U.S. patent number 5,556,961 [Application Number 08/328,079] was granted by the patent office on 1996-09-17 for nucleosides with 5'-o-photolabile protecting groups.
Invention is credited to Robert S. Foote, Richard A. Sachleben.
United States Patent |
5,556,961 |
Foote , et al. |
September 17, 1996 |
**Please see images for:
( Certificate of Correction ) ** |
Nucleosides with 5'-O-photolabile protecting groups
Abstract
Nucleosides with photolabile protecting groups on the
5'-hydroxyl. These nucleosides are useful in the sythesis of
nucleic acids on solid-state arrays.
Inventors: |
Foote; Robert S. (Oak Ridge,
TN), Sachleben; Richard A. (Knoxville, TN) |
Family
ID: |
26815651 |
Appl.
No.: |
08/328,079 |
Filed: |
October 24, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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117783 |
Sep 7, 1993 |
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794723 |
Nov 15, 1991 |
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Current U.S.
Class: |
536/27.1;
536/27.6; 536/27.81; 536/28.5; 536/28.54 |
Current CPC
Class: |
B01J
19/0046 (20130101); B82Y 30/00 (20130101); C07H
19/06 (20130101); C07H 19/16 (20130101); C07H
21/00 (20130101); B01J 2219/00315 (20130101); B01J
2219/00432 (20130101); B01J 2219/00527 (20130101); B01J
2219/00529 (20130101); B01J 2219/00585 (20130101); B01J
2219/0059 (20130101); B01J 2219/00596 (20130101); B01J
2219/00608 (20130101); B01J 2219/00612 (20130101); B01J
2219/00637 (20130101); B01J 2219/00659 (20130101); B01J
2219/00711 (20130101); B01J 2219/00722 (20130101); C40B
40/06 (20130101); C40B 60/14 (20130101); Y02P
20/55 (20151101) |
Current International
Class: |
B01J
19/00 (20060101); C07H 19/16 (20060101); C07H
21/00 (20060101); C07H 19/06 (20060101); C07H
19/00 (20060101); C07H 019/06 (); C07H 019/067 ();
C07H 019/073 (); C07H 019/16 () |
Field of
Search: |
;536/27.1,26.11,27.6,27.81,28.54,28.5,28.4,27.21 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Pillai, Organic Photochemistry, vol. 9, A. Padwa, E. D., Marcel
Dekker, Inc., New York, 1987, pp. 225-323..
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Primary Examiner: Kunz; Gary L.
Attorney, Agent or Firm: Luedeka, Neely & Graham,
P.C.
Government Interests
This invention was made with U.S. government support under Contract
No. 41B-99732 awarded by the Department of Energy. The U.S.
government has certain rights in this invention.
Parent Case Text
This is a continuation, of application Ser. No. 08/117,783, filed
Sep. 7, 1993, now abandoned, which is a division of Ser. No.
07/794,723, filed Nov. 15, 1991, now abandoned.
Claims
What is claimed is:
1. A nucleoside consisting of:
a saccharide selected from the group consisting of ribose and
deoxyribose;
a basic group selected from the group consisting of purines and
pyrimidines attached to the saccharide at the 1'-position of the
saccharide; and
a photolabile protecting group protecting the 5'-O-position of the
saccharide wherein the photolabile protecting groups contains an
ortho-nitrobenzyl moiety and a hydrogen atom on the alpha-carbon
atom of the moiety.
2. The nucleoside of claim 1 wherein the purine or pyrimidine is
selected from the group consisting of adenine, cytosine, guanine,
thymine, uracil, and derivatives thereof.
3. The nucleoside of claim 2 wherein the photolabile protecting
group is selected from the group consisting of 2-nitrobenzyl,
2-nitrobenzyloxycarbonyl and 6-nitroveratryloxycarbonyl.
4. A nucleoside having the following structure: ##STR1## wherein R
is a purine or a pyrimidine group, R.sub.1 is hydrogen or an OH
group, and PLPG is a photolabile protecting group containing an
ortho-nitrobenzyl moiety and a hydrogen atom on the alpha-carbon
atom to the moiety.
5. The nucleoside of claim 4 wherein the purine or pyrimidine group
is selected from the group consisting of adenine, cytosine,
guanine, thymine, uracil, and derivatives thereof.
6. The nucleoside of claim 5 wherein the photolabile protecting
group is selected from the group consisting of 2-nitrobenzyl,
2-nitrobenzyloxycarbonyl and 6-nitroveratryloxycarbonyl.
Description
The present invention relates to solid-state arrays of chemical
products, particularly biopolymers and to methods for making
solid-state arrays.
Recently, micro-scale solid-state arrays of biopolymers (such as
nucleic acids or polypeptides) have been reported in the literature
for various analytical and synthetic uses. For example, Foder, et
al. (Science, Vol. 251, pp. 767-773 [1991]) observes that such
arrays of oligonucleotides would be valuable in gene mapping,
fingerprinting, diagnostics, and nucleic acid sequencing.
Solid-state arrays of biopolymers comprise aligned rows and columns
of, usually, different biopolymers arranged on the surface of a
substrate. These arrays are generally prepared by successively
reacting selected portions of the array substrate with selected
biomonomers (such as nucleotides or amino acids) in the form of
derivatives for solid-phase synthesis.
