 is
selected from the group consisting of hydrogen, alkyl and aryl,
L.sub.1 is a linking chain selected from the group of alkyl linking
chains consisting of an alkyl linking chain from 1 to 30 carbons in
length, wherein one or more carbon is optionally substituted with a
heteroatom selected from the group consisting of N, S, O and P, and
wherein the alkyl linking group optionally includes one or more
sites of unsaturation, and an alkyl linking chain from 1 to 30
carbons in length, wherein one or more carbon is optionally
replaced with a heteroatom selected from the group consisting of N,
S, O and P, and wherein the alkyl linking group optionally includes
one or more sites of unsaturation; L.sub.2 is a linking chain
selected from the group of alkyl linking chains consisting of an
alkyl linking chain from 1 to 30 carbons in length, wherein one or
more carbon is optionally substituted with a heteroatom selected
from the group consisting of N, S, O and P, and wherein the alkyl
linking group optionally includes one or more sites of
unsaturation, and an alkyl linking chain from 1 to 30 carbons in
length, wherein one or more carbon is optionally replaced with a
heteroatom selected from the group consisting of N, S, O and P, and
wherein the alkyl linking group optionally includes one or more
sites of unsaturation; Y is selected from the group consisting of a
dimethoxytrityl protecting group and a photocleavable protecting
group; Z is selected from the group consisting of a dimethoxytrityl
protecting group and a photocleavable protecting group; and X is a
nucleic acid integration element comprising a phosphorous atom.
19. The labeling linker of claim 18, wherein Z is the
photocleavable group MeNPOC.
20. The labeling linker of claim 17, wherein the linker has the
structure ##STR30##
21. The labeling linker of claim 17, wherein the linker has the
structure ##STR31##
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a divisional application which claims
the benefit of the filing dates of U.S. patent application Ser. No.
08/574,461, filed Nov. 30, 1995; and U.S. Provisional Application
Ser. No. 60/003,726, filed Sep. 13, 1995, by Barone et al., which
are hereby incorporated herein by reference in their entirety.
COPYRIGHT NOTICE
[0002] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
[0003] Methods of forming large arrays of oligonucleotides,
peptides and other polymers on a solid substrate are known. Pirrung
et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO
90/15070), McGall et al., U.S. Ser. No. 06/440742, Chee et al.,
USSN PCT/US94/12305, and Fodor et al., PCT Publication No. WO
92/10092 describe methods of forming vast arrays of peptides,
oligonucleotides and other polymers using, for example,
light-directed synthesis techniques.
[0004] In the Fodor et al. PCT application, methods are described
for using computer-controlled systems to direct polymer array
synthesis. Using the Fodor approach, one heterogeneous array of
polymers is converted, through simultaneous coupling at multiple
reaction sites, into a different heterogeneous array. See also,
U.S. Ser. No. 07/796,243 and U.S. Ser. No. 07/980,523 and Fodor et
al. (1991) Science, 251: 767-777. The arrays are typically placed
on a solid surface with an area less than 1 inch.sup.2, although
much larger surfaces are optionally used.
[0005] More recently, U.S. applications U.S. Ser. No. 06/440,742,
U.S. Ser. No. 08/284,064, U.S. Ser. No. 08/143,312, U.S. Ser. No.
08/082,937 and PCT application (designating the United States) SN
PCT/U594/12305, describe methods of making arrays of
oligonucleotide and oligonucleotide analogue probes, e.g., to check
or determine a partial or complete sequence of a target nucleic
acid, or to detect the presence of a nucleic acid containing a
specific oligonucleotide sequence. U.S. application Ser. No.
08/327,687 and U.S. application Ser. No. 06/440,742 describe
methods of creating libraries of nucleic acid probes for the
analysis of nucleic acid hybridization, and for screening nucleic
acid binding molecules, e.g., as potential therapeutic agents.
[0006] Additional methods applicable to polymer synthesis on a
substrate are described in co-pending Applications U.S. Ser. No.
07/980,523, filed Nov. 20, 1992, and U.S. Ser. No. 07/796,243,
filed Nov. 22, 1991, incorporated herein by reference for all
purposes. In the methods disclosed in these applications, reagents
are delivered to the substrate by flowing or spotting polymer
synthesis reagents on predefined regions of the solid substrate. In
each instance, certain activated regions of the substrate are
physically separated from other regions when the monomer solutions
are delivered to the various reaction sites, e.g., by means of
grooves, wells and the like.
[0007] Procedures for synthesizing polymer arrays are referred to
herein as very large scale immobilized polymer synthesis
(VLSIPS.TM.) procedures. The development of VLSIPS.TM. technology
as described in the above-noted U.S. patents and patent
applications is pioneering technology in the fields of
combinatorial polymer synthesis and screening.
SUMMARY OF THE INVENTION
[0008] VLSIPS.TM. procedures are of increasing commercial
importance, providing powerful compositions and methods, e.g., for
detecting genetic disorders, screening potential therapeutics,
facilitating basic research and rapid sequencing of nucleic acids.
Accordingly, the manufacturing processes which produce VLSIPS.TM.
arrays benefit from quality control and synthesis optimization
methods for measuring and improving the efficiency of polymer and
polymer array synthesis and coupling of monomers and polymers to
solid substrates.
[0009] The present invention provides methods and compositions to
monitor the synthesis and coupling of monomers and polymers to
solid substrates, e.g., in VLSIPS.TM. arrays. The methods typically
operate by incorporating a detectable label (typically an isomeric
label, e.g., as provided by the compositions herein) into the
polymers of an array. The polymers are cleaved from the array, and
the efficiency of polymer synthesis assessed by monitoring the
detectable label in an appropriate assay.
[0010] In one class of embodiments, the present invention provides
a method of monitoring polymer array synthesis on a solid substrate
by providing a preselected array of labeled polymers connected to
cleavable linkers on a solid substrate, cleaving the array of
labeled polymers from the solid substrate by cleaving the cleavable
linkers, thereby creating labeled unbound polymers, and detecting
the labeled unbound polymers. In this embodiment, the labeled
polymers each typically comprise a single isomeric label, although
any detectable label can also be used. The polymers cleaved from
the array are separated by physical properties such as size and/or
charge, using known analytical techniques such as HPLC, standard
column chromatography (anion, cation, size exclusion, etc.),
gel-electrophoresis, centrifugation, capillary gel-electrophoresis
and the like.
[0011] The methods are generally suitable to any polymer array,
regardless of the type of polymer. Thus, the efficiency of
synthesis for biological polymers such as proteins, nucleic acids,
antigens, and venoms are monitored using the above method.
Non-biological polymers such as carbon chains, vinyls, alcohols,
and other polymers are similarly monitored. The polymer array is
typically provided by synthesizing the array on the solid
substrate, but the array can also be provided by synthesizing the
polymers to be attached to the array in solution, and subsequently
attaching the polymers to pre-selected sites in the array.
[0012] In a second class of embodiments, the invention provides a
method of measuring and improving the synthesis of polymer arrays,
by synthesizing an array of polymers on a solid support by a first
synthesis protocol, creating a reference array of polymers;
synthesizing an array of polymers on a solid support by a second
synthesis protocol, wherein the second synthesis protocol is
different than the first synthesis protocol, thereby creating a
test array of polymers; cleaving separately the reference array of
polymers and the test array of polymers, thereby creating cleaved
reference polymers and cleaved test polymers; detecting the cleaved
test polymers and the cleaved reference polymers, and comparing the
cleaved test polymers to the cleaved reference polymers. By
repeating the process and altering different synthesis parameters
between the test and the reference array of polymers, the optimal
method of synthesizing a particular array is determined.
[0013] Typically, the polymers of an array comprise a detectable
label to facilitate analysis of the cleaved polymers, although the
polymers themselves are also detectable, and the method can,
therefore, be performed without incorporating a detectable label.
Where the method used for detecting the label discriminates between
optical isomers of a label (e.g., HPLC) the label will most often
comprise a single optical isomer. Although it is most preferred
that a single synthetic parameter is altered for the test polymers
relative to the control polymers, multiple parameters can be
altered in each synthetic protocol. Once again, the method is
generally applicable to biological and artificial polymers, each of
which are typically connected to a solid substrate by a cleavable
linker.
[0014] The methods of the present invention are typically performed
using a detectable monomeric monoisomeric polymer synthesis reagent
with the structure A-B, wherein A comprises a detectable
chromogenic moiety and B comprises a polymer integration element.
The polymer integration element typically includes a group which
can be joined to one end of the polymer, or incorporated into the
polymer as it is synthesized (i.e., as a monomeric unit of the
polymer). The precise nature of the integration element depends on
the polymer which the detectable moiety is to be integrated into.
For instance, where the polymer is a peptide, the integration
element will typically comprise an amino or a carboxy group, or
both, similar to the amino acids which comprise the peptide.
Similarly, where the polymer is an oligonucleotide, the polymer
will typically comprise a phosphate or hydroxyl, or both, similar
to the nucleotides which constitute the oligonucleotide polymer.
The chromogenic moiety is most typically a fluorophore, although
other chromogenic agents are also suitable.
[0015] In one embodiment, the polymer synthesis reagent is a
nucleic acid synthesis reagent, and B (the polymer integration
element) comprises the structure ##STR1## wherein [0016] L is an
alkyl linking group from 1 to 30 carbons in length, wherein one or
more carbon is optionally replaced or substituted with a heteroatom
selected from the group consisting of N, S, 0 and P, and is
optionally part of a ring system, and wherein the alkyl linking
chain optionally includes one or more site of unsaturation; [0017]
R.sub.1 is selected from the group consisting of hydrogen, alkyl,
and aryl; [0018] R.sub.2 is selected from the group consisting of
hydrogen, alkyl, and aryl; [0019] X is a nucleic acid integration
element comprising a phosphorous atom; [0020] Y is selected from
the group consisting of hydrogen, alkyl, and aryl; [0021] Y.sub.2
is an alkyl chain; and [0022] Z comprises a protecting group.
