JP2004509323A - Replicatable probe array - Google Patents

Abstract

For example, generating substantially identical specific binding ligand probe arrays by preparing and replicating an original master array and / or providing a reusable assay array that can be regenerated System to do. In one embodiment, the system comprises: a) a master array surface having immobilized address ligands; b) a binding region complementary to the address ligand; a binding region complementary to the target ligand; Multi-ligand conjugates having a third ligand for use in transferring the conjugate into or onto the surface of an assay array and which can be used with an address and its complementary ligand or upon dissociation thereof , Including the preparation and use of

Description

【０１８７】
【化２】

【０１８８】
【化３】

（式中、ｘ＝０．１〜５モル％、ｙ＝２〜３０モル％、およびｚ＝６５〜９７．９モル％）
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本発明の好ましい実施形態が記載されてきたが、一方で、本発明の精神および添付クレームの範囲から逸脱することなく、種々の変更、適合および修飾がそこでなされ得ることは理解されるべきである。[0001]
This application was filed as a PCT international patent application on July 9, 2001, in the name of a U.S. domestic company, SurModics, Inc., specifying all countries except the United States.
[0002]
Technical field
The present invention relates to the immobilization of specific binding ligands such as nucleic acids and other ligands in a known spatial arrangement. In another aspect, the invention relates to a solid support such as an oligonucleotide array incorporating such nucleic acids. In yet another aspect, the invention relates to photoreactive groups, molecules and / or surfaces derived from such groups, and the attachment of such molecules to the support surface by such groups.
[0003]
Background of the Invention
The development of oligonucleotide probe arrays, more commonly known as "DNA chips" and "Gene chips" (registered trademark of Affymetrix, Inc.), has made significant progress in the past few years. And it is at the core of growing attention and growing importance. See, for example, Stipp, D .; Fortune, p. 56, March 31, 1997. Also, Borman, S.A. , C & EN, p. 42, December 9, 1996, and Travis, J. et al. , Science News 151: 144-145 (1997). These 2- or 3-cm square chips can contain tens of thousands to hundreds of thousands of immobilized oligonucleotides, allowing researchers to witness the behavior of thousands of cooperating genes for the first time. DNA chips are used for observing unique gene expression patterns, properly reading the success of drug treatment, tailoring drugs based on the patient's genetic structure, arranging genes, and conducting research in the field of gene medicine. Useful. Also, “Microchip Arrays Put DNA on the Spot”, R.A. Service, Science 282 (5388): 396-399, 16 October 1998; and "Fomenting a Revolution, in Miniature", I.C. Amato, Science 282 (5388): 402-405, 16 October 1998.
[0004]
In general, oligonucleotide probe arrays display a particular oligonucleotide sequence at a precise location in an information-rich format. In use, the hybridization pattern of a fluorescently labeled nucleic acid target is used to obtain primary structural information for the target. This format can be applied to a wide range of nucleic acid sequencing problems including pathogen identification, forensic applications, monitoring of mRNA expression and de novo sequencing. See, for example, Lipshutz, R.A. J. , Et Al. , BioTechniques 19 (3): 442-447 (1995). Such arrays are sometimes required to carry tens of thousands, or even hundreds of thousands of individual probes. The chip is also required to provide a wide range of sensitivities to detect sequences that can be expressed anywhere from 1 to 10,000 replication levels per cell.
[0005]
A variety of methods have been developed for making and / or using oligonucleotide probe arrays. See, for example, Weaver, et al., Describing a synthetic strategy for the creation of large-scale chemical diversity on a solid support. (WO92 / 10092). This system uses solid-phase chemistry, photolabile protecting groups, and photolithography to achieve light-directed, spatially addressable, parallel chemical synthesis. Using the proper order of masks and chemical sequential reactions, a defined set of oligonucleotides can each be constructed at predefined locations on the array surface.
[0006]
Using this technology, Affymetrix (Santa Clara, CA) has developed a library of single-molecule, double-stranded oligonucleotides on a solid support. See, for example, U.S. Patent No. 5,770,722, which describes an array containing oligonucleotides 4 to 100 nucleotides in length. The array includes a solid support, an optional spacer, a first oligonucleotide, a second oligonucleotide that is first complementary, and a flexible linker or probe. The libraries described are useful for screening for receptors such as proteins, RNA or other molecules that bind to double-stranded DNA. Another array developed by Affymetrix is described in U.S. Patent No. 5,837,832. This document describes a method for making a high-density array of oligonucleotide probes on a silica chip. Oligonucleotide probes are 9-20 nucleotides in length and are synthesized directly on a solid support. The array contains oligonucleotide probes that are complementary to sections of the control sequence.
[0007]
Synteni (Palo Alto, Calif.) Creates an array of cDNAs by applying polylysine to glass slides, followed by printing the cDNA on coated slides, which then crosslink the DNA with polylysine. Exposed to UV light. Next, unreacted polylysine is blocked by reaction with succinic anhydride. These arrays, called "gene expression microarrays" (GEM ™), label mRNA prepared from normal cells with a fluorescent dye, and then label mRNA from abnormal cells with a fluorescent dye of a different color. Used by These two labeled mRNA molecules are simultaneously applied to the microarray, where they competitively bind to the immobilized cDNA molecules. This two-color encoding technique is used to distinguish gene expression differences between two cell samples (Heller, RA, et al., Proc. Natl. Acad. Sci. USA, 94: 2150- 2155 (1997)).
[0008]
At least one group, Cantor et al. (US Pat. No. 5,795,714) describes a method for replicating an array of probes that is said to be useful for large-scale production of diagnostic aids. I have. This patent includes a method for replicating an array of single-stranded probes on a solid support, comprising the steps of:
a) synthesizing a nucleic acid array each comprising a constant sequence of length C at the 3 'end and a variable sequence of length R at the 5'end;
b) fixing the array to the first solid support;
c) synthesizing a set of nucleic acids each comprising a sequence that is complementary to the invariant sequence;
d) hybridizing the set of nucleic acids to the array;
e) enzymatically extending the set of nucleic acids using the variable sequence of the array as a template;
f) denaturing the set of elongated nucleic acids; and
g) Immobilizing the denatured set of nucleic acids on a second solid support to produce a single-stranded probe replication array.