Southern, in PCT Application WO 89/10977, describes the preparation
of an array of oligomeric DNA. The array taught by Southern
comprises a collection of, for example, all 256 of the sequences of
DNA which are four nucleotides long, and contain the nucleotides of
adenine (A), cytosine (C), guanine (G) and thymine (T). The array
is prepared on the reactive surface of a substrate, such as a glass
plate, which is derivatized with an aliphatic linker bearing a
terminal hydroxyl group to which the first base is added. The
biomonomers (in this case nucleoside phosphoramidites, which are
eventually converted to nucleotides) are applied one at a time to
selected portions of the surface. The portions of the surface which
are not selected for receiving a biomonomer during a given step of
the process are protected by the application of a physical mask,
such as silicone rubber, in physical contact with the surface.
For example, Southern teaches that the first four bases of the
array may be laid in four broad stripes on the glass plate. The
second set of bases is then added in four stripes of equal width to
the first and orthogonal to them resulting in a four-by-four array
of dimers (AA, AC, AG, etc.). The third and fourth sets are added
in stripes one-quarter the width of the first stripes and
positioned so that each set of narrow stripes runs within one of
the broad stripes, resulting in an array of all 256
tetranucleotides. The process may be repeated using even narrower
stripes to produce arrays of longer oligonucleotides.
Although the process of Southern is relatively straight-forward,
there are significant practical difficulties in the application of
the technique to producing arrays of very small micro-scale size
(i.e., less than about 2 cm.sup.2) with several tens of thousands
of cells. For example, an array of 256-by-256 is required to
produce an array of all possible sequences of octameric DNA. To
prepare such an array 1.5 cm on a side with physical masks demands
the repeated positioning of the masks with a precision of greater
than 0.06 mm (60 micron). In practice, that level of precision is
very difficult to reproduce and sustain throughout the production
of a micro-scale array. Therefore, no micro-scale arrays are known
to have been produced using the techniques taught by Southern.
A further disadvantage of the array produced in accordance with the
method disclosed by Southern relates to background effects. Arrays
produced by intersecting stripes will contain individual cells
which are surrounded by and border upon regions containing similar,
though shorter sequences. When the array is later contacted by an
unknown reagent, such as with an unknown nucleic acid under
hybridization conditions, interactions may occur to some extent
with the intervening sequences, resulting in background noise and
reduction in discrimination of accurately hybridized sites.
More specifically, the accuracy and positioning of successive masks
during the preparation of arrays on a uniformly functionalized
surface do not precisely define the boundaries between the
individual locations on the surface of the substrate. As the
biomonomers are deposited on the substrate, inaccuracies in and
positioning errors of the masks may produce biopolymers on the
borders between cells which have sequences significantly different
from the sequences in the adjacent cells. These boundary effects
produce "border biopolymers" with sequences that are similar, at
least in part, to cells remote from the border biopolymers.
Therefore, these boundary effects generate "noise" in the array.
For example, two adjacent cells of a nucleic acid sequencing array
prepared of octamers of nucleotides may have the sequences
C-G-T-A-A-G-T-T and C-G-T-A-C-G-A-T. Border biopolymers may have
the sequence C-G-T-A-A-C-G-T, G-T-A-A-C-G-T-A, and T-A-A-C-G-T-A-T.
During the hybridization of a nucleic acid, the boundary effects
will cause the border biopolymers to hybridize with segments of
nucleic acid that ideally should hybridize elsewhere in the array.
The presence of these boundary effects provide a level of
background noise in the array and materially reduces the usefulness
of the array.
It is therefore an object of the present invention to provide an
improved solid-state micro-scale array of chemical products.
It is another object of the present invention to provide an array
of the type described which includes preformed cells having
precisely defined boundaries that separate individual cells.
It is another object of the present invention to provide a
solid-state micro-scale array of oligonucleotides.
It is another object of the present invention to provide an
improved method for producing solid-state micro-scale arrays of
chemical products.
It is a further object of the present invention to provide a method
for the photolithographic production of micro-scale arrays of
chemical products.
It is yet another object of the present invention to provide an
apparatus for the photolithographic production of micro-scale
arrays of chemical products.
It is another object of the present invention to provide a
derivatized nucleic acid monomer which has a photolabile group at
the 5'-O position of the sugar.
In accordance with the present invention there is provided a
solid-state micro-scale array of chemical products comprising a
plurality of discrete cells defined on a surface of a substrate,
each of said cells containing an individual chemical product, the
cells being separated one from the other by boundaries that are
precisely defined to the extent that the chemical reactivity or
interactions of the product in each cell is expressible
independently of and is essentially non-affected by the chemical
reactivity of one or more of the products in neighboring cells. In
a preferred embodiment, the chemical products in the cells are
selected from the group consisting of biomonomers and biopolymers.
Further, in the preferred embodiment, the individual cells of the
array are substantially smaller than those heretofore achievable
using physical masks. The photolithographic method described herein
allows the synthesis of arrays in which individual cells occupy
areas measuring only microns or tens of microns on a side. For
example, an array of all 65,536 octamers of DNA, in which each
octamer is contained within an area 40 microns square, will occupy
a total area of about 1 cm.sup.2. The present invention thus
provides a method for avoiding the inadequacies of the prior art
and producing arrays of very small micro-scale size with thousands
of different chemical products.
In the method of the present invention, a solid-state micro-size
array of chemical products is built up on a solid support, i.e.,
substrate, such as glass in a multi-step process. More
specifically, the present method contemplates the development
initially of a matrix of discrete cells on the surface of the
substrate, each cell having precisely defined boundaries so that
the cells are well defined, individually separated, and at
identifiable locations on the substrate.
