U.S. patent application number 10/322564 was filed with the patent office on 2003-06-19 for combinatorial synthesis on arrays.
Invention is credited to Strathmann, Michael Paul.
Application Number | 20030111356 10/322564 |
Document ID | / |
Family ID | 23338452 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030111356 |
Kind Code |
A1 |
Strathmann, Michael Paul |
June 19, 2003 |
Combinatorial synthesis on arrays
Abstract
The present invention provides methods for synthesizing arrays
of polymers. A barrier to a reaction is applied to select features
of the array thereby limiting the reaction to the remaining
features.
Inventors: |
Strathmann, Michael Paul;
(Mukilteo, WA) |
Correspondence
Address: |
Michael Strathmann
5300 Harbour Pointe Blvd. 302-B
Mukilteo
WA
98275
US
|
Family ID: |
23338452 |
Appl. No.: |
10/322564 |
Filed: |
December 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60341648 |
Dec 17, 2001 |
|
|
|
Current U.S.
Class: |
506/40 ; 204/483;
204/507; 205/414; 205/688 |
Current CPC
Class: |
B01J 2219/00608
20130101; B01J 2219/00443 20130101; B01J 2219/00626 20130101; B01J
2219/00713 20130101; B01J 2219/00612 20130101; B01J 2219/00653
20130101; G01N 2035/00158 20130101; B01J 2219/00659 20130101; B01J
2219/00722 20130101; B01J 2219/00617 20130101; B01J 19/0046
20130101 |
Class at
Publication: |
205/414 ;
205/688; 204/483; 204/507 |
International
Class: |
C25B 003/00 |
Claims
I claim:
1. A method for inhibiting a reaction, comprising: providing a
substrate for the reaction and applying a barrier to the reaction
on the substrate wherein the presence of the barrier is determined
by an electrochemical process.
2. The method of claim 1, wherein the substrate is a chemically
modified electrode.
3. The method of claim 1, wherein the reaction is a chemical
reaction.
4. The method of claim 1, wherein the reaction is an enzymatic
reaction.
5. The method of claim 1, wherein the electrochemical process is
electrodeposition of the barrier.
6. The method of claim 1, wherein the electrochemical process is
electrodissolution of the barrier.
7. The method of claim 1, wherein the substrate is an array
comprising distinct features and the barrier is present on a subset
of the features.
8. The method of claim 7, further comprising performing the
reaction on the array, applying a second barrier to a second
reaction so that the second barrier is present on a second subset
of features, and performing the second reaction on the array.
9. A method for synthesis of an array of separately formed
polymers, comprising: (a) providing an array comprising distinct
features wherein a feature comprises one or more functional groups,
(b) using an electrochemical process to apply a barrier on a subset
of the features wherein the barrier prevents effective coupling
between a monomer having at least one protected functional group
and the functional groups in the features, (c) coupling the
monomers to the array, (d) removing the barrier, (e) if necessary,
deprotecting the functional groups on the monomers coupled to the
array, and (f) repeating steps (b) through (e) until at least two
separate polymers are formed on distinct features of the array.
Description
1. RELATED APPLICATION DATA
[0001] This application claims priority to U.S. Provisional Patent
Application Serial No. 60/341,648, filed Dec. 17, 2001.
2. FIELD OF THE INVENTION
[0002] The present invention is directed to the synthesis and
placement of materials at select locations on a substrate. In
particular, the present invention is directed to a method for
providing separate sequences of chemical monomers at select
locations on a substrate.
3. BACKGROUND
[0003] A variety of methods are currently available for making
arrays of biological macromolecules, such as arrays of nucleic acid
molecules or proteins. One method for making ordered arrays of DNA
on a porous membrane is a "dot blot" approach. In this method, a
vacuum manifold transfers a plurality, e.g., 96, aqueous samples of
DNA from 3 millimeter diameter wells to a porous membrane. A common
variant of this procedure is a "slot-blot" method in which the
wells have highly-elongated oval shapes. The DNA is immobilized on
the porous membrane by baking the membrane or exposing it to UV
radiation. This is a manual procedure practical for making one
array at a time and usually limited to 96 samples per array.
"Dot-blot" procedures are therefore inadequate for applications in
which many thousand samples must be determined.
[0004] An alternate method of creating ordered arrays of nucleic
acid sequences is described by Pirrung, et al. (U.S. Pat. No.