[0023] In one class of preferred embodiments, the polymer synthesis
reagent is a nucleic acid synthesis reagent, and B (the polymer
integration element) comprises the structure ##STR2## wherein
[0024] L is an alkyl linking group from 1 to 30 carbons in length,
wherein one or more carbon is optionally replaced or substituted
with a heteroatom selected from the group consisting of N, S, O and
P, and is optionally part of a ring system, and wherein that alkyl
linking chain optionally includes one or more site of unsaturation;
and [0025] R.sub.1 is selected from the group consisting of
hydrogen, alkyl, and aryl. This polymer integration element is
typically joined to a fluorophore.
[0026] In another class of preferred embodiments, the invention
provides an array of polymers, such as an array of oligonucleotides
or proteins, or non-biological polymers, with a mono-isomeric
detectable label incorporated into each polymer. For instance, in
one embodiment where the array is an oligonucleotide, the invention
provides an array of oligonucleotides attached to a solid
substrate, wherein the label is a mono-isomeric label comprising
the structure ##STR3## wherein [0027] F comprises a fluorescent
group; [0028] L is an alkyl linking group from 1 to 30 carbons in
length, wherein one or more carbon is optionally replaced or
substituted with a heteroatom selected from the group consisting of
N, S, O and P, and is optionally part of a ring system, and wherein
the alkyl linking chain optionally includes one or more site of
unsaturation; [0029] R.sub.1 is selected from the group consisting
of hydrogen, alkyl, and aryl; [0030] R.sub.2 is selected from the
group consisting of hydrogen, alkyl, and aryl; [0031] X is a
nucleotide, nucleic acid or a cleavable linker; [0032] Y is
selected from the group consisting of hydrogen, alkyl, and aryl;
[0033] Y.sub.2 is an alkyl chain; and [0034] Z is a nucleotide or
nucleic acid.
[0035] In one preferred group of embodiments, the nucleic acid
synthesis reagent has the structure ##STR4## wherein R.sub.1 is
selected from the group consisting of alkyl, aryl, and hydrogen;
R.sub.2 is selected from the group consisting of alkyl, and aryl;
and FL is a fluorescent moiety.
[0036] An example compound is fluorescein phosphoramidite 7.
##STR5##
[0037] In another preferred group of embodiments, the reagent has
the structure ##STR6## wherein [0038] L is an alkyl linking group
from 1 to 30 carbons in length, wherein one or more carbon is
optionally replaced or substituted with a heteroatom selected from
the group consisting of N, S, O and P, and is optionally part of a
ring system, and wherein the alkyl linking chain optionally
includes one or more site of unsaturation.
[0039] Most typically, the polymer arrays of the invention further
comprise cleavable linkers, often located proximal to the substrate
which the array is formed upon, to facilitate cleavage of the
polynucleotide from the array.
[0040] In a preferred embodiment of the invention, methods of
post-synthetically labeling an oligonucleotide array are provided.
In these methods, a polymer array which comprises a plurality of
polymers is provided, wherein each polymer in the array, or a
plurality of polymers in the array, include a labeling site to
which a detectable label such as a fluorophore is attached.
[0041] Most typically in this preferred embodiment, the polymers in
the array are synthesized on labeling linkers, which are most
typically attached to cleavable linkers proximal to the surface
upon which the array is synthesized. The labeling linkers include
attachment sites for the detectable label. During polymer synthesis
the labeling linker includes a protected site for the attachment of
the detectable label which is deprotected at a defined point in the
synthesis of the array (typically after the polymers in the array
are completely synthesized, and often after the polymers are
cleaved from the array at the cleavable linker) so that the
detectable moiety can be attached. For instance, where the
detectable reagent is a fluorescent phosphoramidite, the protected
site on the labeling linker will typically comprise an oxygen with
which the phosphate on the phosphoramidite will react to form a
phosphodiester linkage (i.e., after the oxygen is deprotected). DMT
is a preferred protecting group, although many others are also
suitable, depending on the nature of the group to be protected, the
polymer and the detectable moiety.
[0042] The post-synthetic labeling linker used in the method
typically has a site for polymer elongation, a site for attaching a
polymer to a substrate and an attachment site for attaching a
detectable label. Preferred labeling linkers are described herein.
In general, where the labeling linker is a nucleic acid synthesis
reagent, the labeling linker has the structure ##STR7## wherein:
[0043] R.sub.1 is selected from the group consisting of hydrogen,
alkyl and aryl; [0044] R.sub.2 is selected from the group
consisting of hydrogen, alkyl and aryl; [0045] R.sub.3 is selected
from the group consisting of hydrogen, alkyl and aryl; [0046]
L.sub.1 is a linking chain selected from the group of alkyl linking
chains consisting of an alkyl linking chain from 1 to 30 carbons in
length, wherein one or more carbon is optionally substituted with a
heteroatom selected from the group consisting of N, S, O and P, and
wherein the alkyl linking group optionally includes one or more
sites of unsaturation, and an alkyl linking chain from 1 to 30
carbons in length, wherein one or more carbon is optionally
replaced with a heteroatom selected from the group consisting of N,
S, O and P, and wherein the alkyl linking group optionally includes
one or more sites of unsaturation; [0047] L.sub.2 is a linking
chain selected from the group of alkyl linking chains consisting of
an alkyl linking chain from 1 to 30 carbons in length, wherein one
or more carbon is optionally substituted with a heteroatom selected
from the group consisting of N, S, O and P, and wherein the alkyl
linking group optionally includes one or more sites of
unsaturation, and an alkyl linking chain from 1 to 30 carbons in
length, wherein one or more carbon is optionally replaced with a
heteroatom selected from the group consisting of N, S, O and P, and
wherein the alkyl linking group optionally includes one or more
sites of unsaturation; [0048] Y is selected from the group
consisting of a dimethoxytrityl protecting group and a
photoclevable protecting group; [0049] Z is selected from the group
consisting of a dimethoxytrityl (DMT) protecting group and a
photoclevable protecting group; and [0050] X is a nucleic acid
integration element comprising a phosphorous atom.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 provides a synthesis scheme for the synthesis of
fluorescent amidite (7).
[0052] FIG. 2 provides a post-synthetic labeling scheme for
oligonucleotide probe arrays.
[0053] FIG. 3 provides a chromatogram of a fluorescein-labeled
T.sub.16 homopolymer.
DEFINITIONS
[0054] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al. (1994) Dictionary of Microbiology and Molecular
Biology, second edition, John Wiley and Sons (New York) and March
(March, Advanced Organic Chemistry Reactions, Mechanisms and
Structure 4th ed J. Wiley and Sons (New York, 1992) provides one of
skill with a general guide to many of the terms used in this
invention.
[0055] Although one of skill will recognize many methods and
materials similar or equivalent to those described herein which can
be used in the practice of the present invention, the preferred
methods and materials are described. For purposes of the present
invention, the following terms are defined below.
[0056] An "activating agent" refers to those groups which, when
attached to a particular functional group or reactive site, render
that site more reactive toward covalent bond formation with a
second functional group or reactive site. For example, the group of
activating groups which are useful for a carboxylic acid include
simple ester groups and anhydrides. The ester groups include alkyl,
aryl and alkenyl esters, and in particular such groups as
4-nitrophenyl, N-succinimidyl and pentafluorophenyl. Other
activating agents are known to those of skill in the art.
[0057] An "alkyl" or "lower alkyl" group refers to a saturated
hydrocarbon or hydrocarbon radical which includes a straight or
branched chain (for example, methyl, ethyl, isopropyl, t-amyl, or
2,5-dimethylhexyl). Preferred alkyl groups are those containing 1
to 15 carbon atoms or more preferably contain 1-6 carbon atoms
unless otherwise indicated (e.g., for certain alkyl linking groups
described herein, the optimal length of the alkyl chain is from 1
to 30 carbons in length). When "alkyl" or "alkylene" is used to
refer to a linking group or a spacer moiety, it is taken to be a
group having at least two available valences for covalent
attachment, for example, --H.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--,
--H.sub.2CH.sub.2CH(CH.sub.3)CH.sub.2-- and
--CH.sub.2(CH.sub.2CH2).sub.2CH.sub.2--. The hydrocarbon chain is
optionally substituted with heteroatoms such as N, O, P, and S, or
similarly, one or more carbons can be replaced with a heteroatom.
The alkyl chain optionally includes one or more site of
unsaturation (e.g., a C.dbd.C bond).
[0058] An "aryl" group as used herein, refers to an aromatic
substituent which is a single or multiple ring structure, linked
covalently or linked to a common group such as an ethylene or
methylene moiety. The aromatic rings may each contain heteroatoms,
for example, phenyl, naphthyl, biphenyl, diphenylmethyl,
2,2-diphenyl-1-ethyl, thienyl, pyridyl and qyinoxalyl. The aryl
moieties may also be optionally substituted with halogen atoms, or
other groups such as nitro, carboxyl, alkoxy, phenoxy and the like.
Additionally, the aryl radicals may be attached to other moieties
at any position on the aryl radical which would otherwise be
occupied by a hydrogen atom (such as, for example, 2-pyridyl,
3-pyridyl and 4-pyridyl). As used herein, the term "aralkyl" refers
to an alkyl group bearing an aryl substituent (for example, benzyl,
phenylethyl, 3-(4-nitrophenyl)propyl, and the like). All numerical
ranges in this specification and claims are intended to be
inclusive of their upper and lower limits.