To develop such well-defined cells, the present inventors, in a
preferred method, derivatize the substrate by the addition thereto
of a linking group which attaches to the substrate surface and also
bears a functional group, such as a hydroxyl or amino group, which
is blocked by a photolabile protective group. Thereafter, a
photolithographic mask having predetermined areas of transparency
and opacity is positioned over the substrate and light is caused to
pass through the transparent areas of the mask and fall upon those
portions of the underlying layer of photolabile groups which lie
beneath the areas of transparency. This action renders the
photolabile groups ineffective in that they no longer protect their
associated functional groups against chemical reaction with a
further chemical moiety. In this step the areas of opacity are
positioned over the areas of the substrate which are to become the
discrete cells of the array and the areas of transparency are
positioned over the areas which are to become the intervening areas
of the array which separate the cells. Following the
photodeprotection of functional groups in the intervening areas
these same functional groups are reblocked by reaction with a
reagent, e.g. acetic anhydride, which renders the functional groups
inactive and in which the blocking group is substantially stable to
light and chemical reagents used in the later steps of array
synthesis.
These "permanently" blocked areas of the substrate surface
constitute a "primary mask" which separates and defines the borders
of the cells to which biomonomers and biopolymers are attached
during synthesis of the array. In subsequent steps of the array
synthesis, photolithographic masks are positioned such that the
borders of opaque and transparent areas are positioned over areas
of the primary mask of the substrate. This substantially prevents
the background and boundary effects which result from the use of
substrates uniformly derivatized with reactive linkers and from the
use of photolithographic masks which allow contact between
neighboring cells or in which the borders of the individual cells
are defined by multiple masking steps. The presence of nonreactive
zones between the cells of the array allows a degree of tolerance
in the alignment of successive masks to be used in the later stages
of array synthesis. The preparation of substrates bearing a
patterned array of individual cells separated by a nonreactive
primary mask may also be accomplished by other photolithographic
methods. For example, the areas of the substrate which are to
become the discrete cells may be protected by a photoresist layer
while exposed intervening areas of substrate are coated with a
primary masking layer such as a siliconizing agent. Conversely, the
primary mask of a uniformly coated substrate may be etched away at
the sites of the discrete cells while the intervening border areas
are protected by a photoresist layer.
At the completion of the substrate preparation each cell in the
array contains linkers terminating in functional groups blocked
with photolabile moieties as described above. Thereafter, a second
photolithographic mask having predetermined areas of transparency
and predetermined areas of opacity is positioned over the entire
substrate and light is caused to pass through the transparent areas
of the photolithographic mask and fall upon selected cells which
lie beneath the areas of transparency, resulting in the
photodeprotection of the functional groups in the exposed
cells.
Once the functional groups in the selected cells are deprotected,
the surface of the substrate is flooded with a selected chemical
moiety, for example, a solution containing one of the nucleotides
of DNA in a form suitable for solid-phase synthesis, such as the
beta-cyanoethylphosphoramidite, these nucleotides also having
associated therewith a photolabile group, for example blocking the
5'-O position. The nucleotides attach themselves to the exposed
functional groups, for example hydroxyls, and become anchored in
individual cells on the substrate.
Thereafter, a further photolithographic mask is positioned over the
substrate, this further photolithographic mask generally having
selected areas of transparency and selected areas of opacity that
are of a different pattern than the pattern of transparent and
opaque areas of the previous photolithographic mask. Light is
directed through the transparent areas of this further
photolithographic mask onto the substrate surface. Generally, the
pattern of transparency and opacity of this further
photolithographic mask is selected to exclude light from all or
part of those cells which received the first nucleotide and to
deprotect other of the cells that contain functional groups.
Following this deprotection step, the substrate is flooded with
solution containing a second nucleotide having a photolabile group
associated therewith, for example, which attaches itself to those
functional groups or nucleotides which were exposed by the most
recent light treatment through the further mask.
The foregoing steps of masking the substrate, exposing the
substrate to light in selected areas to deprotect such areas,
addition of a further chemical moiety having a photolabile group
associated therewith to those cells which have been exposed by the
most recent light treatment, etc., are repeated for that number of
times required to build up within each cell whatever specific
chemical product is desired, for example a biopolymer comprising
the nucleic acid sequence of GACT, AACT, etc. Each cycle of
nucleotide addition may include additional steps, e.g., oxidation
of phosphite to phosphate, as required by the particular synthesis
chemistry used. Modified bases, as well as modified sugars and
internucleotide phosphate linkages may also be incorporated into
members of the array if desired; for example, to test the effect of
specific modifications on hybridization or for increased stability
of array members.
The present invention thus provides a photolithographic method for
the parallel synthesis of multiple chemical products disposed
individually at specific predetermined positions of an array on a
surface of a substrate. The general method comprises, first,
derivatizing the surface of the substrate with a functional group
for the attachment of a chemical moiety. The functional group may
include a "linker" to space the reactive site away from the surface
of the substrate. The functional group is derivatized with a
photolabile protective group or with a chemical moiety having a
further functional site which is blocked by a photolabile
protective group. Next, first selected areas of the surface of the
substrate are illuminated with light of a wavelength and intensity
and for a sufficient amount of time to deprotect the one other
functional sites at the selected areas without deprotecting the
functional sites not at the selected areas. Subsequently, the
substrate is treated with a chemical moiety having a first
functional site capable of reacting with and attaching to the
photodeprotected site of the functional group while substantially
not attaching to other sites on the substrate. Further, the
chemical moiety has at least a second functional site which is
blocked by a photolabile protective group. The moiety is different
from or the same as the functional group. Subsequently, second
selected areas of the surface of the substrate are illuminated with
light of a wavelength and intensity and for a sufficient amount of
time to deprotect the functional sites at the second selected areas
without deprotecting other functional sites not at the selected
areas. The second selected areas are different from or the same as
the first selected areas. The steps of treating with chemical
moieties and illuminating selected areas are repeated, wherein the
chemical moieties are different from or the same as the functional
group chemical moiety and wherein the selected areas are different
from or the same as the first and second selected areas. Thus, an
array of the desired multiple chemical products is synthesized and
each individual chemical product is located at a specific
predetermined position in the array.