5,143,854, 1992), and also by Fodor, et al. (Science 251:767-773,
1991). The method involves synthesizing different nucleic acid
sequences at different discrete regions of a support. This method
employs elaborate synthetic schemes, and is generally limited to
relatively short nucleic acid sample, e.g., less than 20 bases. A
related method has been described by Southern, et al. (Genomics
13:1008-1017, 1992).
[0005] Montgomery (U.S. Pat. No. 6,093,302, 2000) teaches a method
for making arrays of polymers by employing electrochemically
generated reagents that are confined by buffering and/or scavenging
agents. The method requires substituting standard chemical
reactions that can be used for polymer synthesis (e.g.
oligonucleotide chemistry) with tailored electrochemical
reactions.
[0006] There is a need in the art for a method of synthesizing
high-density arrays of polymers that makes use of the many standard
chemistries already described for synthesizing individual polymers,
including enzymatic techniques. The current invention addresses
this problem by making use a barrier to a reaction that can be
selectively applied to different features in an array. The use of a
barrier minimizes the need to tailor well understood chemical
reactions to fit a specific requirement for constructing arrays
(e.g. the use of photocleavable protecting groups).
4. BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides methods for synthesizing
arrays of polymers. A barrier to a reaction is applied to select
features of the array thereby limiting the reaction to the
remaining features. The locations of the barrier on the array are
determined by an electrochemical process (e.g. electrodeposition,
electrodissolution, etc.). The array preferably comprises
chemically modified electrodes. By repeating the process and
changing the parts of the array to which the barrier is applied, it
is possible to construct arrays of polymers (e.g. oligonucleotides,
peptides, etc.) using standard chemistries.
5. DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention is designed to provide a barrier to a
reaction (e.g. chemical, enzymatic, etc.) at selected locations (or
features) in an array while permitting the reaction to occur at
other locations in the array (an "array refers to a structure that
provides a plurality of spatially addressable locations). After the
reaction is finished, the barrier may be removed and applied to a
different subset of features. Different reactions may be performed
sequentially at different and/or overlapping subsets of features in
the array (i.e. combinatorial synthesis) thereby producing an array
of different compounds (e.g. oligonucleotides, peptides, small
molecules, carbohydrates, materials, etc.).
[0009] The barrier is patterned electrochemically with an array of
addressable electrodes. Each feature in the array is associated
with an electrode. The barrier may be applied everywhere on the
array (all the features and any intervening space between the
features) and then selectively removed from a subset of features.
Alternatively, the barrier may be formed directly on a subset of
the features.
[0010] In many ways, the present invention is analogous to
photolithography in which a barrier is patterned on a surface by
light. The barrier is a photoresist and the pattern is determined
by a photomask. Indeed, photolithography coupled with standard
oligonucleotide synthesis chemistry has been used to synthesize
oligonucleotide arrays (Brock, P. J. et al., U.S. Pat. No.
5,658,734). In the instant invention, the photomask is replaced
with an array of addressable electrodes, light is replaced with
electricity, and the photoresist is replaced with materials that
can be electrodeposited and/or electrodissolved either directly or
by interaction with an electrochemically generated reagent (e.g.
acid, base, radical, etc.).
[0011] Methods for making arrays of individually addressable
electrodes are well known in the art (see for example, Montgomery,
D. D., U.S. Pat. No. 6,093,302; Teoule, R. et al., U.S. Pat. No.
5,837,859; Havens, J. R. et al., U.S. Pat. No. 6,306,348).
Microlithography techniques have been used to make arrays with 1000
addressable electrodes (Caillat, P. et al., Sens. Actuators B,
61:154-162, 1999). Arrays with greater than 100,000 electrodes can
be constructed, and the electrodes can be less than 1 .mu.M in
diameter (Montgomery, D. D., U.S. Pat. No. 6,093,302).