[0009]
With respect to a separate subject, the designated agents of the present invention have previously described a variety of uses for photochemistry, particularly for example the use of photoreactive groups to attach polymers and other molecules to a support surface. . For example, U.S. Patent Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329, 5,563,056, and 5,637,460. No. 5,714,360 and International Patent Application Nos. PCT / US96 / 08797 (Viral Inactivating Coating), PCT / US96 / 07695 (Capillary Endothelialization), and PCT / US97 / 05344. No. (chain transfer agent).
[0010]
Despite various developments to date, there remains a need for methods and reagents that improve the immobilization of nucleic acids on a variety of support materials, for example, to form oligonucleotide probe arrays. Clearly, what is needed is a novel method for replicating a specific binding ligand (eg, nucleic acid) array in a cost-efficient and efficient manner while maintaining accurate and sensitive products. Improved methods and reagents.
[0011]
Summary of the Invention
The present invention, in one embodiment, creates a specific binding ligand (eg, nucleic acid) probe array in substantially the same spatial configuration, for example, by preparing and replicating an original “master” array. To provide a system for In another embodiment, the invention provides a reusable assay array that can be regenerated. The method can be contrasted with the conventional method of preparing each probe array separately and individually. In addition, the present invention allows for duplication of a master array that maintains the spatial arrangement established by the master array. The methods of the present invention can be adapted to be used in conventional arrays to provide their replicas, but preferably in the embodiments described herein for such purposes. Used with master arrays (and other ingredients) specifically designed for
[0012]
In a preferred embodiment, the present invention provides a method and system for reproducibly preparing an assay array, the system comprising:
a) a master array comprising a support surface having a plurality of address ligands immobilized thereon;
b) at least one molecule of the first, wherein each multiligand conjugate comprises: (i) a core; (ii) a ligand selected to bind to a specific address ligand of the master array in a complementary manner. A ligand binding region (iii) at least one molecule of a second ligand binding region comprising a ligand selected to bind to a characteristic target ligand in a complementary manner; and (iv) at least one molecule of a third ligand binding region. A plurality of multi-ligand conjugates comprising a ligand, wherein the first ligand binding region, the second ligand binding region, and the third ligand are bound to a core; and
c) an assay array support comprising a support surface and a binding partner for binding a third ligand;
including.
[0013]
Preferably, the address ligand is patterned and immobilized on a master array, optionally in the form of a random embodiment. In a preferred embodiment, the first and second ligand binding regions of the multi-ligand conjugate comprise a nucleic acid sequence. In this embodiment, the second ligand binding region comprises a nucleic acid sequence that is complementary to a target nucleic acid sequence to be detected in a sample. Optionally, the address ligands are immobilized in a random manner, and the exact location of each address ligand is determined after the address ligand is immobilized on the master array support surface.
[0014]
Alternatively, the present invention provides a method and system for reproducibly preparing an assay array, the system comprising:
a) a master array comprising a support surface having a plurality of address ligands immobilized thereon;
b) at least one molecule of a second ligand, wherein each multi-ligand conjugate comprises: (i) a core; (ii) a ligand selected to bind to the address ligand and a characteristic target ligand in a complementary manner. A plurality of multiligand conjugates comprising a binding region and (iii) a third ligand of at least one molecule, wherein the second ligand binding region and the third ligand are attached to a core;
c) an assay array support comprising a support surface and a binding partner for binding a third ligand;
including.
[0015]
In this embodiment, the use of a first ligand binding region is not required. According to this embodiment, the second ligand binding region is not only complementary to the address ligand of the master array, but also to a target ligand suspected of being present in the sample. The nucleic acid sequence of the assay array produced according to this embodiment is complementary to the address ligand of the master array. If necessary, the assay array created by this embodiment can be replicated to create a duplicate of the original master array. Preferably, the third ligand is selected from a binding ligand and a polymerizable group.
[0016]
A corresponding preferred method of the invention in which the address and the target ligand are both nucleic acid sequences comprises the following steps:
a) Prepare the above-mentioned master array;
b) binding a multi-ligand conjugate thereto by allowing each of them to hybridize to a complementary address ligand of the master array;
c) bringing the assay array support sufficiently close to the master array under conditions suitable to allow the bound multiligand conjugate to bind to the assay array support;
d) Dissociating the hybridized complementary nucleic acid sequence under conditions suitable to allow the assay array support to be recovered and utilized.
[0017]
The resulting assay array comprises an assay array support having a plurality of multi-ligand conjugates attached thereto, preferably having a third ligand present thereon in a pattern established by the master array.
[0018]
In another embodiment, in which the multi-ligand conjugate comprises a polymerizable group (eg, in addition to or instead of a third ligand), the multi-ligand conjugate comprises a space defined by the address of the master array. To form a polymeric backing sufficient to allow the resulting polymerized layer to be supported and used, e.g., transferred to an assay array support, while maintaining the proper configuration. These groups can be maintained in their orientation on the master array surface by polymerizing them in situ.
[0019]
In yet another embodiment, the multiligand conjugate comprises only a second ligand binding region and a third ligand (ie, there is no first ligand binding region) and the address ligand and the target ligand are both nucleic acids The method comprises the following steps:
a) Prepare the above-mentioned master array;
b) binding a multi-ligand conjugate thereto by allowing each of the second ligand binding regions to hybridize to a complementary address ligand of the master array;
c) bringing the assay array support into close proximity to the master array under conditions suitable to allow the bound multiligand conjugate to bind to the assay array support;
d) Dissociate the hybridized complementary nucleic acid sequence under conditions suitable to allow the assay array support to be recovered and used.