In a preferred embodiment of the present invention, the chemical
moieties comprise nucleotide derivatives bearing a photolabile
protecting group on the 5'-oxygen and the chemical products
comprise oligonucleotides. A number of photolabile
hydroxyl-protecting groups are available for this purpose,
including 2-nitrobenzyl, 6-nitroveratryl, 2-nitrobenzyloxycarbonyl,
6-nitroveratryloxycarbonyl, and analogs having comparable chemical
and photochemical properties. The preferred substrate for the
synthesis of oligonucleotide arrays is one functionalized with
hydroxyl groups. Where the chemical products comprise peptides, the
preferred chemical moieties comprise amino acid derivatives bearing
a photolabile protective group on the amino function. A number of
photolabile blocking groups are also available for the amino
function, including the 2-nitrobenzyloxycarbonyl,
6-nitroveratryloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,
alpha,alpha-dimethyl-3,5-dimethoxybenzyloxylcarbonyl, various
arenesulphonyl groups, and analogs having comparable chemical and
photochemical properties. The preferred substrate for the synthesis
of peptide arrays is one functionalized with amino groups.
The illumination of the selected areas is carried out, in a
preferred embodiment, by illuminating the substrate through a
photolithographic mask or by illuminating the selected areas with a
laser or focused beam of light. The substrate in a preferred
embodiment is a plate of a material selected from the group
consisting of soda-lime glass, borosilicate glass, quartz, or
silicon.
The present invention also provides for a method for the parallel
synthesis of multiple chemical products disposed individually at
specific predetermined positions in discrete cells of an array on a
surface of a substrate. The discrete cells of the array are
separated one from another by border areas. The method comprises
preparing the surface of the substrate by derivatizing the surface
with a functional group for the attachment of a chemical moiety.
The functional group may include a linker to space the reactive
site away from the surface of the substrate. The functional group
is derivatized with a photolabile protective group or with a
chemical moiety having a further functional site which is blocked
by a photolabile protective group. First selected areas of the
surface of the substrate are illuminated with light of a wavelength
and intensity and for a sufficient amount of time to deprotect the
functional sites at the selected areas without deprotecting the
functional sites not at the selected areas. The first selected
areas substantially correspond to the border areas separating the
discrete cells of the array. The substrate is then treated with a
chemical agent capable of reacting with and attaching to the
photodeprotected sites at the selected areas while substantially
not reacting with other sites on the substrate. Examples of such
chemical agents include acylating agents, such as acetic anhydride,
for reaction with hydroxyl and amino functions. The resulting
blocked functional sites are substantially non-reactive and
non-photolabile. Next, second selected areas of the surface of the
substrate are illuminated with light of a wavelength and intensity
and for a sufficient amount of time to deprotect the functional
sites at the second selected areas without deprotecting other
functional sites not at the selected areas. The second selected
areas correspond to at least one of the discrete cells of the
array. Then the substrate is treated with a chemical moiety having
a first functional site capable of reacting with and attaching to
the deprotected site of the linker or functional group while
substantially not attaching to other sites on the substrate. The
third chemical moiety has at least a second functional site which
is blocked by a photolabile protective group. The moiety different
from or the same as the functional group. Third selected areas of
the surface of the substrate are then illuminated with light of a
wavelength and intensity and for a sufficient amount of time to
deprotect the functional sites of those linkers or chemical
moieties having photolabile protecting groups at the selected areas
without deprotecting other functional sites not at the selected
areas. The third selected areas correspond to at least one discrete
cell of the array. The third selected areas are different from or
the same as the second selected areas. The steps of treating with
chemical moieties and illuminating selected areas are then repeated
a sufficient number of times to produce the desired array of
chemical products. During the repeated treatment steps, the
chemical moieties are different from or the same as the chemical
moieties. Also, the selected areas are different from or the same
as the previous selected areas. Thus, an array of the desired
multiple chemical products is synthesized and each individual
chemical product is located in a discrete cell at a specific
predetermined position in the array.
The synthesis of many different products may thus be carried out in
parallel in the different cells of the array with the product
contained in each cell being determined by the pattern of masking
and addition steps. The present invention is therefore particularly
useful in providing micro-scale arrays of biopolymer sequences,
such as oligonucleotides and peptides having a large number of
members and capable of being synthesized on solid supports from
monomers. The photolithographic method of parallel synthesis may
also, of course, be carried out in the absence of a "primary mask",
though with certain of the boundary effects mentioned above.
Upon completion of the masking and addition steps, the entire array
is subjected to light and/or other treatments as necessary, e.g.,
ammonium hydroxide, to deprotect all of the chemical products in
the several cells without cleaving the products themselves from the
support. The entire array may then be exposed to a solution
containing molecules which interact with specific members of the
array, causing those members to become associated with detectable
reporter groups, such as fluorescent moieties or radioisotopes.
Inasmuch as the identity of such substrate member may be determined
from its position in the array, these interactions may be used to
identify specific properties of the solute and/or substrate
molecules. For example, exposure of an array of oligonucleotides to
a solution of labeled DNA or RNA under conditions which allow
hybridization of substrate members to complementary sequences in
the labeled molecules may be used to simultaneously identify many
such sequences in the labeled molecules. Oligonucleotide arrays
produced by the methods of the present invention therefore have
many applications to DNA mapping, sequencing, fingerprinting and
diagnostics.