[0059] The term "capping" in the context of synthesizing an array
of polymers refers to a step in which unreacted groups that fail to
condense and successfully couple with the next polymer synthesis
reagent (e.g., a monomer such as a phosphoramidite or amino acid)
are blocked. This insures that subsequent reactions proceed only by
propagating chains of desired sequence. For instance, capping
typically involves the acetylation of 5'-hydroxyl functions on
oligonucleotides. This is accomplished, e.g., using acetic
anhydride catalyzed by 4-dimethylaminopyridine (DMAP). Other
reagents known to those of skill in the art are also suitable.
[0060] The term "label" refers to a composition detectable by
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. For example, useful labels include .sup.32p,
.sup.35S, fluorescent dyes, chromophores, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin,
or haptens and proteins for which antisera or monoclonal antibodies
are available.
[0061] A "labeling linker" is a polymer synthesis reagent which
contains a labeling site and a polymer attachment site. The
chemical nature of the polymer attachment site is selected to be
compatible with the polymer to be synthesized. Thus, where the
polymer is an oligonucleotide, the polymer attachment site
comprises an oxygen or phosphate for attachment to the 3' OH or 5'
phosphate of the oligonucleotide. A "polymer labeling site" or
"labeling site" in the context of the labeling linker, or polymers
in a polymer array is a chemical site which is reactive with the
reactive groups in a label or synthetic labeling reagent. Thus, the
precise chemical composition of the site depends on the chemical
nature of the reactive site on the label. Thus, where the label
comprises a phosphate, the labeling site would optionally include
an oxygen (e.g., a double-bond O or hydroxyl), or other
phosphate-reactive moiety. It is assumed that one of skill is
familiar with many chemical sites which react to form a chemical
bond, including those which occur during nucleic acid synthesis and
polynucleotide synthesis. In many embodiments, the labeling site is
protected with a protecting group. It is generally understood that
nucleic acid reagents carry a protected phosphate or hydroxyl group
in order to form a phosphodiester linkage.
[0062] In preferred embodiments, the labeling linker comprises two
polymer attachment sites, such that the polymer is extended from
the labeling linker. In many embodiments, the labeling linker is
attached to a cleavable linker at one end, and to a polymer at the
other end. Thus, in this embodiment, the labeling linker has three
attachment sites: one site for forming a chemical bond to the
cleavable linker, one site for forming a chemical bond to a monomer
from which the polymer is elongated, and one site for forming a
chemical bond to the detectable label.
[0063] A "nucleic acid reagent" is a molecule which can be used for
oligonucleotide synthesis. The molecules typically carry protected
phosphates and/or protected oxygen moieties. For instance, nucleic
acid reagents include nucleotide reagents, nucleoside reagents,
nucleoside phosphates, nucleoside-3'-phosphates, nucleoside
phosphoramidites, phosphoramidites, nucleoside phosphonates,
phosphonates, methyl phosphonates, O-methyl phosphates etc. It is
generally understood that nucleotide reagents carry a protected
phosphate or hydroxyl group in order to form a phosphodiester
linkage.
[0064] A "nucleoside" is a pentose glycoside in which the aglycone
is a heterocyclic base; upon which the addition of a phosphate
group the compound becomes a "nucleotide." The major biological
nucleosides are .beta.-glycoside derivatives of D-ribose or
D-2-deoxyribose. Nucleotides are phosphate esters of nucleosides
which are generally acidic in solution due to hydroxyl groups on
the phosphate. The nucleosides of the polymeric nucleic acids DNA
and RNA are connected together via phosphate units attached to the
3' position of one pentose and the 5' position of the next pentose.
Nucleotide analogues and/or nucleoside analogues are molecules with
structural similarities to the naturally occurring nucleotides or
nucleosides. Means of converting a nucleoside to a phosphoramidite
are well known to those of skill in the art. See, for example,
Atkinson et al., chapter 3, in Gait, ed., Oligonucleotide
Synthesis: A Practical Approach (IRL Press, Washington, D.C.,
1984), which is incorporated herein by reference, and McBride and
Caruthers, Tetrahedron Lett., 24:245 (1983). See also Blackburn and
Gait (eds.) (1990) Nucleic Acids in Chemistry and Biology, IRL
press, New York.
[0065] A "nucleic acid" is a deoxyribonucleotide or ribonucleotide
polymer in either single- or double-stranded form, and unless
otherwise limited, encompasses known analogs of natural nucleotides
that function in a manner similar to naturally occurring
nucleotides (See, copending application U.S. Ser. No. 06/440742 for
a description of nucleic acid analogues).
[0066] An "oligonucleotide" is a nucleic acid polymer composed of
two or more nucleotides or nucleotide analogues. An oligonucleotide
can be derived from natural sources but is often synthesized
chemically. It is of any size. Copending application U.S. Ser. No.
06/440742 describes a variety of oligonucleotide analogues.
[0067] A "polymer" refers to a chain of monomers, typically
connected through chemical or electrostatic interactions. Monomers
include, but are not limited to, biological monomers such as
L-amino acids, D-amino acids, synthetic amino acids, nucleotides,
nucleosides, phosphoramidites, and carbohydrates, as well as
non-biological monomers which are connected to form polymers. As
used herein, monomer refers to any unit of a polymer. For example,
dimers of the 20 naturally occurring L-amino acids form 400
monomeric units for synthesis of polypeptides. Different monomeric
units are optionally used at any site in the polymer. Each
monomeric unit optionally includes protected members which are
optionally modified after polymerization. Biological polymers
include, but are not limited to, agonists and antagonists of cell
membrane receptors, toxins and venoms, viral epitopes, hormones
(e.g., opiates, steroids, etc.), hormone receptors, peptides,
retro-inverso peptides, polymers of .alpha.-, .beta.-, or
.omega.-amino acids (D- or L-), enzymes, enzyme substrates,
cofactors, drugs, lectins, sugars, nucleic acids (both linear and
cyclic polymer configurations), oligosaccharides, proteins,
phospholipids and antibodies. Synthetic polymers such as
heteropolymers in which a known drug is covalently bound to any of
the above, such as polyurethanes, polyesters, polycarbonates,
polyureas, polyamides, polyethyleneimines, polyarylene sulfides,
polysiloxanes, polyimides, and polyacetates are also included.
Other polymers will also be apparent to one of skill upon review of
this disclosure.
[0068] A "peptide" or "polypeptide" or "protein" refers to a
polymer of amino acids. Typically the monomers are alpha amino
acids which are joined together through amide bonds. In the context
of this specification, the L-optical isomer and the D-optical
isomer are both contemplated, unless otherwise indicated. Standard
abbreviations for amino acids are used (e.g., P for proline). These
abbreviations are included, e.g., in Stryer, Biochemistry, Third
Edition, 1988.
[0069] A "polymer array" is a spatially defined pattern of polymers
on a solid support. A "preselected array of polymers" is a
spatially defined pattern of polymers on a solid support which is
designed before being constructed (i.e., the arrangement of
polymers on solid substrate during synthesis is deliberate, and not
random).
[0070] A "protecting group" as used herein, refers to any of the
groups which are designed to block one reactive site in a molecule
while a chemical reaction is carried out at another reactive site.
More particularly, the protecting groups used herein can be any of
those groups described in Greene, et al., Protective Groups In
Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y.,
1991, which is incorporated herein by reference. The proper
selection of protecting groups for a particular synthesis is
governed by the overall methods employed in the synthesis. For
example, in "light-directed" synthesis, discussed herein, the
protecting groups are typically photolabile protecting groups such
as NVOC, MeNPoc, and those disclosed in co-pending Application
PCT/US93/10162 (filed Oct. 22, 1993), incorporated herein by
reference. In other methods, protecting groups are removed by
chemical methods and include groups such as FMOC, DMT and others
known to those of skill in the art.
[0071] The term "protected amino acid" refers to an amino acid,
typically an .alpha.-amino acid having either or both the amine
functionality and the carboxylic acid functionality suitably
protected by one of the groups described above. Additionally, for
those amino acids having reactive sites or functional groups on a
side chain (i.e., serine, tyrosine, glutamic acid), the term
"protected amino acid" is meant to refer to those compounds which
optionally have the side chain functionality protected as well.
[0072] A "solid substrate" has a fixed organizational support
matrix, such as silica, polymeric materials, or glass. In some
embodiments, at least one surface of the substrate is partially
planar. In other embodiments it is desirable to physically separate
regions of the substrate to delineate synthetic regions, for
example with trenches, grooves, wells or the like. Example of solid
substrates include slides, beads and polymeric chips. A solid
support is "functionalized" to permit the coupling of monomers used
in polymer synthesis. For example, a solid support is optionally
coupled to a nucleoside monomer through a covalent linkage to the
3'-carbon on a furanose. Solid support materials typically are
unreactive during polymer synthesis, providing a substratum to
anchor the growing polymer. Solid support materials include, but
are not limited to, glass, polyacryloylmorpholide, silica,
controlled pore glass (CPG), polystyrene, polystyrene/latex, and
carboxyl modified teflon. The solid substrates are biological,
nonbiological, organic, inorganic, or a combination of any of
these, existing as particles, strands, precipitates, gels, sheets,
tubing, spheres, containers, capillaries, pads, slices, films,
plates, slides, etc. depending upon the particular application. In
light-directed synthetic techniques, the solid substrate is often
planar but optionally takes on alternative surface configurations.