[0020]
Once transferred to the assay array support, the second ligand binding region is then used in a conventional manner to determine the presence of one or more target ligands in a sample in absolute or relative amounts. be able to. The order and arrangement of the second ligand binding regions is predetermined and is retained during the replication procedure described herein. For example, the resulting assay array can be prepared in a conventional manner, for example, by contacting the array with a sample suspected of containing the target ligand under conditions suitable to allow any target ligand to bind and be detected. Can be used. In other aspects, the invention provides methods for using such systems; various components for use in such systems, including kits or combinations of one or more components; and assay arrays formed by the methods of the invention. I do.
[0021]
Detailed description
The present invention provides methods and systems for reproducibly preparing specific binding ligand arrays, such as nucleic acid arrays. As used herein, “nucleic acid” refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), or generally the ability to base pair or hybridize to a complementary chemical structure. Macromolecules such as any sequence meaning bases bound by the chemical backbone possessed. The term includes oligonucleotide, nucleotide, or polynucleotide sequences, and fragments or portions thereof. The nucleic acid can be provided in any suitable form, e.g., isolated from natural sources, recombinantly produced, or artificially synthesized, and can be single-stranded or double-stranded. Yes, it is possible to indicate sense or antisense strands. While the invention is specifically described with respect to nucleic acids (and their ability to specifically “bind” via hybridization), the invention is directed to immunological binding pairs or other ligand / antiligand binding pairs, such as It is understood that it has application to specific binding ligands.
[0022]
The term "oligonucleotide" is then used to describe generally short chains (e.g., prepared using techniques available in the art, such as solids-supported nucleic acid synthesis, or using DNA replication or reverse transcription, for example). , Less than about 100 nucleotides in length, generally 20-50 nucleotides in length). The exact size of the oligonucleotide will depend on many factors, in turn, dictated by the ultimate function or use of the oligonucleotide.
[0023]
As used herein, “target ligand” or “target” refers to a ligand, such as a nucleic acid sequence, suspected of being contained in a sample and detected and / or quantified in a method or system of the invention. In one embodiment, the nucleic acid comprises a gene or gene fragment to be detected in a sample.
[0024]
The term "sample" is used in its broadest sense. The term includes specimens or strains (eg, microbial strains), and biological samples.
[0025]
As used herein, the terms "complementary" or "complementarity," when used in reference to a polynucleotide (ie, a sequence of nucleotides such as an oligonucleotide or a target nucleic acid), are used by Watson and Crick. Refers to sequences that are correlated by the base pairing rules developed by the Company. For example, for the sequence "TGA", the complementary sequence is "ACT". Complementarity can be "partial," in which only some of the nucleobases conform according to the base pairing rules. Alternatively, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization between nucleic acid strands.
[0026]
The terms "complementary" or "complementarity," when used in reference to molecules other than nucleic acids, refer to molecules that can bind to a binding partner, such as a molecule that is a member of a specific binding pair.
[0027]
The term "hybridization" is used in connection with the pairing of complementary nucleic acids. The degree of hybridization and the strength of hybridization (ie, the binding strength between nucleic acids) depends on the degree of complementarity between the nucleic acids, the stringency of the conditions incorporated, the melting point (Tm) of the hybrid formed, and the G: C It is affected by factors such as the ratio.
[0028]
The term "binding site" as used herein in the context of an assay array refers to the region on which a particular ligand is bound. As described in more detail below, the binding site preferably contains a molecule that is a member of a specific binding pair for forming a bond with a multiligand conjugate.
[0029]
In one embodiment, the invention provides a master array having immobilized thereon a plurality of address ligands, the pattern of which can be controlled and / or determined by any suitable technique. Thereafter, the composition comprising the plurality of multi-ligand conjugates is treated under conditions suitable to allow the binding region of the multi-ligand conjugate to bind to a complementary address ligand on the master array support. It is brought to the coupling distance with the master array. In a preferred embodiment, the multi-ligand conjugate includes a third ligand comprising members of a specific binding pair. In this embodiment, the third ligand serves to transfer the resulting conjugate to the assay array support. The master array containing the bound conjugation molecules is brought close to the assay array support. The assay array support then contains a binding site available for binding to the third ligand of the multiligand conjugate. The binding site of the assay array support binds to the corresponding binding ligand on the multiligand conjugate, thus providing a temporary "sandwich" structure in the form of a master array / multiligand conjugate / assay array support To form
[0030]
Once a "sandwich" structure has been formed, the base pairs between the address ligand of the master array and the complementary ligand of the multiligand conjugate are then removed along with the multiligand conjugate to which the assay array is attached. It can be dissociated to make it possible. The conjugates bind in a spatial relationship corresponding to the spatial positioning set by their initial binding to the master array surface, thereby allowing the location of the target ligand to be maintained and determined. The master array can then be reused repeatedly, providing additional assay arrays in a similar manner. In another aspect, the invention provides a plurality of substantially identical assay arrays formed by recreating a common master array or by sequentially replicating one or more assay arrays.
[0031]
In yet another aspect, the invention is reusable without the need to first prepare or recreate a master array, for example, where the assay array surface is provided in the form of a fiber optic array. Methods and systems for directly preparing various nucleic acid arrays are provided. In one such embodiment, each fiber is provided with a nucleic acid adapted to hybridize in a detectable manner to its complementary nucleic acid. Fiber arrays suitable for use in such embodiments are described, for example, in K. K., whose disclosure is incorporated herein by reference. Michael et al. , Analytical Chem. 70 (7): 1242 (1998).
[0032]
Next, the assay arrays of the invention are preferably adapted to detect a wide variety of nucleic acids in a biological sample. In the course of its use, the assay array contains one or more target ligands under conditions suitable to allow the target ligands to hybridize to their corresponding binding region complement on the assay array. Can be exposed to samples that are believed to be The presence or absence of the target nucleic acid on the assay array can be measured by a selected signal generation and detection system. Such detection methods are known in the art.
[0033]
The system of the present invention offers many advantages over current methods, including, for example, an optimal combination of properties such as nucleic acid probe density, ease of use, reproducibility, sensitivity, accuracy, and reduced cost. The present invention provides, for example, higher nucleic acid probe densities than commercial methods that currently rely on photolithography. In one embodiment, such probe density is achieved by using microbeads that are even smaller than the wavelength of the photolithographic system. Further, the present invention provides improved assay specificity by the use of longer (and thus more specific for binding) nucleic acids than are currently available, for example, photolithographically or through solid phase synthesis. Can be provided.