[0012] Methods for chemically modifying electrodes are also well
known (Fujihira, M., Topics in Organic Chemistry, Plenum, 255-294,
1986). The modified electrodes ("functionalized electrodes")
provide functional groups on which solid-phase synthesis can occur
(e.g. hydroxyl groups for oligonucleotide synthesis, etc.). For
example, chlorosilane and alkoxysilane reagents will react with
surface hydroxyls on metal oxide electrodes (e.g., RuO.sub.2, doped
SnO.sub.2, doped TiO.sub.2, doped InO.sub.2, etc.) and partially
oxidized metal electrodes (e.g., platinum, etc.) to provide a
variety of functional groups tethered to the electrode by a linker
moiety (Murray, R. W., Techniques of Chemistry, Vol. 22, John Wiley
& Sons, 1-48, 1992). A wide variety of conducting polymers
(e.g. polypyrrole, polyaniline, polythiophene, etc.) with pendant
functional groups can be electrochemically deposited on electrodes
(Chandrasekhar, P., Conducting Polymers: Fundamentals &
Applications, Kluwer, 1999). Composite materials, consisting of a
conducting component (e.g. metal, conducting polymer, conducting
metal oxide, etc.) and a particulate functionalized component (e.g.
SiO.sub.2, polystyrene beads, TiO.sub.2, etc.) may be
electrochemically codeposited on electrodes (Ferreira, C. A. et
al., J. Appl. Electrochem. 31:49-56, 2001; Gangopadhyay, R. &
De, A., Chem. Mater., 12:608-622, 2000; Musiani, M., Electrochim.
Acta, 45:3397-3402, 2000; and Hovestad, A. et al., J. Appl.
Electrochem., 29:331-338, 1999). A variety of polymers (e.g.
acrylate/polyvinyl alcohol, polysaccharides, polyacrylamides, etc.)
may be cast over the entire array of electrodes (Bard, A. J. &
Faulkner, L. R., Electrochemical Methods, John Wiley & Sons,
580-589, 2001; Montgomery, D. D., U.S. Pat. No. 6,093,302). Clays,
zeolites and other porous structures, such as CPG and sol-gel
materials may be coated over the array of electrodes to provide
functional groups (Montgomery, D. D., U.S. Pat. No. 6,093,302;
Bard, A. J. & Faulkner, L. R., Electrochemical Methods, John
Wiley & Sons, 580-589, 2001; Bard, A. J. & Mallouk, T.,
Techniques of Chemistry, John Wiley & Sons, 271-312, 1992;
Havens, J. R. et al., U.S. Pat. No. 6,306,348).
[0013] An important element of the invention is the ability to
cover the functional groups with a barrier that effectively
minimizes the participation of the functional groups and/or
appended molecules in a solid-phase synthesis reaction. In this
way, only those features in the array that are not covered by the
barrier will participate. Whether or not the barrier covers a
feature is determined by the voltage (or current) applied to the
associated electrode. As mentioned above, an analogy can be drawn
between the present invention and photolithography. Much effort has
gone into the development of many chemically amplified resists as
is known by those skilled in the art. In general, a photoresist is
spincast on a surface and exposed to light. A photo-acid generator
(PAG) produces acid that catalytically affects the structure of the
photoresist so it becomes either more or less stable to subsequent
exposure to a developer. An analogous procedure can be utilized in
the present invention. A resist may be spin-cast on an array of
functionalized electrodes. Acid may be generated electrochemically
(e.g. through oxidation of water) over select electrodes. In the
case of a PAG, acid diffusion is limited by exposure of the resist
in the solid phase. The diffusion of electrochemically generated
acid may be limited by including a buffer in the solvent
(Montgomery, D. D., U.S. Pat. No. 6,093,302). Subsequent treatment
of the resist after exposure to acid may follow closely the steps
utilized in photolithography (e.g. a post-exposure bake and
development). The end result can be a highly cross-linked polymer
that provides an effective barrier to solid-phase synthesis (e.g.
oligonucleotide chemistry, see McGall, G. et al., Proc. Natl. Acad.
Sci. USA, 93:13555-60, 1996). The entire process must be repeated
(including stripping the developed resist) for each step in the
synthesis (e.g. each nucleotide to be added to the growing
oligonucleotide). The above procedure is somewhat cumbersome since
each step requires spin-casting and in some cases, baking.