For example, the solid substrate optionally contains raised or
depressed regions on which synthesis takes place. In some
embodiments, the solid substrate is chosen to provide appropriate
light-absorbing characteristics. For example, the substrate may be
a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge,
GaAs, GaP, SiO.sub.2, SiN.sub.4, modified silicon, or any one of a
variety of gels or polymers such as (poly)tetrafluoroethylene,
(poly)vinylidendifluoride, polystyrene, polycarbonate, or
combinations thereof. Other suitable solid substrate materials will
be readily apparent to those of skill in the art. Preferably, the
surface of the solid substrate will contain reactive groups, such
as carboxyl, amino, hydroxyl, thiol, or the like. More preferably,
the surface is optically transparent and has surface Si--OH
functionalities, such as are found on silica surfaces. A substrate
is a material having a rigid or semi-rigid surface. In spotting or
flowing VLSIPS.TM. techniques, at least one surface of the solid
substrate is optionally planar, although in many embodiments it is
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 regions upon which polymer synthesis occurs.
DETAILED DESCRIPTION
[0073] The present invention provides methods and compositions for
monitoring and optimizing polymer array synthesis. The methods
presented herein typically proceed by incorporating a detectable
label into polymers in arrays on a solid substrate. The labeled
polymers in the array are then cleaved from the solid surface,
typically by cleaving a cleavable linker which attaches the
polymers of the array to the solid surface. The polymers are then
analyzed by monitoring the detectable label in an appropriate
assay, i.e., determined by the label. A variety of analytic assays
such as HPLC, gel electrophoresis or capillary gel electrophoresis
(CGE) are contemplated.
[0074] One aspect of interest during the manufacture of a
VLSIPS.TM. array is the efficiency of polymer synthesis in the
array, including the length distribution of synthesized species and
the presence, nature and extent of truncated species. Measuring the
size and/or electrostatic charge of polymers cleaved from an array,
and comparing the measurements to the predicted size and/or charge
of the polymers provides a measure of the efficiency of polymer
synthesis. Varying synthesis protocols and comparing the resulting
polymer arrays also provides an efficient empirical strategy for
optimizing the procedures for producing polymer arrays.
[0075] The effect of environmental, synthetic, or experimental
conditions on polymer arrays is also determined by the methods
presented herein. In one class of embodiments, a reference polymer
array and a test polymer array are synthesized using identical
procedures. The reference polymer array is cleaved from the solid
support after synthesis and analyzed as described herein. The test
polymer array is subjected to defined additional environmental
conditions such as immersion in an aqueous, acidic, or basic
solution, or exposure to nucleases or peptidases, and then
analyzed. By comparing the polymers of the array (or of identical
sets of arrays) before and after exposure to the defined
environmental conditions, the stability and durability of the array
is determined.
[0076] Although essentially any detectable label can be used, in
preferred embodiments the label is mono-isomeric, i.e., the label
has only a single optical isomer. The use of mono-isomeric labels
avoids any ambiguity in monitoring the size or charge of polymers
in an array caused by having an enantiomeric or diastereomeric
label. The use of mono-isomeric labels is particularly useful when
the detection method is extremely sensitive. For instance, the use
of mono-isomeric labels when the detection method is HPLC is
particularly preferred.
Synthesis of Polymer Arrays
[0077] The present invention relates to the creation of labeled
polymer arrays. The synthesis of polymer arrays generally is known.
The development of very large scale immobilized polymer synthesis
(VLSIPS.TM.) technology provides methods for arranging large
numbers of polymer probes in very small arrays. Pirrung et al.,
U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070),
McGall et al., U.S. Ser. No. 06/440,742, Chee et al., USSN
PCT/US94/12305, and Fodor et al., PCT Publication No. WO 92/10092
describe methods of forming vast arrays of peptides,
oligonucleotides and other polymers using, for example,
light-directed synthesis techniques. See also, U.S. Ser. Nos.
07/796,243 and 07/980,523, Fodor et al., (1991) Science
251:767-777, and U.S. Ser. No. 08/327,687.
[0078] As described above, diverse methods of making polymer arrays
are known; accordingly no attempt is made to describe or catalogue
all known methods. For exemplary purposes, light directed
VLSIPS.TM. methods are briefly described below. One of skill will
understand that alternate methods of creating polymer arrays, such
as spotting and/or flowing reagents over defined regions of a solid
substrate, bead based methods and pin-based methods are also known
and applicable to the present invention (See, Holmes et al. (filed
Jan. 17, 1995) U.S. Ser. No. 08/374,492).
[0079] Light directed VLSIPS.TM. methods are found, e.g., in U.S.
Pat. No. 5,143,854. The light-directed methods discussed in the
'854 patent typically proceed by activating predefined regions of a
substrate or solid support and then contacting the substrate with a
preselected monomer solution. The predefined regions are activated
with a light source, typically shown through a photolithographic
mask. Other regions of the substrate remain in active 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
contacting different monomer solutions with the substrate, a
diverse array of polymers is produced on the substrate. Other
steps, such as washing unreacted monomer solution from the
substrate, are used as necessary.
[0080] The surface of a solid support is typically modified with
linking groups having photolabile protecting groups (e.g., NVOC or
MeNPOC) and illuminated through a photolithographic mask, yielding
reactive groups (e.g., typically hydroxyl groups when the polymer
array is an oligonucleotide array) in the illuminated regions. For
instance, during oligonucleotide synthesis, a 3'-O-phosphoramidite
(or other nucleic acid synthesis reagent) activated deoxynucleoside
(protected at the 5'-hydroxyl with a photolabile group) is then
presented to the surface and coupling occurs at sites that were
exposed to light in the previous step. Following capping and
oxidation, the substrate is rinsed and the surface illuminated
through a second mask, to expose additional hydroxyl groups for
coupling. A second 5'-protected, 3'-O-phosphoramidite activated
deoxynucleoside (or other monomer as appropriate) is then presented
to the resulting array. The selective photodeprotection and
coupling cycles are repeated until the desired set of
oligonucleotides (or other polymers) is produced.
Making Polymers To Be Coupled Into Arrays
[0081] As described above, several methods for the synthesis of
polymer arrays are known. In preferred embodiments, the polymers
are synthesized directly on a solid surface as described above.
However, in certain embodiments, it is useful to synthesize the
polymers and then couple the polymers to the solid substrate to
form the desired array. In these embodiments, polymers are
synthesized (in vitro or in vivo, in solution, or using solid phase
chemistry) and then attached to a solid substrate in a desired
pattern to form the desired array on the solid substrate.
[0082] Molecular cloning and expression techniques for the
synthesis of biological and synthetic polymers in solution are
known in the art. A wide variety of cloning and expression and in
vitro methods suitable for the construction of polymers are
well-known to persons of skill. Examples of techniques and
instructions sufficient to direct persons of skill through many
cloning exercises for the expression and purification of biological
polymers (DNA, RNA, proteins) are found in Berger and Kimmel, Guide
to Molecular Cloning Techniques, Methods in Enzymology volume 152
Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.
(1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3,
Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York,
(Sambrook); and Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(1994 Supplement) (Ausubel).
[0083] Examples of techniques sufficient to direct persons of skill
through in vitro methods of polymer synthesis in solution,
including enzymatic methods such as the polymerase chain reaction
(PCR), the ligase chain reaction (LCR), Q.beta.-replicase
amplification (QBR), nucleic acid sequence based amplification
(NASBA), strand displacement amplification (SDA), the cycling probe
amplification reaction (CPR), branched DNA (bDNA) and other DNA and
RNA polymerase mediated techniques are known. Examples of these and
related techniques are found in Berger, Sambrook, and Ausubel, as
well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR
Protocols A Guide to Methods and Applications (Innis et al. eds)
Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim &
Levinson (Oct. 1, 1990); WO 94/11383; Vooijs et al. (1993) Am J.
Hum. Genet. 52: 586-597; C&EN 36-47; The Journal Of NIH
Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad.
Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;
Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990)
Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560;
Sooknanan and Malek (1995) Bio/Technology 13, 563-564; Walker et
al. Proc. Natl. Acad. Sci. USA 89, 392-396), and Barringer et al.
(1990) Gene 89, 117. Improved methods of cloning in vitro amplified
nucleic acids are described in Wallace et al., U.S. Pat. No.
5,426,039.
[0084] Methods of producing polymers in vitro and in vivo, such as
polyclonal and monoclonal antibodies are also known to those of
skill in the art. See, e.g., Coligan (1991) Current Protocols in
Immunology Wiley/Greene, New York; and Harlow and Lane (1989)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, New York;
Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange
Medical Publications, Los Altos, Calif., and references cited
therein; Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.) Academic Press, New York, N.Y.; and Kohler and
Milstein (1975) Nature 256: 495-497. Other suitable techniques for
antibody preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse et al. (1989)
Science 246: 1275-1281; and Ward, et al. (1989) Nature 341:
544-546.
[0085] Solid phase synthesis of polymers, including biological
polymers is also known. See, e.g., Merrifield (1963) J. Am. Chem.
Soc. 85: 2149-2154. Solid-phase synthesis techniques have also been
provided for the synthesis of peptide sequences on, for example, a
number of "pins." See e.g., Geysen et al. (1987) J. Immun. Meth.
102: 259-274 and Holmes et al (filed Jan. 17, 1995) Ser. No.
08/374, 492. Other solid-phase techniques involve, for example,
synthesis of various peptide sequences on cellulose disks supported
in a column. See Frank and Doring (1988) Tetrahedron 44: 6031-6040.
Still other solid-phase techniques are described in U.S. Pat. No.
4,728,502 (Hamill) and WO 90/00626 (Beattie).
[0086] Oligonucleotide synthesis is optionally performed on
commercially available solid phase oligonucleotide synthesis
machines (see, Needham-VanDevanter et al. (1984) Nucleic Acids Res.
12:6159-6168) or manually synthesized using the solid phase
phosphoramidite triester method described by Beaucage et al.