Photolithographic masks are easily prepared and positioned with
great precision. Therefore, the method of the present invention
makes the production of micro-scale arrays of biopolymers, such as
octamers of DNA, a relatively direct process.
A laser or focused light beam might also be used to deliver light
to specific locations in the array for photodeprotection. Light
beams may be accurately and repeatably directed onto the substrate
of a micro-scale array by one of a number of methods. For example,
a confocal scanning microscope would move the sample while holding
the focused beam steady while a laser scanning microscope uses
lenses and mirrors to accurately direct a laser beam over a
substantially stationary sample.
The photolabile groups used in the present invention are well known
in the art (Pillai, in Organic Photochemistry, Vol. 9, A Padwa,
ed., Marcel Dekker, Inc., New York, 1987, pp. 225-323). The
2-nitrobenzyl and related groups have been used to protect hydroxyl
function. Notably, the 2-nitrobenzyl group has been used to protect
the 2'-hydroxyl of ribonucleotides during oligoribonucleotide
synthesis; efficient removal of the protective function from the
synthesized oligomers has been accomplished by exposure in solution
to ultraviolet light of a wavelength greater than 320 nm, without
damage to the nucleoside bases. The 6-nitroveratryl group (i.e.,
4,5-dimethoxy-2-nitrobenzyl) has been used as a photolabile
protective group for the hydroxyl function in synthetic
carbohydrate chemistry and was efficiently removed by irradiation
at wavelengths greater than 320 nm. Additional photocleavable
protective groups for the hydroxyl function include the
2-nitrobenzyloxycarbonyl and related groups. Analogs of these
groups which neither substantially affect the reactivity of the
blocking group nor substantially affect the photolability of the
blocking group are also acceptable for use as photolabile blocking
groups.
The 2-nitrobenzyloxycarbonyl, 6-nitroveratryloxycarbonyl,
3,5-dimethoxybenzyloxycarbonyl, alpha,
alpha-dimethyl-3,5-dimethoxylbenzyloxycarbonyl and arenesulphonyl
groups have all been used as photolabile protecting groups for the
amino-terminus during peptide synthesis. These groups are generally
removed by irradiation with light of wavelength greater than about
320 nm. Photolabile blocking or photoactivating groups are also
known for other functions, including the carboxyl, thiol and
carbonyl groups and photolytic deprotection or photoactivation
reactions have been employed in the synthesis of a variety of
chemical products.
In the preferred method of the present invention photolabile
blocking groups are used in place of the normal terminal blocking
groups used in solid-phase synthesis, which are removed by chemical
treatments. Thus, for example, the 5'-O'-dimethoxytrityl group
normally used in oligonucleotide synthesis is replaced by a
photosensitive hydroxyl-protecting function and the
tertiary-butoxycarbonyl function used to protect the amino function
in solid-phase peptide synthesis is replaced by a photolabile
amine-protecting group. This principal may be extended to the
solid-phase synthesis of other types of polymers, such as
oligosaccharides, in which the subunits bearing a terminal blocking
group are incorporated stepwise onto the support. More generally,
the combination of photolithographic methods with chemical
syntheses employing photolabile blocking or photoactivating groups
may be used to prepare arrays of molecular variants of a variety of
chemical products on solid supports.
The present invention provides for an apparatus for the
photolithographic production of a solid-state micro-scale array of
selected biomonomers and biopolymers. The apparatus comprises a
base member including at least one generally open concave cavity,
and at least one aperture through the base member into the cavity.
There is also a substrate member having a length and width being
such as to at least completely cover the open cavity in the base
member, and being treated such that the substrate member has
photolabile protected functional groups available in cells of an
array along at least one surface of the substrate member. The
substrate member consists of soda-lime glass, borosilicate glass
(e.g., PYREX, Dow Corning) or other material, such as quartz or
silicon, which is substantially transparent to the wavelengths of
light used to remove the photolabile blocking groups employed in
the oligomer synthesis. The functional groups are reactive with the
selected biomonomers when the functional groups are deprotected. A
set of photolithographic masks is provided, each of which hag
different transparent portions and different opaque portions.
Further, there is a source of light of a wavelength and intensity
sufficient to labilize a photolabile chemical group. The apparatus
operates such that the transparent substrate member is joined to
the base member, wherein the cavity and the substrate member form a
reaction chamber. One of the photolithographic masks is placed
between the substrate member and the source of light. The
transparent portions of the mask substantially correspond to those
cells of the array where the selected biomonomer is to be added to
the array. The source of light is operated to labilize the
photoprotecting groups exposed to the light resulting in the cells
of the array having exposed reactive groups. The labilized groups
are rinsed from the reaction chamber via the aperture in the base
member. The reaction chamber is supplied with a solution, again via
the aperture, which includes a selected photolabile protected
biomonomer which is reactive to the exposed reactive groups
available in the cells of the array along the one side of the
substrate member. The biomonomer reacts with the linking group and
forms a layer of photolabile protected groups in the array. A
different photolithographic mask is placed between the substrate
member and the source of light, the source of light is again
operated, and the reaction chamber is again supplied with a
solution including a selected photolabile protected biomonomer
which is reactive to the exposed reactive groups available in the
cells of the array along the one side of the substrate member. The
process is repeated until the desired micro-scale array is
produced.