[0014] Preferably, the barrier can be deposited directly on the
array in solution. Then all subsequent steps in the chemical
synthesis may be performed in the same reaction vessel. This
process is easily automated by simply moving fluids into and out of
the reaction chamber (assuming subsequent reactions are performed
in solution). Electrodeposition of polymers is a preferred means
for creating a barrier over select, functionalized electrodes. The
polymers may be electrochemically polymerized from monomers (e.g.
pyrrole, thiophene, aniline, 4-vinyl pyridine, 5-nitroindole, etc.)
or the barrier may be formed by electrodeposition of polymers in
solution, such as for example polyvinyl ferrocene, polyvinyl
alcohol/borate, etc. (Murray, R. W., Techniques of Chemistry, Vol.
22, John Wiley & Sons, 1-48, 1992; Bruckenstein, S. &
Pater, E., Anal. Chem., 72:1598-1603, 2000; Zhitomirsky, T. &
Petric, A., Mater. Sci. Engin. B, 78:125-130, 2000; Jennings, P. et
al., J. Chem. Soc., Faraday Trans., 93:3791-3797, 1997). The
electropolymerization of monomers is preferred because the
resulting barriers are typically free from defects. During
polymerization, pinholes in the film (barrier) are efficiently
filled due to increased current fluxes and thus concentration of
activated monomer at the pinhole (Murray, R. W., Techniques of
Chemistry, Vol. 22, John Wiley & Sons, 1-48, 1992).
[0015] After the first solid-phase synthesis step, the
electrodeposited polymer must be removed and redeposited on a
different set of features (functionalized electrodes) in
preparation for the next synthesis step. The barrier must remain
insoluble (or at least effectively intact) during the synthesis
steps, but must be soluble (or at least removable) under a
different set of conditions. Of course, many polymers (and other
deposits in general) can be removed simply by changing the solvent
or by chemical etching.
[0016] Considerable effort has been applied to modifying the
solubility properties of conducting polymers (e.g. polypyrrole,
polyaniline, polythiophene, etc., see Chandrasekhar, P., Conducting
Polymers: Fundamentals & Applications, Kluwer, 1999). Many
conducting polymers have increased solubility in their reduced
state, such as polyaniline, poly(3-alkylthiophenes),
poly(alkoxyethylene dioxythiophene, etc. (Chandrasekhar, P.,
Conducting Polymers: Fundamentals & Applications, Kluwer, 1999;
Izou, K. T. & Gregory, R. V., Synth. Met., 69:109-112, 1995;
Czardybon, A. & Lapkowski, M., Synth. Met., 119:161-162, 2001;
Hosseini, S. H. et al., Iran Polymer J., 9:255-261,2000). Indeed,
the ability to electrochemically modify the solubility and/or
attachment of the polymer barrier is a preferred embodiment of the
invention. A preferred monomer is 2,5-di-(2-thienyl)-pyrrole (SNS).
The monomer is readily oxidized in acetonitrile and 0.1M
LiClO.sub.4 to form a film that is insoluble in a variety of
organic and aqueous solvents. Upon reduction in
acetonitrile/LiClO.sub.4, the film electrodissolves back to
monomers and soluble oligomers (Brillas, E. et al., J. Electroanal.
Chem., 392:55-61, 1995; Carrasco, J. et al., J. Electrochem. Soc.,
148:E19-E25, 2001).
[0017] The oxidation/reduction of thiol/disulfide groups can be
exploited for the reversible electrodeposition or cross-linking of
polymers on an electrode (see for example, Naoi, K. et al., J.
Electrochem. Soc., 142:354-360, 1995; Endo, K. & Bu, H.-B., J.
Electroanal. Chem., 506:155-161, 2001; Bernkop-Schnurch, A. et al.,
Int. J. Pharmaceutics, 226:185-194, 2001). Electrochemically
reversible polymers may be formed from monomers comprising a
disulfide bond flanked by groups that can be anodically coupled
(see for example, Saito, M. et al., Electrochim. Acta,
45:3021-3028, 2000). In many cases, dissolution of the polymers can
be performed chemically in addition to or instead of
electrochemically (e.g. a reducing agent such as
.beta.-mercaptoethanol may be added to the solution over the
deposits). Some deposits (polymers, etc.) may also simply lose
adhesion to the surface by varying the voltage of the
functionalized electrodes (e.g. potential cycling).