(Beaucage et al. (1981) Tetrahedron Letts. 22 (20): 1859-1862)
prior to attachment on a solid substrate. Bead-based synthetic
techniques are described in copending application U.S. Ser. No.
07/762,522 (filed Sep. 18, 1991); U.S. Ser. No. 07/946,239 (filed
Sep. 16, 1992); U.S. Ser. No. 08/146,886 (filed Nov. 2, 1993); U.S.
Ser. No. 07/876, 792 (filed Apr. 29, 1992); PCT/US93/04145 (filed
Apr.28, 1993); and Holmes et al. (filed Jan. 17, 1995) U.S. Ser.
No. 08/374,492. Finally, as described above, polymers are
optionally synthesized using VLSIPS.TM. methods in arrays, or
optionally cleaved from the array and then reattached to a solid
substrate to form a second array.
Cleavable Linkers
[0087] Cleavable linking groups used in VLSIPS.TM. and other solid
phase synthetic techniques are known. Typically, linking groups are
used to attach polymers or labeled polymers during the organic
synthesis of polymer arrays. In addition, in some embodiments,
polymer arrays are used prior to cleavage from the arrays,
typically in aqueous hybridization experiments. Thus, preferred
linkers operate well under organic and aqueous conditions, but
cleave readily under specific cleavage conditions. The linker is
typically provided with a spacer having active cleavable sites. In
the particular case of oligonucleotides, for example, the spacer is
selected from a variety of molecules which can be used in organic
environments associated with synthesis as well as aqueous
environments, e.g., associated with nucleic acid binding studies.
Examples of suitable spacers are polyethyleneglycols, dicarboxylic
acids, polyamines and alkylenes, substituted with, for example,
methoxy and ethoxy groups. Linking groups which facilitate polymer
synthesis on solid supports and which provide other advantageous
properties for biological assays are known. In some embodiments,
the linker provides for a cleavable function by way of, for
example, exposure to an acid or base.
[0088] Additionally, the linkers have an active site on the distal
end, relative to the attachment of the linker to the solid
substrate. The active sites are optionally protected during polymer
synthesis using protecting groups. Among a wide variety of
protecting groups which are useful are nitroveratryl (NVOC)
.alpha.-methylnitroveratryl (Menvoc), allyloxycarbonyl (ALLOC),
fluorenylmethoxycarbonyl (FMOC),
.alpha.-methylnitro-piperonyloxycarbonyl (MeNPOC), --NH-FMOC
groups, t-butyl esters, t-butyl ethers, and the like as described,
e.g., by Holmes et al. (id). Various exemplary protecting groups
are described in, for example, Atherton et al., (1989) Solid Phase
Peptide Synthesis, IRL Press, and Greene, et al. (1991) Protective
Groups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New
York, N.Y. The proper selection of protecting groups for a
particular synthesis is governed by the overall methods employed in
the synthesis. For example, in "light-directed" synthesis,
discussed herein, the protecting groups are photolabile protecting
groups such as NVOC, MeNPoc, and those described in co-pending
Application PCT/U593/10162 (filed Oct. 22, 1993). See also, Holmes
et al. (supra); Wang (1976) J. Org. Chem. 41:3258; and Rich, et al.
(1975) J. Am. Chem. Soc. 97:1575-1579. In other methods, protecting
groups are removed chemically, and include groups such as FMOC,
di(p-methoxyphenyl)phenyl (DMT) and others known to those of skill
in the art. See, Holmes et al. (supra).
[0089] Some of the linking groups are hydrophilic and provide a
"wettable" surface which aids in synthesis of the polymers as well
as in screening of the polymers for activity. Other linking groups
are more hydrophobic. A variety of hydrophobic and hydrophilic
cleavable linkers suitable for use in polymer array synthesis are
described in the patents and applications relating to VLSIPS.TM.
synthesis described supra.
[0090] The linker attaches to the solid substrate through a variety
of chemical bonds. For example, the linker is optionally attached
to the solid substrate using carbon-carbon bonds, for example via
substrates having (poly)trifluorochloroethylene surfaces, or
siloxane bonds (using, for example, glass or silicon oxide as the
solid substrate). Siloxane bonds with the surface of the substrate
are formed in one embodiment via reactions of derivatization
reagents bearing trichlorosilyl or trialkoxysilyl groups.
[0091] The particular linking group is selected based upon, e.g.,
its hydrophilic/hydrophobic properties where presentation of an
attached polymer in solution is desirable. Groups which are
suitable for attachment to a linking group include amine, hydroxyl,
thiol, carboxylic acid, ester, amide, isocyanate and
isothiocyanate. Preferred derivatizing groups include
aminoalkyltrialkoxysilanes, hydroxyalkyltrialkoxysilanes,
polyethyleneglycols, polyethyleneimine, polyacrylamide,
polyvinylalcohol and combinations thereof.
Labeling of Polymers
[0092] In preferred embodiments, the present invention proceeds by
labeling polymers in arrays, which are then cleaved from the arrays
and analyzed. A variety of labels are appropriate and known. A
"label" comprises a moiety which is detectable by spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. For
example, useful labels include .sup.32P, fluorescent dyes,
electron-dense reagents, enzymes (e.g., as commonly used in an
ELISA), biotin, dioxigenin, haptens and proteins. In preferred
embodiments, the label is detectable spectroscopically, i.e., is
chromogenic. Suitable chromogens include molecules and compounds
which absorb light in a distinctive range of wavelengths so that a
color may be observed, or emit light when irradiated with radiation
of a particular wavelength or wavelength range (e.g., a fluorescent
label). In preferred embodiments, labels of the present invention
have the structure A-B, where A is a detectable moiety, and B is a
"linking" or "bridging" group which comprises one or more
functional regions which allow the detectable moiety to be
incorporated into a polymer, or attached to one end of the polymer,
using chemistry similar to that used to connect monomers into the
polymer. Examples of suitable bridging regions include alkyl and
substituted alkyl carbon chains with 1-30 carbons, or more
preferably 3-10 carbons, with functional groups such as oxygen and
phosphate (i.e., when the polymer is an oligonucleotide) or amino
and carboxyl (i.e., where the polymer is a peptide).
[0093] For example, in preferred embodiments where the label is a
nucleic acid synthesis reagent to be incorporated into a nucleic
acid, the bridging group has the structure ##STR8## wherein [0094]
L is an alkyl linking group from 1 to 30 carbons in length, wherein
one or more carbon is optionally replaced or substituted with a
heteroatom selected from the group consisting of N, S, O and P, and
is optionally part of a ring system, and wherein the alkyl linking
chain optionally includes one or more site of unsaturation; [0095]
R.sub.1 is selected from the group consisting of hydrogen, alkyl,
and aryl; [0096] R.sub.2 is selected from the group consisting of
hydrogen, alkyl, and aryl; [0097] X is a nucleic acid integration
element comprising a phosphorous atom; [0098] Y is selected from
the group consisting of hydrogen, alkyl, and aryl; [0099] Y.sub.2
is an alkyl chain; and [0100] Z comprises a protecting group.
[0101] In more preferred embodiments in which the label is a
nucleic acid synthesis reagent to be incorporated into a nucleic
acid, the bridging group has the structure ##STR9## wherein [0102]
L is an alkyl linking group from 1 to 30 carbons in length, wherein
one or more carbon is optionally replaced or substituted with a
heteroatom selected from the group consisting of N, S, O and P, and
is optionally part of a ring system, and wherein the alkyl linking
chain optionally includes one or more site of unsaturation.
Typically L is joined to a detectable moiety such as a fluorophore.
R.sub.1 is selected from the group consisting of hydrogen, alkyl,
and aryl, and more preferably is selected from the group consisting
of hydrogen or methyl.
[0103] In preferred embodiments, the nucleic acid synthesis reagent
(A-B, i.e., the detectable moiety and the integration or bridging
element) has the structure ##STR10## wherein L is an alkyl linking
group from 1 to 30 carbons in length, wherein one or more carbon is
optionally replaced or substituted with a heteroatom selected from
the group consisting of N, S, O and P, and is optionally part of a
ring system, and wherein the alkyl linking chain optionally
includes one or more site of unsaturation; R.sub.1 is selected from
the group consisting of hydrogen, alkyl, and aryl; R.sub.2 is
selected from the group consisting of hydrogen, alkyl, and aryl; X
is a nucleic acid integration element comprising a phosphorous
atom; Y is selected from the group consisting of hydrogen, alkyl,
or aryl; Y.sub.2 is an alkyl chain from 1 to 30 carbons in length;
Z comprises a protecting group; and F comprises a fluorescent
group.
[0104] In a still more preferred embodiment, the nucleic acid
synthesis reagent has the structure ##STR11## wherein R.sub.1 is
selected from the group consisting of alkyl, aryl, and hydrogen;
R.sub.2 is selected from the group consisting of hydrogen, alkyl
and aryl; and FL is a fluorescent moiety. An example of such a
nucleic acid synthesis reagent label is the isomeric nucleic acid
synthesis reagent with the structure ##STR12##
[0105] In particularly preferred embodiments, the label is
mono-isomeric (or more simply "isomeric"), i.e., when incorporated
into a polymer does not provide multiple isomeric species, e.g., as
detected by HPLC. The Examples below describe a novel fluorescent
label (fluorescein phosphoramidite 7, See below) which is isomeric.