The present invention also provides for a nucleoside comprising a
saccharide selected from the group consisting of ribose and
deoxyribose. Further, there is a base component selected from the
group consisting of purines and pyrimidines attached to the
saccharide at the 1'-position of the saccharide. In addition, there
is a photolabile protecting group at the 5'-position of the
saccharide. In a preferred embodiment of the invention, the purine
or pyrimidine is selected from the group consisting of adenine,
cytosine, guanine, thymine, uracil, and derivatives thereof.
The present invention may be better understood by reference to the
following detailed description of exemplary embodiments when
considered in conjunction with the drawings in which:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of the preparation of a
deoxyribonucleic acid base with a photolabile group in the
5'-position for use in the present invention;
FIG. 2 is a diagram depicting a preferred embodiment of the present
invention wherein a primary mask is chemically attached to the
surface of a substrate;
FIGS. 3-1, 3-2, and 3-3 are is diagrams illustrating the generation
of a micro-scale solid-state array of biopolymers according to one
embodiment of the present invention;
FIGS. 4-1, 4-2, 4-3, and 4-4 are is diagrams illustrating the
generation of a micro-scale solid-state array of biopolymers
according to another embodiment the present invention;
FIG. 5 is a perspective view of a flow cell to be used in producing
micro-scale arrays of the present invention; and
FIG. 6 is a cross-sectional diagram of the flow cell of FIG. 8
taken through 6--6.
Deoxyribonucleoside derivatives in which the 5'-hydroxyl is
protected as a photosensitive carbonate ester are readily prepared
by reaction of 2-nitrobenzyloxycarbonyl chloride and related
compounds such as 6-nitroveratryloxycarbonyl chloride with
thyroidinc or the N-protected derivatives of deoxyadenosine,
deoxycytidine and deoxyguanosine. The chloroformates react
preferentially at the primary hydroxyl group of the nucleoside.
Analogously, the corresponding 5'-carbonate esters are prepared
from N-protected ribonucleoside derivatives in which the
2'-hydroxyl is also blocked, e.g. as a silyl ether.
Nucleosides in which the 5'-hydroxyl is protected as a
photocleavable ether are prepared by reaction of the primary
hydroxyl with 2-nitrobenzyl bromide or 6-nitroveratryl bromide in
the presence of sodium hydride. In the case of nucleosides in which
the purine or pyrimidine is substantially reactive (e.g., thymine
and guanine) leading to undesired products, a multistep synthesis
is used as indicated in FIG. 1. Initially, the unsubstituted sugar
is reacted with the photolabile protecting reagent, and the
resulting protected sugar is then reacted (after conversion to its
corresponding pentosyl chloride) with the appropriate purine or
pyrimidine derivative, via published glycosylation procedures, to
give the 5'-O-protected nucleoside. The glycosylation procedure
includes the initial steps of protecting the 3-hydroxyl and
converting the sugar to a 1-chloro derivative, not shown. In the
case of ribonucleoside the 2- and 3-hydroxyls of the unsubstituted
sugar are initially protected as the isopropylidene derivative.
The 5'-O-protected nucleoside derivatives are then converted to
3'-O derivatives such as the phosphoramidites or H-phosphonates by
standard procedures for use in solid-phase oligonucleotide
synthesis.
Referring now to the drawings in which like reference characters
designate like or corresponding parts throughout the Figures, there
is shown in FIG. 2 a substrate 10. For purposes of clarity in
explaining the present invention, the following description refers
to an array of biopolymers, i.e., sequences of nucleic acids, built
up from biomonomers, i.e., individual nucleic acids, and
specifically to sequences of the four DNA nucleotides of guanine
(G), adenine (A), thymine (T) and cytosine (C). This description
could just as easily refer to an array of different biopolymers
(such as RNA, peptides, or oligosaccharides) built up from
different biomonomers (such as the RNA nucleotides, amino acids or
saccharides). A suitable material for such a substrate 10 would be
a plate of soda-lime glass, borosilicate glass or quartz. The
substrate 10 has been previously prepared to be reactive to a
photolabile group, X, or to a moiety bearing a functional group
blocked by X, as well as various biomonomers, represented here as
AX, and a blocking group, represented by B. The substrate 10 is
treated with a photolabile group, X, or the aforesaid moiety
bearing X, which reacts with the functional groups on the surface
12 of the substrate 10 to form a layer 14 on the substrate 10. A
photolithographic mask 16, with transparent regions 18 and an
opaque region 20, is placed between the layer 14 and a source of
light, not shown. Light from the source of light penetrates the
transparent regions 18 to strike a portion of the layer 14. The
protecting groups are labilized from those exposed portions of the
layer 14 and the labilized groups are removed leaving a remaining
protected portion 22 of the layer 14. The substrate 10 is treated
with a non-photolabile blocking group, B, which reacts with the
functional groups on the surface 12 of the substrate 10 to form
layers 24 on the substrate 10. The layers 24 act as non-reactive
non-photolabile boundary areas between the cells of the array. The
substrate 10 is then illuminated with the light source to labilize
the remains of the layer 14. The protecting groups are removed
leaving an exposed cell area 26 which is reactive toward the
selected biomonomer with a photolabile group, AX. The substrate 10
is next treated with the selected biomonomer, AX, which attaches to
the substrate 10 in the area 28. Thus, selected biomonomers may be
added to the cells of the micro-scale array without attachment to
the boundaries between the cells, as shown for the equivalent array
illustrated by FIGS. 4-1, 4-2, 4-3, and 4-4.