[0018] Other types of electrodeposited materials may also provide
suitable barriers to synthetic reactions. Electroplating of metals
is a well developed art. Metals such as copper, aluminum, gold
iron, tin, lead, etc. and their alloys may be electrodeposited from
ions in solution (Leith, S. D. et al., J. Electrochem. Soc.,
146:1431-1435, 1999; Legrand, L. et al., Electrochim. Acta
40:1711-1716, 1995; Bakos, I., J. Solid State Electrochem.,
4:80-86, 2000; Ramos, A. et al., J. Electrochem. Soc.,
148:C315-C321, 2001; Donten, M. et al., Electrochim. Acta,
45:3389-3396, 2000; Krumm, R. et al., Electrochim. Acta,
45:3255-3262, 2000). Metals may be removed from the functionalized
electrodes by chemical etching or more preferably by
electrodissolution (Datta, M. & Harris, D., Electrochim. Acta,
42:3007-3013,1997). Similarly, metal oxide/hydroxide barriers may
be electrodeposited from solution (e.g. Cu.sub.2O, MnO(OH),
CoO(OH), NiO(OH), V.sub.2O.sub.5, PbO.sub.2, TiO.sub.2, ZnO,
ZrO.sub.2, etc., see Therese, G. H. A. & Kamath, P. V., Chem.
Mater., 12:1195-1204) and subsequently removed by chemical and/or
electrochemical means (Bakardjieva, S. et al., J. Solid State
Electrochem., 4:306-313, 2000; Isaacs, H. S. et al., Electrochem.
Meth. Corrosion, 247:19-24, 1997).
[0019] Some barriers may be deposited by electrochemically
generated reagents (e.g. base) that can diffuse from the electrode
and may lead to deposition at nearby electrodes. To minimize this
occurrence, buffers and/or scavengers may be used as taught by
Montgomery in U.S. Pat. No. 6,093,302.
[0020] Clearly, any material can function as a barrier as long as
it satisfies two criteria: 1) the deposition and/or removal of the
material at the functionalized electrode is affected by a change in
voltage (or current) applied to the electrode, and 2) the material
effectively prevents or minimizes a solid-phase reaction at the
functionalized electrode on which the material is deposited, but
the material does not prevent the reaction at other electrodes that
lack deposits. The preferred barrier materials described above are
affected by electrochemical processes but more generally, barrier
materials may be affected by other processes that can occur at the
electrodes such as the generation of light (e.g., light emitting
diodes) and heat (e.g. resistors, see for example Caillat, P. et
al., U.S. Pat. No. 6,255,677).
[0021] The maximum allowable porosity or permeability of the
barrier is determined by the solid-phase reaction to be blocked.
The diffusion (or movement) of a large enzyme can be minimized by a
more porous barrier than is required to block the diffusion of a
small molecule like pyridine. In some cases, a reaction component
(reagent) may be coupled to a bulkier molecule to prevent diffusion
through a very porous barrier (e.g. polyvinyl pyridine may be able
to replace pyridine in a reaction, see for example, Sanghvi, Y. S.,
et al., Organic Process Res. Dev., 4:175-181, 2000). Some reactions
will have multiple components of which only one need be blocked by
the barrier to prevent the reaction from occurring at the
functionalized electrode.
[0022] The diffusion of a reaction component (e.g. Fe.sup.2+)
through a barrier may be influenced by the redox state of the
barrier. For example, the charge and/or porosity of several
conducting polymer films can change depending on whether the film
is reduced (insulating state) or oxidized (conducting state), see
Stockert, D. et al., Synth. Met., 55:1323, 1993; Maysymiuk, K.
& Doblhofer, K., Synth. Met., 55:1382, 1993; Maysymiuk, K.
& Doblhofer, K., Electrochem. Acta, 39:217, 1994. In this case,
the invention may be practiced without actually physically removing
the barrier material from the functionalized electrode. To remove
or deposit such a barrier, one simply cycles between the reduced
and oxidized forms of the barrier material. This barrier could also
provide the functional groups on which synthesis occurs, which
would make it part of the functionalized electrode. For example, a
polymer membrane used to functionalize an electrode may contain
both functional groups and a redox reversible cross-linker (e.g.
thiol groups) so that the membrane is highly cross-linked and
impermeable under one set of conditions (e.g. oxidized) but is
porous under another set of conditions (e.g. reduced).
[0023] As described above, the preferred array for the solid-phase
synthesis reactions comprises an array of functionalized
electrodes. The functional groups are directly attached to the
electrode (e.g. silane groups) or they are directly attached to a
material that is physically contacting, or proximate to, the
electrode (e.g. a membrane coating over the electrode array).