A variety of suitable labels are constructed by converting existing
detectable groups, including quinoline, triarylmethane, acridine,
alizarine, phthaleins, azo labels, anthraquinoid tags, cyanine,
phenazathionium, and phenazoxonium into mono-isomeric forms. In one
set of preferred embodiments (see, e.g., Example 1, below), a
mono-isomeric detectable moiety is connected to a mono-isomeric
bridging region and appropriate functional groups which allow for
incorporation of the label into a polymer by the same chemistry as
that used to connect the monomers of a polymer. For instance, amino
acids are optionally used to form the linking regions of labels,
with suitable groups being added for incorporation of the label
into the polymer (e.g., hydroxy and phosphate groups are attached
to the linking region for incorporation into oligonucleotides (see,
Example 1 below), or attached to amino and carboxy groups for
incorporation into peptides). Amino acid linking regions are
preferred because of the ready availability of mono-isomeric amino
acids (e.g., D or L isomers) and the presence of reactive
functional groups.
[0106] A variety of fluorescent groups are suitable, or are
rendered suitable by converting the group to a .mono-isomeric form,
either alone or in conjunction with quencher molecules. Suitable
fluorescent labels are typically characterized by one or more of
the following: high sensitivity, high stability, low background,
low environmental sensitivity and high specificity in labeling. As
described herein, suitable labels are also preferably
mono-isomeric.
[0107] Fluorescent moieties, which are incorporated into the labels
of the invention, or are converted into mono-isomeric forms and
then incorporated into the present invention, are generally are
known, including 1- and 2-aminonaphthalene, p,p'-diaminostilbenes,
pyrenes, quaternary phenanthridine salts, 9-aminoacridines,
p,p'-diaminobenzophenone imines, anthracenes, oxacarbocyanine,
merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole,
bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,
bis-3-aminopyridiium salts, hellebrigenin, tetracycline,
sterophenol, benzimidazolylphenylamine, 2-oxo-3-chromen, indole,
xanthen, 7-hydroxycoumarin, phenoxazine, calicylate,
strophanthidin, porphyrins, triarylmethanes and flavin. Individual
fluorescent compounds which have functionalities for linking or
which can be modified to incorporate such functionalities include,
e.g., dansyl chloride; fluoresceins such as
3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate;
N-phenyl 1-amino-8-sulfonatonaphthalene; N-phenyl
2-amino-6-sulfonatonaphthalene;
4-acetamido-4-isothiocyanatostilbene2,2'-disulfonic acid;
pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6sulfonate;
N-phenyl-N-methyl-2-aminoaphthalene-6-sulfonate; ethidium bromide;
stebrine; auromine-0,2-(9'-anthroyl)palmitate; dansyl
phosphatidylethanolamine; N,N'-dioctadecyl oxacarbocyanine;
NN,N'-dihexyl oxacarbocyanine; merocyanine, 4-(3'pyrenyl)stearate;
d-3-aminodesoxy-equilenin; 12-(9'-anthroyl)stearate;
2-methylanthracene; 9-vinylanthracene;
2,2'(vinylene-p-phenylene)bisbenzoxazole;
p-bis(2-(4-methyl-5-phenyl-oxazolyl))benzene;
6-dimethylamino-1,2-benzophenazin; retinol; bis(3'-aminopyridinium)
1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin;
chlorotetracycline;
N-(7-dimethylamino-4-methyl-2-oxo-3chromenyl)maleimide;
N-(p-(2-benzimidazolyl)-phenyl)maleimide;
N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin;
4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin;
rose bengal; and 2,4-diphenyl-3(2H)-furanone. Many fluorescent tags
are commercially available from SIGMA chemical company (Saint
Louis, Mo.), Molecular Probes, R&D systems (Minneapolis,
Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH
Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich
Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL
Life Technologies, Inc. (Gaithersberg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
and Applied Biosystems (Foster City, Calif.) as well as other
commercial sources known to one of skill.
[0108] Desirably, fluorescent labels should absorb light above
about 300 nm, preferably about 350 nm, and more preferably above
about 400 nrm, usually emitting at wavelengths greater than about
10 nm higher than the wavelength of the light absorbed. It should
be noted that the absorption and emission characteristics of the
bound label may differ from the unbound label. Therefore, when
referring to the various wavelength ranges and characteristics of
the labels, it is intended to indicate the labels as employed and
not the label which is unconjugated and characterized in an
arbitrary solvent.
[0109] Fluorescent labels are generally preferred, in part because
by irradiating a fluorescent label with light, one can obtain a
plurality of emissions. Thus, a single label can provide for a
plurality of measurable events. Detectable signal may also be
provided by chemiluminescent and bioluminescent sources.
Chemiluminescent sources include a compound which becomes
electronically excited by a chemical reaction and may then emit
light which serves as the detectible signal or donates energy to a
fluorescent acceptor. A diverse number of families of compounds
have been found to provide chemiluminescence under a variety or
conditions. One family of compounds is 2,3-dihydro-
1,4-phthalazinedione. The most popular compound is luminol, which
is the 5-amino compound. Other members of the family include the
5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog.
These compounds can be made to luminesce with alkaline hydrogen
peroxide or calcium hypochlorite and base. Another family of
compounds is the 2,4,5-triphenylimidazoles, with lophine as the
common name for the parent product. Chemiluminescent analogs
include para-dimethylamino and -methoxy substituents.
Chemiluminescence may also be obtained with oxalates, usually
oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g.,
hydrogen peroxide, under basic conditions. Other useful
chemiluminescent compounds are also known and available, including
--N-alkyl acridinum esters (basic H.sub.2O.sub.2) and dioxetanes.
Alternatively, luciferins may be used in conjunction with
luciferase or lucigenins to provide bioluminescence.
Isomerism
[0110] If a molecule is not superimposable on its mirror image, it
is chiral (optically active). If a molecule can be superimposed on
itself, it is achiral (optically inactive). Mirror image molecules
are referred to as enantiomers. A molecule can have one or more
chiral center, typically carbon, nitrogen or phosphorous atoms with
unique substituents (i.e., none of the substituents on a chiral
atom can be the same as another substituent on the same atom).
Molecules in compounds with more than one chiral center are
referred to as diastereomers. The properties of diastereomers are
typically similar, but not identical. For instance, diastereomers
typically interact with other compounds in different ways
(particularly chiral compounds, and particularly at chiral centers
within compounds), and migrate through size or charge matrices at
different rates. One effect of the difference between properties of
diastereomers is that they often migrate through HPLC columns at
different rates. Thus, the use of diastereomer labels in the
present invention makes resolution by size of polymers after
cleavage from a solid substrate more difficult using techniques
such as column chromatography, HPLC, capillary electrophoresis, or
gel electrophoresis.
[0111] Accordingly, a preferred embodiment of the present invention
utilizes a single optical isomer of all the possible diastereomers
of a particular molecule as a label. Methods of purifying
diastereomers, and methods of purifying enantiomers used to make
diastereomers (i.e., "asymmetric synthesis") are known in the art.
March (1992) Advanced Organic Chemistry: Reactions, Mechanisms and
Structure Fourth Edition, John Wiley and Sons and the references
therein, particularly chapter 4, and Lide (ed) CRC Handbook of
Chemistry and Physics 75.sup.th edition and the references therein
provide a general guide for the purification of stereoisomers.
Briefly, a pair of enantiomers can be separated by reaction with a
stereoselective reagent, reactions in the presence of circularly
polarized light, or, most commonly, by conversion to diastereomers
and subsequent purification. Typically, enatiomers are converted to
diastereomers by reaction with an optically active acid or base to
form a diastereomeric salt. The diastereomeric salt is crystalized
into distinct diastereomers, and separated by distillation, gas
chromatography, HPLC, or preparative liquid chromatography as
appropriate. Similarly, diasteromers in general are separated by
distillation, gas chromatography, HPLC or preparative liquid
chromatography.
Post-Syntheti Labeling of Polymers
[0112] In one preferred class of embodiments, the present invention
provides for post-synthetic labelling of polymer arrays. As
described herein, the labeling of polymers in arrays is optionally
performed by adding the label at the end of the polymer proximal to
the surface to which the polymers of the array are attached, to the
end distal to the surface, or as a monomeric building block of the
polymer at any position within the polymer. In certain
applications, it will be preferable to label the polymers of the
array at the end of the array distal to the attachment surface,
i.e., after the synthesis of the polymers in the array. This is
especially useful where the chemistry used for synthesizing the
polymer is not compatible with the addition of monomers of the
polymer to the labeling reagent.
[0113] In one preferred class of embodiments, a second method for
adding the detectable moiety after the polymer is synthesized is
provided. In this method, a labeling linker is incorporated into
the polymer at a chosen site, and a detectable moiety (e.g.,
fluorophore or chromogenic agent) is added to the polymer by
attachment to the labeling linker. FIG. 2 shows one application of
this strategy. ##STR13##
[0114] In this preferred embodiment, a labeling linker is attached
to a cleavable linker bound to a solid substrate. The labeling
linker has a site with appropriate chemistry to link with the
cleavable linker, a second site with appropriate chemistry for
adding monomers for polymer elongation, and a third site with
appropriate chemistry for linking with the detectable moiety. After
polymer synthesis, the detectable fluorophore is added to the site
within the labeling linker with the appropriate chemistry for
binding the fluorophore.
[0115] In this preferred embodiment, the site in the labeling
linker which accepts the fluorophore is protected by an appropriate
protecting group such as DMT until the fluorophore is added. The
protecting group is then removed, for example by reduction, and the
fluorophore is added, for example by a condensation reaction. The
polymer is then optionally cleaved from the array by cleaving the
cleavable linker, and then analyzed by the methods described herein
(HPLC, capillary gel electrophoresis, etc.).
[0116] For clarity, FIG. 2 is directed to a post-synthetic labeling
scheme for oligonucleotide arrays; however, one of skill will
readily appreciate that the same strategy (i.e., the use of a
labeling linker with sites for polymer elongation, attachment to a
surface or cleavable moiety, and for binding a fluorophore or other
S detectable moiety), is also useful for in the synthesis and
detection of polypeptide arrays, as well as other polymer arrays.