[0034]
In addition, assay arrays according to the present invention can be used with conventional array readers to provide a variety of target nucleic acid capabilities, for example, depending on the areal density of the immobilized array. The system of the present invention can also provide a significant reduction in the cost of manufacturing high density array slides. The cost of preparing a master array can itself be reduced, for example, through the use of patterned deposition and immobilization of the address nucleic acid sequence, and optionally through the use of microbeads.
[0035]
As can be seen from the description herein, the present invention replicates a probe array that provides a universal "master array" that can be used with a wide variety of multiligand conjugates to assemble any number of replication assay arrays. Provide an improved way to The versatile nature of the master array is provided by address ligands that have no specificity for a particular assay array. Rather, address ligands can be used with any number of specific multiligand conjugates to assemble a unique replication assay array containing probes to detect a given target ligand, or target ligand system. . In this way, the master array provides a spatial arrangement for creating any number of replicated assay arrays.
[0036]
Master Array
According to the present invention, a master array includes a support surface having a plurality of address ligands immobilized thereon. The address ligand can be directly or indirectly immobilized on the master array support surface. Direct immobilization of the address oligonucleotide is achieved by derivatization of the address oligonucleotide, the support surface of the master array, or both. Indirect immobilization involves the use of a binding agent to attach the address oligonucleotide to the surface, as described in more detail below.
[0037]
The support surface of the master array is manufactured from any suitable material to provide an optimal combination of desirable properties such as stability, size, shape, and surface smoothness. Preferred materials should not interfere with nucleic acid hybridization and should not be subject to high amounts of non-specific binding of nucleic acids. Suitable materials include living or non-living, organic or non-organic substances. For example, the master array can be made from any suitable plastic or polymer, silicon, glass, ceramic, or metal, and is provided in the form of a solid, resin, gel, rigid film, or flexible membrane. be able to. Suitable polymers include polystyrene, poly (methacrylic acid (alkyl), poly (vinylbenzophenone), polycarbonate, polyethylene, polypropylene, polyamide, vinylidene fluoride resin, and the like. Preferred materials include glass and silicon.
[0038]
The size of the master array is determined based on factors such as the size of the assay array to be made and the size of the desired address oligonucleotide diversity. Preferably, the master array is provided in a planar dimension between about 0.5 cm and about 7.5 cm long, and between about 0.5 cm and about 7.5 cm, preferably between about 1 cm and about 2 cm wide. The array can also be placed singly or in multiples on another support such as a microscope slide (eg, having dimensions of about 7.5 cm × about 2.5 cm). Those skilled in the art, given the teachings herein, will be able to readily adapt the dimensions of the master array for a particular application. The master array can be provided in any suitable configuration including, for example, capillaries, membranes, wafers, pins or needles. In addition, as described in more detail below, the master array may be provided in the form of one or more optical fibers.
[0039]
The address oligonucleotide is attached to the master array support surface, either directly or via a linker. In one embodiment, the address oligonucleotide is directly supported by providing and / or derivatized one or more reactive groups on either the master array surface, the oligonucleotide, or both. Is bound to the body surface. In another embodiment, the address oligonucleotide is indirectly attached to the master array support surface through the use of a linker. In this embodiment, the master array support surface is coated with a plurality of linkers, such as linked microbeads having oligonucleotides attached thereto. Thus, when used in combination with linked microbeads, the master array support surface is preferably such that topographical irregularities are smaller than the radius of the linked microbeads (eg, bumps are formed in the connected microbeads of the master array). (Less than 50% of the bead diameter), providing sufficient smoothness.
[0040]
As used herein, the term "address oligonucleotide" is used to refer to a nucleic acid sequence that is immobilized on a master array to provide a template array for use in the present invention. Thus, the template is an ordered array of address sequences that allows the replication assay array to be created using the positional information set by the master array. The address oligonucleotide is selected to be non-complementary to the target nucleic acid sequence potentially present in the biological sample to be tested. As a result, the address oligonucleotide does not base pair or otherwise hybridize to any target nucleic acid found in the particular biological sample being tested, thereby allowing for background or false positive reads Expected to avoid or minimize gender. The oligonucleotide is sufficiently long to provide a suitable diversity of "addresses" on the master array while avoiding substantial complementarity to the target nucleic acid (e.g., naturally occurring) sequence to be detected by the assay array at the same time. Provided by Preferably, these address oligonucleotides are between 10 and 100 nucleotides in length, and most preferably between 20 and 50 nucleotides in length.
[0041]
As used herein, the term "immobilized" means that the address oligonucleotide is attached to the support surface in a sufficiently stable manner for the purposes herein. Such a bond is preferably a covalent bond, although other suitable stable bonds are also contemplated. Suitable linkages will be apparent from the teachings herein.
[0042]
According to the present invention, the deposition of the address oligonucleotide on the master array surface is accomplished in any suitable manner, including, for example, by direct binding or by indirect binding through a linking agent (eg, linking microbeads). Is done. For example, if the address oligonucleotide is bound to microbeads that are then to be bound to the master array, the deposition will cause the microbead specific gravity to cause the individual microbeads carrying the address oligonucleotide to be attached to the master array surface. This can be achieved by a specific gravity based method that causes the contacting. This deposit is ordered on the master array but forms an irregular pattern. Preferably, the microbeads are treated to provide a photoreactive surface function. The resulting surface is then irradiated under conditions suitable for activating the photoreactive groups and causing bond formation between the microbeads carrying the address oligonucleotide and the surface of the master array support.