FIGS. 3-1, 3-2, and 3-3 are diagrams illustrating the generation of
a micro-scale solid-state array of biopolymers according to one
embodiment of the present invention. A substrate 310 has a surface
312 for attaching biomonomers. In the first step, a, of the
generation of the array, a biomonomer, represented in this case by
A, with a photolabile protecting group, represented in this case by
X, is attached to the surface 312 of the substrate 310. This
produces a carpet of protected biomonomers attached to the surface.
A mask 314 with transparent areas 316 and opaque areas 318 is
placed over the substrate and biomonomers in step b. Light 320 is
projected on to the mask and through the transparent areas 316. The
light 320 penetrates through the transparent areas 316 onto a
portion of the photolabile groups attached to the biomonomers. The
photolabile groups, X, which have been illuminated by the light 320
are removed from the biomonomers in step c. In step d, the
substrate is then treated with another biomonomer with a
photolabile protecting group, AX. Another mask 322 is placed over
the array and light 324 is projected on to the mask 322 in step e.
The light 324 penetrates the transparent area 326 of the mask 322.
In step f, those photolabile groups, X, which were illuminated by
the light 324 are removed. The matrix is then treated with another
biomonomer with a photolabile protecting group, TX, in step g. The
process of masking, illumination, and treatment with biomonomers
with photolabile protective groups is repeated a number of times in
steps h through s, to produce the final array 328. The array 328
contains a series of different biopolymers which occupy the
positions of the elements of the array 328. The elements
illustrated in the final array 328 of FIG. 3 represent eight
different polynucleotide sequences where A represents adenine and T
represents thymine. The sites of the final array 328 are 330 for
the sequence AAA; 332 for the sequence AATA, 334 for the sequence
AAAT; 336 for the sequence AATT, 338 for the sequence ATAA; 340 for
the sequence ATTA; 342 for the sequence ATAT; and 344 for the
sequence ATTT. An example of the boundary effect is demonstrated by
the sequences AATATAT 346 and AAATT 348 which have been formed by
misalignments of the masks, in steps b, e, h, k, n, and q, during
the formation of the final array 328. The sequences 346 and 348 are
situated between the elements 336 and 338. In a hypothetical
experiment, if the final array 328 was treated with tetrameric
nucleic acids, several of the tetrameters hybridizing with the
sequence 346 would have no relationship to the sequence 336 or the
sequence 338. For example, a sequence complementary to the sequence
ATAT, represented at the site 342, would bind at the upper end of
the sequence 346. In a similar way, a nucleic acid sequence the
sequence AATA, the same sequence as is seen at site 332, would
hybridize with the lower portion of the sequence 346. Therefore,
the presence of sequences similar to the sequence 346 or the
sequence 348 contribute to noise in the background of the final
array 328. (These hypothetical examples of hybridization are given
for illustrative purposes only; in practice, arrays of longer
oligonucleotides are used.)
FIGS. 4-1, 4-2, 4-3, and 4-4 are diagrams illustrating the
generation of a micro-scale solid-state array of biopolymers
according to another embodiment of the present invention. A
substrate 410 has a surface 412 which has a primary mask 414. The
primary mask 414 divides the surface 412 into areas 416 to which
biomonomers are capable of being attached and areas 418 to which
biomonomers cannot attach. When the surface 412 of the substrate
410 is flooded in step a with a biomonomer having an attached
photolabile protecting group (AX) the biomonomer attaches only in
those areas 416 where they are capable of attaching. In step b, a
secondary mask 420 is placed over the surface 412 of the substrate.
Light 422 is projected on to the photolithographic mask 420. The
light penetrates the transparent portion 414 of the mask and is
blocked by the opaque 426 of the mask 420. Only those photolabile
groups in the cells 416 of the substrate 410 exposed to the light
422 are deprotected in step c. The substrate 410 is then treated
with additional biomonomer having a photolabile protecting group
(AX) which forms the first set of biopolymers 428 in step d. In
step e, a second photolithographic mask 430 is placed over the
substrate 410 and light 432 is projected on to the surface 412 of
the substrate 410 through the transparent areas 434 of the
photolithographic mask 430. The light 432 is blocked by the opaque
areas 436 of the photolithographic mask 430. In steps f and g, the
exposed photolabile groups are removed and the substrate 410 is
then treated with another biomonomer having a photolabile
protecting group (TX) which forms a second set of biopolymers 438.
The process of masking, projecting of light, and treatment with
protected biomonomers is repeated several times, in steps h through
s until the desired array is produced. The final array 438 contains
eight different sets of biopolymers 440, 442, 444, 446, 448, 450,
452, and 454. These sets of biopolymers exist in discrete cells
416. In the array of the present invention, there are none of the
boundary effects as are seen in an array prepared according to the
previous embodiment. When an oligonucleotide array of the present
invention is treated with nucleic acid sequences, hybridization may
occur only at the discrete sites 416 and not in the boundary
regions bordering each cell 418.
A top view of a flow cell 510 for preparing solid-state micro-scale
arrays is shown in FIG. 5; a cross-sectional view of the flow cell
510 is shown in FIG. 6. The flow cell 510 comprises a base member
512 having a concave cavity 514 formed, milled or otherwise
structured into the base member 512. The base member 512 is
preferably constructed of a generally non-chemically reactive
polymeric material such as TEFLON (DuPont) although other
nonreactive materials such as siliconized glass, may be used. Two
apertures 516 and 518 through the base member 512 provide direct
connection between the cavity 514 and the exterior of the base
member 512. The apertures 516 and 518 are in direct communication
with connectors 520 and 522, respectively. The connectors 520 and
522 are adapted for connection with tubing for the delivery or
removal of liquid materials to and from the cavity 514.