Southern teaches a method for electrochemically patterning a
surface with an array of electrodes that does not physically
contact the surface (U.S. Pat. No. 5,667,667). The electrode array
lies adjacent to the surface, separated by electrolyte.
Electrochemically generated reagents diffuse or migrate to the
surface. Southern's method may be used in the instant invention to
affect deposition and/or removal of a barrier on the surface
adjacent to the array of electrodes. The functional groups for
solid-phase synthesis reside on the surface. For example, a base
soluble polymer may be spin cast on the surface to act as a
barrier. The array of electrodes is used to generate base (e.g. by
reducing water) in order to dissolve the membrane at certain
locations on the surface. The surface (or more correctly, the
exposed surface) is then subjected to a reaction.
[0024] The instant invention lends itself to a wide variety of
solid-phase reactions. Indeed, an array of different compounds can
be synthesized from essentially any solid-phase reactions that can
be combined in a combinatorial manner (see for example, Montgomery,
D. D., U.S. Pat. No. 6,093,302; Lebl, L. et al., U.S. Pat. No.
6,045,755; Horlbeck, E. G., U.S. Pat. No. 5,880,972 and references
therein). The only requirement is that the barrier be compatible
with the chemistry, which typically means the barrier does not
break down in the solvents--at least during the time frame of the
reaction. In contrast to other array-based combinatorial synthesis
methods (Montgomery, D. D., U.S. Pat. No. 6,093,302; Fodor, S. P.
A. et al. U.S. Pat. No. 5,445,934), the instant method does not
require well-established chemistries (e.g. oligonucleotide
synthesis) to be altered in order to function in an array format.
In addition, because the synthesis reagents are not generated in
situ, combinatorial synthesis methods that utilize enzymes are
readily adapted to the present invention (for example, carbohydrate
synthesis, Takayama, S., Chem Soc. Rev., 26:407-415, 1997; Nicolau,
K. C. & Mitchell, H. J., Angew. Chem. Int. Ed., 40:1576, 2001;
Koeller, K. M. & Wong, C.-H., Glycobiology, 10:1157-1169,
2000).
6. EXAMPLES
6.1 Example 1
[0025] The following example demonstrates the synthesis of three
different dinucleotides (AC, AG, TT) on an array (at positions 1, 2
& 3, respectively).
[0026] An array of four electrodes is made by insertion of four
platinum wires (diameter 0.6 mm) into a glass cylinder (diameter 5
mm.times.height 10 mm). One electrode serves as a counter
electrode. The array is inserted into a reaction chamber with a
reference electrode (see Teoule, R. et al., U.S. Pat. No. 5,837,859
for details). The three working electrodes are functionalized with
a polypyrrole film that contains dimethoxytrityl (DMT) protected
hydroxyl groups according to the method taught by Teoule (ibid,
Example 4). Briefly, pyrrole and aminoethylpyrrole are
copolymerized in the presence of 0.1M LiClO.sub.4. The incorporated
amine functional groups are coupled to an activated nucleoside.
Secondary alcohol groups and unreacted amine functions are blocked
with acetic/anhydride/N-methylimidazole in pyridine.
[0027] To prepare the electrodes for solid-phase oligonucleotide
synthesis, the DMT protected hydroxyls must be deblocked. The array
is washed with acetonitrile and transferred to Deblock Solution (3%
trichloroacetic acid in dichloromethane) for one minute. The array
is washed with acetonitrile and transferred to the electrochemical
reaction chamber described above.
[0028] In preparation for coupling the first phosphoramidite (C), a
barrier must be deposited at positions 2 and 3 of the array, since
these positions will possess different residues at the 3'-end
(note, synthesis occurs in the 3' to 5' direction). The reaction
chamber is filled with the Barrier Solution (5 mM
2,5-di-(2-thienyl)-pyrrole (SNS) and 0.1M LiClO.sub.4 in
acetonitrile). The barrier is electrodeposited by oxidizing the SNS
monomer at electrodes 2 and 3 (that is, the electrodes at position
2 and 3) at constant current (0.5 mA cm.sup.-2) for 100 seconds at
room temperature. The array is washed with acetonitrile and exposed
to the "C" Coupling Solution (50 mM DMT-protected deoxycytidine
phosphoramidite and 0.25M tetrazole in anhydrous acetonitrile) for
2 minutes at room temperature. Under these conditions, almost all
of the exposed hydroxyls at position 1 are coupled to C
phoshoramidite while the poly(SNS) barrier minimizes reaction to
the hydroxyls at positions 2 and 3.