[0043]
Preferably, the deposition of the address oligonucleotides is ordered, but irregular. In this way, the address oligonucleotides are placed on the surface of the master array without initial mapping or otherwise determining the exact location of each unique address oligonucleotide. In other words, the address oligonucleotides are placed on the surface of the master array and immobilized on the surface, after which their location on the master array is determined using known detection techniques. In this way, the present invention provides an efficient method of creating a master array that can be used in a versatile way to create an assay array of any desired composition. For example, once a master array has been assembled and the surface has been "mapped" to determine the location of each address oligonucleotide, the master array can be used in conjunction with any selection of multiligand conjugates. An assay array for a particular purpose can be prepared. This is evident through the following discussion.
[0044]
Direct attachment of the address oligonucleotide to the master array support surface can be achieved by derivatizing the nucleic acid, the support surface of the master array, or both the nucleic acid and the support surface. Modifications of the nucleic acid should preferably avoid substantially altering the function of the nucleic acid attributable to Watson-Crick base pairing. Preferably, the modification of the nucleic acid does not substantially impair the ability of the nucleic acid sequence to hybridize to its complement.
[0045]
Suitable methods for immobilizing address oligonucleotides include chemical modification of the oligonucleotide (eg, amine modification) and non-covalent attachment with a high affinity binder (eg, avidin-biotin). Next, the surface of the master array support can be derivatized (eg, with a carboxyl or epoxy group) to attach to the oligonucleotide itself or the corresponding reactive group provided by a suitable crosslinking reagent. Alternatively, a binding partner (eg, streptavidin) can be bound directly to the master array support surface to allow for interaction between the address oligonucleotide and the master array support surface. Other methods include adsorption of unmodified or modified oligonucleotides, and inkjet or "needle" printing onto a support surface using thermochemical immobilization of soluble probes on an activated master array support surface. Immobilization of the oligonucleotide. While those skilled in the art will readily recognize that other techniques can also be used, exemplary embodiments for direct attachment of address oligonucleotides to a master array support surface will now be described. .
[0046]
Modification of the nucleic acid can be accomplished in any suitable manner that allows for binding to the master array support surface. In one embodiment, the nucleic acid is derivatized to contain at least one reactive moiety that can react with the surface of the master array. Alternatively, nucleic acids are synthesized with modified bases. Other techniques known in the art include modification of the sugar moiety of the nucleotide or nucleobase, such as by using N7- or N9-deazapurine nucleosides. Alternatively, the nucleic acid backbone can be modified such that a reactive group can be attached to the nitrogen center provided by the modified phosphate backbone (eg, phosphoramidite DNA). Given the teachings herein, one of ordinary skill in the art can readily employ these and other standard nucleic acid modification techniques to bind nucleic acid sequences to the support surface of a master array.
[0047]
One embodiment in which the photoreactive nucleic acid binds to a surface is described, for example, in US patent application Ser. No. 09 / 028,806 (filed Feb. 24, 1998, Guire et al.). This application is commonly owned by the designated agent of the present application, and the entire disclosure of this application is incorporated herein by reference. As described in that application, a photoactivatable nucleic acid derivative can be provided in the form of a nucleic acid having one or more photoreactive groups attached thereto. The photoreactive group is preferably covalently attached, directly or indirectly, at one or more points along the nucleic acid. Photoreactive groups are derivatives that can be selectively and specifically activated to bind nucleic acids to a support and in a manner that substantially retains its intended chemical or biological function. The present invention provides an activated nucleic acid. According to this embodiment, "direct" attachment of the photoreactive group means that the photoreactive compound is directly attached to the nucleic acid. On the other hand, an "indirect" linkage refers to the binding of photoreactive compounds and nucleic acids to common structures, such as synthetic or natural polymers. The resulting photo-derivatized nucleic acids can be covalently immobilized on a variety of substrate surfaces by the application of suitable irradiation, and usually without the need for surface pretreatment. The method of this embodiment involves both thermochemical attachment of one or more photoreactive groups to the nucleic acid and photochemical immobilization of the nucleic acid derivative on the substrate surface.
[0048]
Preferably, the oligonucleotide is immobilized on the master array surface using a photoactivatable compound to form a bond between the address oligonucleotide and the master array support surface. These photoactivatable compounds can be provided in any suitable manner, for example by means of a master array support surface or address oligonucleotides themselves.
[0049]
In one embodiment, the reagent composition is for binding a biomolecule, such as a nucleic acid, as described in US patent application Ser. No. 09 / 227,913 (filed Jan. 8, 1999, Chappa et al.). Used to modify the surface. This application is commonly owned by the designated agent of the present invention, the entire disclosure of which is incorporated herein by reference. As described in that application, the reagent composition is a biomolecule (eg, a nucleic acid) that can then be used for a specific binding reaction (eg, hybridizing a nucleic acid to its complementary strand). Used to covalently bind a target molecule such as The reagent composition comprises one or more thermochemically reactive groups, ie, groups that have a temperature dependent reaction rate. Suitable groups are selected from the group consisting of activated esters (eg, N-oxysuccinimide or "NOS"), epoxides, azlactones, activated hydroxyl and maleimide groups. Optionally and preferably, the composition further comprises one or more photoreactive groups. In addition, the reagents can include one or more hydrophilic polymers to which thermochemically and / or photoreactive groups can hang. Photoreactive groups can be used to attach reagent molecules to the surface of the support, for example, when applying a suitable energy source such as light. The thermochemically reactive group can then be used to form a covalent bond with a suitable complementary functional group on the target molecule.
[0050]
Generally, according to this embodiment, the reagent molecules are first attached to the surface by activation of the photogroups. The oligonucleotide is then contacted with a binding reagent under conditions suitable to allow it to approach during the binding distance with the binding polymer. The nucleic acid is thermochemically bound to the binding reagent by a reaction between a reactive group of the binding reagent and a suitable functional group on the nucleic acid molecule. The thermochemically reactive group can be present either on the same polymer or on various polymers that are co-immobilized, for example, on a surface. Optionally and preferably, the target molecule is prepared or provided with a functional group adapted to the group of the reagent molecule. During their synthesis, for example, oligonucleotides can be prepared with functional groups such as amine or mercapto groups.