A substrate member 524 is placed on the base member 512. A gasket
of non-reactive material, such as TEFLON (DuPont), may be used as a
seal between the two members 524 and 512. The substrate member 524
has a length and width such that it completely covers the open
cavity 514. Thus, the cavity 514 and one side 526 of the substrate
member 524 form a reaction chamber 528. The substrate member 524
preferably comprises a soda-lime glass, borosilicate glass or
quartz plate, but any suitable material, which is transparent to
the wavelengths of light required for removal of photolabile
blocking groups and which may be functionalized for the attachment
of monomers, may be advantageously used with the present invention
depending on the nature and identity of the biomonomers used and
the biopolymers desired.
A photolithographic mask 530 is placed adjacent the second side 532
of the substrate member 524. The mask 530 has opaque portions 534
and transparent portions 536. The opaque portions 534 correspond to
the areas of the substrate member 524 which will not be
photolabilized and the transparent portions 536 correspond to those
areas which will be photolabilized. The opaque portions 534 of the
mask 530 may, as shown, be circular and aligned in a close packed
hexagonal arrangement to maximize the usable space on the substrate
member 524 while maintaining the border areas. However, it should
be noted that the opaque portions 534 may be of any convenient
shape and arrangement.
A source of light 538 is placed on the opposite side of the mask
530 from the substrate member 524. The source of light generally
comprises an ultraviolet lamp 540 which produces light of a
wavelength sufficient to labilize a photolabile group. A filter 542
may also be placed between the lamp 540 and the mask 530 in order
to remove other wavelengths of light which may be otherwise
damaging to the chemical moieties of the array. The light is
distributed across the mask 530 in a substantially uniform and
collimated manner. As noted above, a laser or focused light beam
might also be used for sequential illumination of the cells of the
array.
In the practice of the invention using the flow cell 510, the
substrate member 524 is initially treated to provide a linking
group on the one side 526 which will form, along with the cavity
514, a portion of the reaction chamber 528. The substrate member
524 is then placed onto the base member 512 and aligned with the
substrate alignment members 544. The substrate member 524 is
secured to the base member 512 by clamps 546 and screws 548. The
mask 530 is then placed on the second side 532 of the substrate
member 524, aligned with the mask alignment members 550, and
secured to the base member 512 by clamps 552 and screws 554. The
repeatable alignment of the substrate member 524 and the mask 530
is very important in the practice of this invention using the flow
cell 510. As was discussed above with respect to FIGS. 3 and 4, the
improper or unrepeatable alignment of the mask 530 with respect to
the substrate member 524 will lead to increased background noise in
the use of the final array. Therefore, it is important to be able
to repeatably align the mask 530 and the substrate member 524. It
will be recognized by a practitioner of the art that the present
illustration is not the only effective means of repeatably aligning
the mask 530 and the substrate member 524. In the present
illustration, the mask 530 is held substantially in contact with
the second side 532 but it is not strictly necessary to do so.
However, as the mask 530 is moved away from the substrate member
524, the light from the source 538 becomes diffuse as it falls upon
the substrate member 524.
Once the flow cell 510 is fully assembled, it is connected to
sources of reagents for preparing the array, such as an automated
DNA or peptide synthesizer (not shown). The reaction chamber 528 is
initially filled with a solution containing a compound which has
two functional sites. One site is reactive with the linking group
on the one side 526 of the substrate member 524 and the other site
is protected with a photolabile protecting group. This compound
may, in some cases, consist of an initial biomonomer or biomonomer
analog where its presence in each member of the array as well as
the border areas does not substantially degrade the performance of
the array. The compound reacts with the linking groups and the
residual solution is then rinsed from the reaction chamber 528. The
light source 538 is then activated and the exposed photolabile
groups are lost from the substrate member 524 and are taken up by
the solution in the reaction chamber 528. Again, the chamber 528 is
rinsed. A solution having a non-photolabile blocking group is then
introduced into the chamber 528. The blocking group reacts with the
non-protected linking groups on the substrate member 524 forming
the boundary areas of the array. The blocking group solution is
also rinsed out of the chamber 528.
The first mask 530 is then removed and second mask 530 having a
different pattern of transparency and opacity is aligned and
secured to the base member 512. The light source 538 is then
activated and the exposed photolabile groups are lost from the
substrate member 524 and are taken up by the solution in the
reaction chamber 528. Again, the chamber 528 is rinsed. A solution
having the first selected biomonomer is then introduced into the
chamber 528. The biomonomer reacts with the non-protected groups on
the substrate member 524 forming the first layer of biomonomers at
the selected sites of the array. The biomonomer solution is then
also rinsed out of the chamber 528. The process is repeated in a
manner similar to that illustrated in FIG. 4. Additional reagents,
as required by the oligomer synthesis, are added and removed from
the chamber 528 at each step and the result is a solid-state
micro-scale biopolymer array.
Therefore, an array prepared according to the present invention
does not suffer the effects that limit the usability of arrays
prepared according to prior art methods. The present invention
provides for the preparation of micro-scale solid-state arrays.
These arrays may contain tens of thousands of cells in a matrix
less than 2 cm on a side. In addition, the micro-scale arrays of
the present invention do not suffer the boundary effects of prior
art arrays. The background noise is minimized since each cell in
the array contains only that polymer which was designed to be
there.
Arrays prepared in accordance with the present invention will
provide discrete cells of substrate for the attachment of
biomonomers. There will be substantially no boundary effect in such
arrays.
The features of the invention which are believed to be new are set
forth in the appended claims.
* * * * *