One of skill will also appreciate that the labeling linker is
optionally incorporated into the polymer chain, or located at the
end of the polymer distal to the synthesis surface, depending on
the desired application.
[0117] The labeling linker optionally has additional sites. For
example, the labeling linker can incorporate a cleavable
functionality, i.e., the labeling linker can be constructed to
incorporate a cleavable site to allow separation from an array
substrate. The labeling linkers can also have more than one site
for the attachment of detectable moieties.
[0118] In one preferred embodiment, the labeling linker has the
structure shown below.
Linker For Post Synthetic Fluorescent Labeling of Probe Arrays On
the VLSIP Surface
[0119] ##STR14##
[0120] A synthesis scheme for synthesizing the labeling linker is
provided below. ##STR15##
[0121] Another exemplar linker for post-synthetic labeling of
oligonucleotide arrays has the chemical structure shown below.
Linker For Post Synthetic Fluorescent Labeling of Probe Arrays
[0122] ##STR16## A synthetic scheme for the post-synthetic labeling
linker is provided below. ##STR17## Purification of Polymers
Cleaved From Arrays
[0123] Polymers cleaved from VLSIPS arrays are purified according
to a variety of known techniques, including, but not limited to,
gel electrophoresis, column chromatography, immunopurification,
precipitation, crystallization, dialysis, filtration, high pressure
liquid chromatography (HPLC), flash chromatography, paper
chromatography and affinity chromatography. See, e.g., Sambrook,
supra; Ausubel, supra; R. Scopes, Protein Purification,
Springer-Verlag, New York (1982); Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of recombinant
proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, New
York; Crowe, et al. (1992) QLAexpress: The High Level Expression
& Protein Purification System QUIAGEN, Inc., Chatsworth,
Calif.; Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook (supra); Ausubel (supra); Innis (supra); Harlow and Lane
(supra); Deutscher (1990) "Guide to Protein Purification" in
Methods in Enzymology vol. 182, and other volumes in the Methods in
Enzymology series; and manufacturer's literature on use of protein
purification products, e.g., Pharmacia, Piscataway, N.J., and
Bio-Rad, Richmond, Calif.,
[0124] In preferred embodiments, the polymer cleaved from a
VLSIPS.TM. array is labeled with a detectable label, facilitating
purification. HPLC is a preferred method of purifying polymers
cleaved from a VLSIPS array. HPLC typically allows for the
simultaneous detection and quantitation of a label as it exits an
HPLC column, e.g., by monitoring the efflux from an HPLC column
with a photomultiplier tube, providing a method of rapidly
determining the quantity and size of polymers cleaved from an
array. However, other methods such as capillary gel electrophoresis
and standard column chromatography provide similar information,
particularly when coupled with a photomultiplier or scintillation
counter.
[0125] Once a polymer is purified, it is optionally analyzed
further, for example, to determine the precise sequence of the
monomers which comprise the polymer. Many appropriate techniques
are known, including nuclear magnetic resonance spectroscopy (NMR),
chemical sequencing (e.g., the Maxam-Gilbert chemical sequencing
reaction used to sequence nucleic acids), enzymatic sequencing
(e.g., cloning and dideoxy sequencing used to sequence nucleic
acids) crystallography, circular diachroism, stopped flow
spectroscopy etc. Where the polymer is a nucleic acid, it is also
possible to clone the nucleic acid using standard techniques (see,
e.g., Sambrook, supra).
EXAMPLES
[0126] The following examples are provided by way of illustration
only and not by way of limitation. One of skill will recognize a
variety of parameters which can be changed or modified to yield
essentially similar results.
Materials and Nomenclature
[0127] 5-carboxyfluorescein was obtained from Applied Biosystems,
Foster City Calif. ##STR18##
[0128] Pivalic anhydride, pyridine, dicyclohexylcarbodiimide (DCC),
N-hydroxysuccinimide (NHS), borane-tetrahydrofuran complex
(BH.sub.3THF), acetic acid, methanol, 4,4'-dimethoxytrityl
chloride, piperidine, dimethylformamide (DMF), triethylamine
(Et.sub.3N), 2-cyanoethyl N,N,N',N'-tetraisopropylphosphoramidite,
and diisopropylammonium tetrazolide were all obtained from The
Aldrich Chemical Company, Milwaukee Wis.
[0129] L-threonine methyl ester hydrochloride and
N-Fmoc-.gamma.-aminobutyric acid were obtained from Bachem, Calif.
N-Fmoc-.gamma.-aminobutyryl pivolate was synthesized from
N-Fmoc-.gamma.-aminobutyric acid and pivoloyl chloride.
##STR19##
Example 1
Procedure For the Synthesis of A Novel Fluorescent Phosphoramidite
(Fluorescein Phosphoramidite 7)
[0130] For the example below, see also FIG. 1 which provides a
reaction scheme for the synthesis of fluorescein phosphoramidite 7.
The fluorescent phosphoramidite was used in conjunction with a
cleavable linker at the 3' end of the oligonucleotide polymer for
synthesizing an oligonucleotide array on a chip using the
VLSIPS.TM. light-directed synthesis procedure. The oligonucleotides
were then cleaved from the array at the cleavable linker and
analyzed by HPLC. The general strategy for synthesis is depicted
below (see also, FIG. 1). ##STR20## The novel fluorescent
phosphoramidite 7 ##STR21## is synthesized from a fluoresceinated
linker (6), ##STR22## which is synthesized from two units,
5-carboxyfluorescein N-hydroxysuccinimidyl ester (5) and
(2S,3R)-4-aminobutaneamido-1,3-butanediol trityl ether (6),
##STR23## each of which exists as a single isomer (See also FIG.
1).
[0131] This ensures that, contrary to existing systems used as an
oligonucleotide label, the attachment of the fluorescent
phosphoramidite 7 results in an oligonucleotide with only one
isomer. This allows oligonucleotides with the fluorescent label 7
to be fractionated by size (e.g., by HPLC as shown below) without
the complication of isomeric mixtures of single size classes.
(2S, 3R)-2-amino-1,3-butaneodiol (1)
[0132] To 15 g (88.4 mmol, 1 eq) of L-threonine methyl ester
hydrochloride in 100 mL of dry THF at 0.degree. C. under argon was
added, dropwise over 1 hr, 265.3 mL (265.3 mmol, 3 eq) of
borane-THF (1 M). The ice bath was removed and the reaction was
stirred at room temperature overnight (18 hr). The solution was
cooled to 0.degree. C. and quenched slowly with 180 mL of 10%
acetic acid in methanol. The solution was then evaporated to a
brown viscous oil and the oil co-evaporated three times with 100 mL
of methanol. The crude material was purified by flash
chromatography on silica gel using a step gradient of 1% to 5%
conc. Ammonium hydroxide in methanol/dichloromethane 3:7 to afford
7.5 g (81%) of (2S,3R)-2-amino-1,3-butanediol as a viscous oil.
This material was dissolved in 50 mL of dry DMF and precipitated
with hexanes in the cold to give 1 as a white solid.
(2S-3R)-2-[4'-N-[fluoren-9-ylmethoxy)-carbonyl]-4'-aminobutanamido]-1,3-bu-
tanediol (2)
[0133] To 5 g (15.4 mmol, 1 eq) of N-Fmoc-4-aminobutyric acid and 8
mL (46.1 mmol, 3 eq) of dry diisopropylethylamine in 60 mL of dry
THF at 0.degree. C. under argon was added 2 mL (16.1 mmol, 1.05 eq)
of pivaloyl chloride. The solution was stirred for 1 hr at
0.degree. C. and then 1.8 g (16.9 mmol, 1.1 eq) of 1 was added in 8
mL of dry DMF. The solution was allowed to warm to room temperature
and the solvent removed under vacuum. The oil was dissolved in 100
mL of ethyl acetate and washed with 100 mL of sat. aq. NaHCO.sub.3
and 100 mL of brine and dried over anhydrous Na.sub.2SO.sub.4.
Filtration and solvent removal gave 7 g of an oil. The crude
product was purified by flash chromatography on silica gel using
ethyl acetate/hexanes/1% triethylamine as eluent to afford 3.4 g
(53%) of 2 as a yellow foam.
Protection of the Primary Hydroxyl With DMTC1 To Give 3
[0134] To 3.4 g (8.2 mmol, 1 eq) of 2 in 30 mL of dry pyridine
under argon at ambient temperature was added 3.1 g (9.1 mmol, 1.1
eq) of 4,-4'-dimethoxytrityl chloride. The reaction was stirred for
18 hr and then the solvent removed under vacuum. The oil was taken
up in 50 mL of ethyl acetate and washed twice with 50 mL of
saturated aqueous NaHCO.sub.3 and 50 mL of brine and dried over
anhydrous Na.sub.2SO.sub.4. Filtration and removal of solvent gave
about 7 g of an orange oil. The crude product was purified by flash
chromatography on silica gel using ethyl acetate/hexanes 3:2 and 1%
triethylamine as eluent to afford 4.7 g (80%) of 3 as a white
foam.
Deprotection of the Fmoc Group To Give 4
[0135] The FMOC group was removed by treatment of 4.7 g (6.6 mmol)
of 3 with 50 mL of 20% piperidine in DMF for 2 hr at ambient
temperature. The solvent was removed under vacuum to give a white
solid. The solid was dissolved in 200 mL of ethyl acetate and
washed twice with 100 mL of saturated aqueous NaHCO.sub.3 and 100
mL of brine and dried over anhydrous Na.sub.2SO.sub.4. Filtration
and evaporation of the solvent gave a white solid which was
purified through a plug of silica gel using ethyl acetate/1%
triethylamine to remove the fulvene followed by elution with 60%
methanol/ethyl acetate/1% triethylamine to afford 2.5 g (89%) of 4
as a white foam.