[0051]
In an alternative embodiment, the surface of the master array is modified with an epoxide-based reagent to allow oligonucleotide binding. One such embodiment is described, for example, in U.S. Patent Application Serial No. 09 / 521,545 (filed March 9, 2000, Swan et al.). This patent application is commonly owned by the designated agent of the present application, and the entire disclosure of this application is incorporated herein by reference. As described in this application, epoxide-based reagent compositions are used for covalent attachment of target molecules onto the surface of a substrate. The methods and reagents can be used to covalently immobilize either derivatized or underivatized nucleic acids. As contemplated in this embodiment, examples of derivatized nucleic acids include amine-derivatized nucleic acids, and non-derivatized nucleic acids have groups added for the purpose of thermochemical reaction with epoxide groups. Some do not. The method involves coating the support with reagents, printing the nucleic acid array, culturing the slides in a humid environment, blocking excess epoxide groups, washing the support, and then allowing the hybridization assay to proceed. Ready for use. In particular, the method provides a solid support having a surface; providing a reagent comprising one or more epoxide groups, and optionally also comprising one or more photoreactive groups; coating the reagent on the support surface (E.g., covalently attaching a polymeric reagent to a support surface by activation of a photoreactive group); providing a biopolymer having a corresponding thermochemically reactive group; and attaching the corresponding reactive group. Binding the biopolymer to the support by reacting with epoxide groups. Optionally, the method further comprises blocking residual epoxide groups (eg, using an amine reagent).
[0052]
According to this embodiment, the reagent preferably provides one or more epoxide groups (also known as "oxirane" groups) depending on a polymeric backbone, such as a hydrophilic polyacrylamide backbone. Optionally, the reagent composition further comprises a pendant photoreactive group. Photoreactive groups can be used to attach reagent molecules to the support surface upon exposure to a suitable energy source such as light. The epoxide group can then be used to form a covalent bond with a suitable functional group on the target molecule.
[0053]
In another embodiment, the support surface of the master array is prepared by uniformly distributing the photoreactive bi- or multi-functional reagents on the surface, and then attaching these reagents to the surface. In one embodiment, a silicon chip having sufficient smoothness is cleaned and reacted with a photoreactive organosiloxane reagent. This reagent can be prepared, for example, by combining a commercially available organosiloxane reagent (such as aminopropyldimethylmethoxy siloxane) with a thermochemically reactive photoreagent such as benzoyl benzoic acid acid chloride. The resulting photoreactive siloxane reagent then reacts with the silicon oxide surface of the master array support to provide a smooth surface primed with photoactivatable groups. The photoreactive groups on the resulting primed surface are activated and can be used to coat the oligonucleotide directly or indirectly on the master array support surface with a linker.
[0054]
This embodiment allows a solution containing a plurality of address oligonucleotides to be applied to a master array support surface. The master array is illuminated with long wavelength ultraviolet or visible light to photochemically fix the address oligonucleotide on the master array support surface. In an alternative embodiment, the address oligonucleotides themselves are photoreactive. Suitable photoreactive groups are discussed in further detail below. Optionally, if patterned deposition is required, a mask or suitable beads could be used. Patterned deposition can be achieved in any suitable manner. In one embodiment, the oligonucleotides are deposited on the substrate surface in the patterned array (eg, using a printing technique), and the substrate can then be uniformly illuminated with light of a suitable wavelength. In another embodiment, the oligonucleotides can be uniformly deposited on the substrate surface, followed by patterned irradiation (eg, using a masking technique).
[0055]
In an alternative embodiment, a molten or concentrated solution of a reagent having both latently reactive (eg, photoreactive) and ligand withdrawing (eg, charged, cationic) groups can be used. For example, polystyrene derivatives containing one or more groups of both types (eg, thiocholine derivatives of polyvinyl benzoic acid) can be deposited on a master array surface to form a reactive surface film for immobilizing address oligonucleotides. Can be provided. The surface of the master array can be illuminated to produce a smooth photoreactive surface. Optionally, ligand-withdrawing groups (eg, cation / thiocholine) are removed if necessary.
[0056]
Alternatively, the address oligonucleotide is indirectly attached to the master array support surface through the use of a linker or other suitable spacer molecule. "Linker" or "spacer molecule" when used in connection with the immobilization of an address oligonucleotide refers to a moiety that is attached to a solid support and is attached to an address oligonucleotide. The linker serves to attach the address oligonucleotide to the master array support surface in a spaced-apart manner to allow the address oligonucleotide to contact the multiligand conjugate of the invention.
[0057]
In one embodiment, the linker is provided in the form of microbeads and is referred to as "linked microbeads". Beads suitable for use as linked microbeads include any three-dimensional structure that can be immobilized on a solid support as described herein. Preferably, the linked microbeads are selected to have sufficient specific gravity to allow them to settle on the surface of the master array by gravity while avoiding the effects of Brownian motion. In some cases, the microbeads are porous. Microbeads can be provided in any suitable size, and nanobeads and microbeads are contemplated in the present invention. Linked microbeads suitable for use in the present invention are desirably spherical and uniform in size. Also, preferred beads have a chemical composition, or at least a surface, that readily binds organic reagents. For example, a uniform silica sphere between 0.5 microns and about 5 microns (μ) diameter contains a thermochemically reactive group (eg, an epoxy group) to attach the reagent to the corresponding group of the binding ligand. Can react with organosiloxane reagents. Microbeads can be installed by techniques such as, for example, electrostatic attraction, electrodes or magnetic probes, or engineering tweezers.
[0058]
As contemplated in the present invention, the beads can be made from virtually any insoluble or solid material. For example, beads can be silica gel, glass, nylon, resin, Sephadex, Sepharose, cellulose, magnetic beads, Dynabeads, metal surfaces (eg, steel, gold, silver, aluminum, silicon) Or copper), and plastic materials (eg, polyethylene, polypropylene, polyamide, polyester, vinylidene fluoride resin (PVDF)), and the like, and combinations thereof. Examples of suitable microbeads are described, for example, in US Pat. No. 5,900,481 (May 4, 1999, Lough et al.). Other examples of suitable microbeads are, for example, Interfacial Dynamics that provide latex microspheres that can be fluorescent, stained, and / or provide the desired surface functionality for a particular application. Commercially available from Dynamics Corporation (Portland, Oregon).