[0029] The array is washed with acetonitrile and the barrier at
position 2 is removed by reducing the poly(SNS) film in the
Reducing Solution (0.1M LiClO.sub.4 in acetonitrile) at constant
current (-0.2 mA cm.sup.-2) until the barrier is completely
dissolved (indicated by a rapid drop in voltage relative to the
reference electrode). The array is washed with acetonitrile and
exposed to the "G" Coupling Solution (50 mM DMT-protected
deoxyguanosine phosphoramidite and 0.25M tetrazole in anhydrous
acetonitrile) for 2 minutes at room temperature. Here, mainly the
exposed hydroxyls at position 2 are coupled because hydroxyls at
position 1 are already couple to "C" (thereby protected by the DMT
group) and hydroxyls at position 3 are still protected by the
poly(SNS) barrier.
[0030] The array is washed with acetonitrile and the barrier is
removed from position 3 as described above. The array is rinsed
with acetonitrile and then exposed to "T" Coupling Solution (50 mM
DMT-protected deoxythymidine phosphoramidite and 0.25M tetrazole in
anhydrous acetonitrile). Here, "T" couples mainly with the exposed
hydroxyls at position 3.
[0031] The array is washed with acetonitrile. The phosphite
linkages formed in the previous steps are oxidized to the more
stable phosphotriester linkage by exposing the array to Oxidizing
Solution (0.1M Iodine in water/pyridine/THF 2/20/80) for 1 minute
at room temperature. The array is washed with acetonitrile and then
exposed to Deblock Solution for one minute in order to remove the
DMT protecting groups, thereby exposing hydroxyl groups for
coupling the second phosphoramidite in the dinucleotide
sequence.
[0032] The poly(SNS) barrier is deposited at position 3 as
described above. The array is washed with acetonitrile and then
exposed to "A" Coupling Solution (50 mM DMT-protected
deoxyadenosine phosphoramidite and 0.25M tetrazole in anhydrous
acetonitrile).
[0033] The array is washed with acetonitrile, the barrier is
removed from position 3 as above and the array is exposed to "T"
Coupling Solution.
[0034] Finally, the array is washed with acetonitrile, exposed to
Oxidizer Solution, washed again, exposed to Deblock Solution and
washed again to yield the final array of dinucleotides.
6.2 Example 2
[0035] A dinucleotide array is synthesized as described in Example
1, except whenever the barrier is removed from one position, it is
redeposited at the position (or positions) that just underwent
coupling. This step is accomplished by replacing the Reducing
Solution with the Barrier Solution. Now, in addition to reducing
the poly(SNS) barrier at a selected electrode, the SNS monomer is
simultaneously oxidized at another electrode to redeposit the
poly(SNS) barrier. Following Example 1, just after "C" is deposited
at position 1, the array is washed with acetonitrile. The barrier
at position 2 is removed by reducing the poly(SNS) film in the
Barrier Solution at constant current (-0.2 mA cm.sup.-2) while the
barrier is electrodeposited by oxidizing the SNS monomer at
electrode 1 at constant current (0.5 mA cm.sup.-2). Now, when the
array is exposed to the "G" Coupling Solution, only position 2 is
exposed. In this way, if any hydroxyls at position 1 did not couple
to "C" during the first step (typically coupling efficiencies are
98-99%), then they will not be exposed to "G".
[0036] After the first base has been coupled at all three positions
in the array, the poly(SNS) barrier is removed from all the
electrodes by reduction in Reducing Solution. The array is washed
with acetonitrile and exposed to Capping Solution (8.8% w/v
N-methyl imidazole in acetic anhydride/lutidine/THF 1/1/16). This
step blocks all the unreacted hydroxyls from coupling to
phosphoramidite. As above, the array is exposed to Oxidizer
Solution, washed, exposed to Deblock Solution and washed again in
preparation for coupling the second phosphoramidite at each
position.
[0037] The process described above is then repeated for the second
phosphoramidite coupled at each position to yield the final
dinucleotide array.
* * * * *