Reaction of 5-Carboxyfluorescein N-hydroxysuccinimide With 4 To
Give 5
[0136] 4 g (10.6 mmol, 1 eq) of 5-carboxyfluorescein was
co-evaporated twice under vacuum with 30 mL of dry pyridine and the
mixed with 1.2 g (10.6 mmol, 1 eq) of N-hydroxysuccinimide and 2.3
g (10.6 mmol, 1 eq) of dicyclohexylcarbodiimide in 100 mL of dry
THF under argon. The reaction was stirred at ambient temperature
for 18 hr and then filtered to remove the insoluble urea. The
solvent was removed under vacuum to afford 5 g of an orange solid.
To the crude NHS-ester in 50 mL of 10% pyridine in dichloromethane
was added amine 4 in 20 mL of dichloromethane under argon. The
reaction was stirred overnight (18 hr) at ambient temperature. The
reaction was poured into 100 mL of brine and the aqueous layer was
extracted twice with 100 mL of dichloromethane/isopropyl alcohol
1:1. The organic fractions were combined and dried over anhydrous
Na.sub.2SO.sub.4. Filtration and removal of solvent gave an orange
solid which was purified by flash chromatography on silica gel
using a stepwise gradient of 5% to 30% methanol/dichloromethane to
afford 7.5 g (83%) of 5 as a yellow solid.
Protection of 5 As the Pivaloate 6
[0137] To 7.5 g (8.2 mmol, 1 eq) of 5 in 30 mL of dry
dichloromethane under argon at ambient temperature was added 12.3
mL (16.4 mmol, 2 eq) of triethylamine and 0.2 g (1.6 mmol, 0.2 eq)
of dimethylaminopyridine followed by 6.4 mL (16.4 mmol, 2 eq) of
pivaloic anhydride. The reaction was stirred for 20 hr and washed
twice with 100 mL of dilute aqueous NaHCO.sub.3 (1/10 from
saturation) and 100 mL of brine dried over anhydrous
Na.sub.2SO.sub.4. Filtration and evaporation of the solvent under
vacuum gave a pale yellow foam which was purified by flash
chromatography on silica gel using a methanol/dichloromethane/ethyl
acetate mixture to afford 5.5 g (66%) of 6 as a white solid.
Phosphitylation of 6
[0138] The procedure of Barone et al. (See, Barone et al., (1984)
Nucleic Acids Research, 12:4051 and Reynolds et al. (1992)
Bioconjugate Chemistry, 3:366) was followed to form the cyanoethyl
phosphoramidite 7 in 80% yield after chromatography on silica gel
using 30% haxanes/ethyl acetate as eluent.
Example 2
Analysis of Probe Arrays
[0139] This example describes the quantitative analysis of an array
synthesized by the VLSIPS.TM. method (See, (1994) Proc. Natl. Acad.
Sci. USA, 91:5022), by cleavage of the array from the surface of a
test pool of oligonucleotide probes. The probes were labeled at the
3'-end with a fluorophore (using the fluorescein phosphoramidite 7,
see supra), which were fractionated by HPLC. The information
obtained from this assay includes overall yield of full-length
probe (P) (pmol), density of full-length probe (D) (in
pmol/cm.sup.2), coupling efficiency for a given nucleoside
phosphoramidite used in the synthesis of the probe array, and
density of available sites for reaction on the substrate (in this
case a silane surface) Ds (pmol/cm.sup.2).
Synthesis of Fluorescein-Labeled Deoxyoligonucleotide Calibration
Standard (T.sub.16)
[0140] Calibration sequences were synthesized on a 1.mu. mole scale
using standard DMT-protected, cyanoethyl nucleoside phosphoramidite
reagents at 100 mM and the fluorescein phosphoramidite 7, and SL
phosphoramidites at 50 mM. The sequence of the calibration standard
had the following construction:
CPG-T-SL-FL-3'TTTTTTTTTTTTTTTT5'-DMT Where CPG is a controlled pore
glass substrate, SL is the commercially available sulfone linker
phosporamidite linker, and FL is fluorescein phosphoramidite 7.
[0141] The DMT-on poly-16mer was cleaved automatically from the CPG
support on the synthesizer with 2 mL of conc. NH.sub.4OH into the
collection vial containing 50 mL of 1 M NaOH (final concentration
of NaOH is 25 mM), and allowed to stand in the dark at room
temperature for 15 hr. The solution volume was reduced to about 0.5
mL in a speed-vac. The concentration of the oligonucleotide in
A.sub.495 units (au) per mL was determined by dilution of the crude
solution to obtain an absorbance reading between 0.1 au and 1
au/mL. The solution was stored at -20.degree. C. in the dark.
HPLC Purification of Calibration Oligonucleotide
[0142] The sample was microfuged (14,000 rpm) for 5 min to remove
particulate matter, and then analyzed in small aliquots (0.25 au)
by HPLC on a PRP-1 reverse-phase column at a flow rate of 2 mL/min,
using 0-60% B linear gradient over 45 min (R.sub.t=28 min) where
1.times.TEAA pH7=buffer A, and CH.sub.3CN=buffer B with detection
performed at 260/495 nm absorbance.
[0143] The remaining crude oligo was purified under the same
conditions, except that detection was at 290/510 nm and 1 mL
fractions were collected in 13.times.100 nm glass culture tubes
across the main peak. The fractions spanning the peak were pooled
and evaporated to dryness in a speed-vac. The residue was suspended
in 1 mL of 80% acetic acid in an Eppendorf tube and allowed to
stand for 1 hr. at room temperature. The solution was evaporated to
dryness in a speed-vac and the residue suspended in 350 .mu.L of
sdiH.sub.2O. The sample was then microfuged (14,000 rpm) for 5 min
to pellet any particulate material.
[0144] The sample was then purified by HPLC on a tentacle
ion-exchange column in a similar fashion as above at a flow rate of
1 mL/min and a 0-100% B linear gradient over 40 min R.sub.t
(analytical)=17.4 min and R.sub.t (preparative)=13.5-17.5 min
(broad peak) where 0.25.times. buffer A=buffer A and 1.times.
buffer B=buffer B as described above. Detection was carried out at
260/495 nm absorbance for the analytical peak and 290/510 nm
absorbance for the preparative peak. Fractions were collected in
0.5 ml fractions in 13.times.100 mm glass culture tubes.
[0145] Fractions (40-50 .mu.L) were analyzed on a dionex
ion-exchange column with a flow rate of 1 mL/min, a 0-100% B linear
gradient over 30 min (R.sub.t=24.3 min), using 0.25.times. buffer A
and 1.times. buffer B. Fractions were monitored at 260/495 nm
absorbance
[0146] The purest fractions (>93% pure) were pooled, neutralized
with acetic acid (tested against litmus paper) and evaporated to
dryness in a speed-vac. The residue was suspended in 1 mL of
sdiH.sub.2O and desalted on a sep-pak plus C18 cartridge using the
manufacturer's recommended procedure, except that the
oligonucleotide was eluted with 80% aqueous acetonitrile. The
combined fractions were evaporated to dryness and suspended in 200
.mu.L of sdiH.sub.2O. Two aliquots of the pool were again tested by
dionex chromatography for final purity (93%), first by detecting
one aliquot at 260/495 nm absorbance and second by detecting the
second aliquot at 520 nm fluorescence emission (495 nm excitation).
The combined fractions were then pooled at -20.degree. C. in the
dark.
Chip Synthesis And Cleavage
[0147] The test probe sequence (below) was synthesized on a glass
VLSIPS.TM. substrate employing standard VLSIPS.TM. techniques using
DMT nucleodside phophoramidites.
SURFACE-silane-Peg-SL-FL-3'-TTTTTTTTTTTTTTTT5'-OH
[0148] After synthesis, the chip was placed in a 15 mL amber-glass
bottle. 2 mL of fresh, cold conc. NH4OH (stored at -20.degree. C.)
was added, and the bottle sealed tightly with a screw-cap and
allowed to stand at room temperature for a minimum of 15 hrs. The
solution was transferred with a pipetman to a 6 mL glass culture
tube and the bottle/chip rinsed twice with 1 mL of sdiH.sub.2O and
the rinse portions were added to the tube. The solution was
evaporated to dryness in a speed-vac at medium heat and the residue
suspended in 1 mL of sdiH.sub.2O (2.times.0.5 mL rinses).
[0149] The above test samples and the calibration samples were spun
in a microfuge (14,000 rpm) for 5 min. 1 mL of the calibration set
and test samples were added to injection vials and analyzed in
triplicate on a dionex HPLC ion-exchange column with a flow rate=1
mL/min, 0-100% B linear gradient over 30 min, 0.25.times. buffer A
and 1.times. buffer B. Detection of the test and reference
(calibration) oligos was performed at 520 nm fluorescence (495 nm
excitation), with the photomultiplier tube (PMT) sensitivity set to
medium and injection volume=100 .mu.L (full-loop mode).
General Features of the HPLC Chromatogram of the Homopolymer
T.sub.16 Synthesized Using DMT Chemistry
[0150] FIG. 3 shows a typical HPLC chromatogram of a
fluorescein-labeled T.sub.16 homopolymer. The predominant peak at
25.77 min. corresponds to the full length 16-mer with a number of
smaller truncated species that trail.
[0151] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, modifications can be made thereto without
departing from the spirit or scope of the appended claims.
[0152] All publications and patent applications cited in this
specification are herein incorporated by reference for all purposes
as if each individual publication or patent application were
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
* * * * *