[0059]
Preferably, the linked microbeads containing the address oligonucleotide are immobilized in spaced relation on the master array support surface. In such embodiments, woven screens with spacings that are only slightly larger than the bead diameter are used to promote the deposition of microbeads on the surface of discrete, but ordered, structural shapes, and to reduce the surface of the master array. Or can be located on its surface.
[0060]
Linked microbeads are commercially available and can carry oligonucleotides using commercially available manufacturing methods and materials. Binding of the address oligonucleotide to the linked microbeads is preferably achieved by formation of a covalent bond between the oligonucleotide and the beads. Methods for attaching address oligonucleotides include those described herein for attaching address oligonucleotides directly to a master array support surface. In one preferred embodiment, the surface of the ligated microbeads reacts with an organosiloxane reagent containing a thermochemically reactive group (eg, an epoxy group). Glycidoxypropyltriethoxysiloxane can, for example, react with microbeads to provide an epoxy surface. Next, the 5 'end of the address oligonucleotide is modified to create an end containing an alkylamine group on the spacer. The epoxy siloxane on the microbead surface reacts with the alkylamine located at the end of the address oligonucleotide to form a bond between the address oligonucleotide and the microbead surface. Each ligated microbead can contain one unique address oligonucleotide. Alternatively, each linked microbead can contain multiple identical address oligonucleotide sequences.
[0061]
The array of microbeads is optionally deposited from an aqueous suspension containing a block copolymer having both photoreactive groups and ligand-withdrawing groups (eg, ionic groups such as quaternary amines). Can be. Monodisperse microbeads (e.g., between about 1.5 microns and 5 microns in diameter) previously carrying the address oligonucleotide are applied to a master array support surface from a suspension of the copolymer in an aqueous solution. You. Cationic reactive groups serve to bind oligonucleotide sequences on the beads, while aromatic blocks (eg, oligostyrene) bind to and bind to the photoreactive surface. In a particularly preferred embodiment, the linked microbeads are provided in a slight excess.
[0062]
The smooth, flat photoreactive master array support surface has address oligos immobilized thereon in a sufficient number to provide at least a monolayer of tethered microbeads on the master array support surface. It is exposed to a slurry of linked microbeads having a nucleotide sequence. The slurry consists of a number of beads approximately equal to each of the intended address oligonucleotide sequences plus a number (eg, at least three times) of beads containing no oligonucleotide. The number of oligonucleotide-coated beads is sufficient to provide a small (eg, at least 3-fold) redundancy of each target nucleic acid-specific site.
[0063]
The master array is cultured in a solution containing linked microbeads in the dark or in dim light to avoid premature activation of the optical groups. As the solvent (preferably aqueous) evaporates from the slurry pool on the photoreactive surface, the uniformly sized beads are optionally spaced or positioned using a microscreen as described herein. Packed into a two-dimensional "crystal" arrangement.
[0064]
This ordered arrangement can then be illuminated, for example, in the dry state, with carbon-carbon bonds between photoreactive groups on the surface of the master array support and organic groups (mostly oligonucleotides) on linked microbeads. Can be "fixed" in place. Patterned arrays of beads fixed on a photoreactive surface by specific gravity can be photochemically fixed to the surface by irradiation with long-wave ultraviolet or visible light. This irradiation activates the photoreactive organosiloxane reagent which then forms a bond with the address oligonucleotide. Optionally, the unbound block copolymer can be rinsed from the master array once the arrayed linked microbeads have been photobound to the master array support surface. This rinse removes subsequent interference (eg, during hybridization) with oligonucleotide sequences located around the beads but not immobilized on them.
[0065]
Although the use of any form of linker is not required in the present invention, the use of microbeads in the present invention will increase the density of address oligonucleotides immobilized on the surface of the master array, The ordered spacing of the address oligonucleotides to form the individual locations of the address, and the increasing density of address oligonucleotide deposits at each individual location on the master array surface (address oligonucleotides greater than one are less than (If provided in connection with a linker).
[0066]
A typical master array of the present invention includes a number of address ligands, each at a separate location or address, immobilized on its surface. The term “address” as used in this context refers to a spatially distinct surface on a support that is sufficiently identifiable to allow the ligand (eg, oligonucleotide) bound thereto to be identified and distinguished. Refers to the determined position. The result of this immobilized crystalline array is to provide a master array containing a number of unique "addresses" on its surface.
[0067]
A master array support surface of this type is preferably provided in the form of a planar, non-porous solid support having at least a first surface. The plurality of heterologous address oligonucleotides is 100 heterologous oligonucleotides / cm ² And preferably 1000 heterologous oligonucleotides / cm ² And each heterologous oligonucleotide binds to the surface of the solid support in a different predetermined region, has a different defined sequence, and has at least 4 It is nucleotides in length, and preferably at least 10 nucleotides, and more preferably 30 nucleotides in length.
[0068]
In a preferred embodiment, the master array is at least 1,000, more preferably at least 10, each located in a predetermined area physically separated from the other areas and bonded to the first surface of the solid support. Contains 2,000 heterologous address oligonucleotides. Given the selection for multiple sites of each oligonucleotide, the total number of oligonucleotide sites in a single array can often exceed 10,000.
[0069]
In a preferred embodiment of the present invention, the master array support surface contains excess address oligonucleotides immobilized thereon such that a particular address appears at multiple spatially defined locations on the master array. I do. As a result of this redundancy, a particular multiligand conjugate containing a sequence complementary to a particular address oligonucleotide can bind to any of a number of locations on the master array. This redundancy of any particular address on the master array helps to increase the sensitivity and accuracy of the assay for detecting the corresponding target nucleic acid in the sample. For example, in one embodiment, the master array contains an address sequence that is immobilized on the support surface at between 1 and 100, preferably between 2 and 10, different locations. In such a preferred embodiment, detection of target nucleic acids that bind to extra addresses is performed at a number of different locations on the master array.
[0070]
Once the address oligonucleotides have been immobilized on the surface of the master array, then the spatially defined position of each immobilized address oligonucleotide can be determined. Suitable methods for determining the location of each address oligonucleotide include sequential addition of fluorescently labeled complementary sequences to the array, followed by fluorescent hybridization including rinsing and scanning between each addition. Fluorescently labeled oligonucleotides complementary to each address oligonucleotide sequence are available, or generally comprise several (eg, 10 or more) different fluorescent compounds known to be distinguishable from each other. It can be synthesized to contain one of them. Multiple (eg, 10) different oligonucleotides can be prepared, each having a different known sequence complementary to the address sequence on the surface, and each having a corresponding identifiable fluorophore. The labeled probe can be simultaneously applied to the surface and hybridized, for example, at 55 ° C. for 30 minutes. Next, the surface is rinsed and scanned to identify the locations of each of the ten arrays. Next, another set of 10 sequences is hybridized to the surface and scanned to identify a second set of 10 sequences. This process continues until all addresses have been identified, after which the master array is stripped from the hybridized oligonucleotides, for example by immersion in boiling water for 2 minutes, to prepare a replication assay array. Used for One skilled in the art will readily recognize that other detection methods can also be used.
[0071]
Preferably, the address oligonucleotides of the present invention comprise one or more pendent, potentially reactive (preferably photoreactive) groups covalently attached thereto, directly or indirectly. Photoreactive groups are defined herein and preferred groups are sufficiently stable that they are stored under conditions that retain such properties. See, for example, US Pat. No. 5,002,582, the disclosure of which is incorporated herein by reference. Potentially reactive groups can be selected that are responsive to various portions of the electromagnetic spectrum, and are particularly responsive to the ultraviolet and visible portions of the spectrum (referred to herein as "photoreactive").
[0072]
Photoreactive groups respond to a specific applied external stimulus to effect active species generation, for example, by covalent bonds obtained to adjacent chemical structures, such as provided by the same or different molecules. Photoreactive groups are atomic groups in molecules that retain their covalent bonds unchanged under storage conditions, but they form covalent bonds with other molecules when activated by an external energy source .
[0073]
Photoreactive groups generate free radicals and active species such as certain nitrenes, carbene, and ketones in excited states upon absorbing electromagnetic energy. Photoreactive groups can be selected to be reactive to various portions of the electromagnetic spectrum, and for example, photoreactive groups that are reactive to the ultraviolet and visible portions of the spectrum are preferred and are described herein. At times can be referred to as a "photochemical group" or an "optical group".
[0074]
Acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles (ie, heterocyclic analogs of anthrones such as those having N, O, or S at the 10-position), or substitutions thereof (eg, ring substitution). Photoreactive aryl ketones such as derivatives are preferred. Examples of preferred aryl ketones include the heterocyclic derivatives of anthrone, including acridone, xanthone, and thioxanthone, and their ring-substituted derivatives. Particularly preferred are thioxanthones, having an excitation wavelength greater than about 360 nm, and derivatives thereof.
[0075]
Such ketone functional groups are preferred because they can easily perform the activation / deactivation / reactivation cycle described herein. Benzophenone is a particularly preferred photoreactive moiety because it allows photochemical excitation by the initial formation of an excited singlet state, which makes the system crossover to the triplet state. The excited triplet state can snap into a carbon-hydrogen bond by abstraction of a hydrogen atom (e.g., from the support surface), thus creating a radical pair. The continuous breakdown of radical pairs leads to the formation of new carbon-carbon bonds. If a reactive bond (e.g., carbon-hydrogen) is not available for bond formation, UV-induced excitation of the benzophenone group is reversible and the molecule returns to the ground state energy level upon removal of the energy source. Photoactivatable aryl ketones such as benzophenone and acetophenone are of particular interest because these groups are susceptible to multiple reactivation in water, thus providing increased coating efficiency.
[0076]
Azides constitute a preferred class of photoreactive groups and include aryl azides such as phenyl azide and especially 4-fluoro-3-nitrophenyl azide (C ₆ R ₅ N ₃ ), Benzoyl azide and acyl-azide such as p-methylbenzoyl azide (-O-CO-N ₃ ), Ethyl azidoformate, phenyl azidoformate and the like (-O-CO-N ₃ ), Sulphonyl azide such as benzenesulphonyl azide (-SO ₂ -N ₃ ) And phosphoryl azides (RO) such as diphenyl phosphoryl azide and diethyl phosphoryl azide ₂ PON ₃ including. Diazo compounds constitute another class of photoreactive groups and include diazoalkanes such as diazomethane and diphenyldiazomethane (-CHN ₂ ), Diazoacetophenone and diazoketones such as 1-trifluoromethyl-1-diazo-2-pentanone (-CO-CHN ₂ ), Diazoacetates such as t-butyl diazoacetate and phenyl diazoacetate (—O—CO—CHN ₂ ) And beta-keto-alpha-diazoacetates such as t-butyl alpha-diazoacetoacetate (-CO-CN ₂ —CO—O—). Other photoreactive groups include diazirines such as 3-trifluoromethyl-3-phenyldiazirine (-CHN ₂ ), And ketene such as ketene and diphenylketene (-CH = C = O).
[0077]
Upon activation of the photoreactive group, the reagent molecules are covalently attached to each other and / or to the surface of the material by covalent bonds through the residues of the photoreactive group. Representative photoreactive groups and their residues upon activation are shown below.
[0078]
Photoreactive group
Aryl azide amine
Acyl azide amide
Azido formate Carbamate
Sulfonyl azide sulfonamide
Phosphoryl azide phosphoramide
Diazoalkane New CC bond
Diazoketones New CC Bonds and Ketones
Diazoacetate Novel CC bond and ester
Beta-keto-alpha-new CC bond and beta-
Diazoacetate ketoester
Aliphatic azo Novel CC bond
Diazirine, a new CC bond
Ketene New CC bond
Photoactivated ketones New CC bonds and alcohols
[0079]
The photoactivatable nucleic acids of the present invention can be applied to any surface having a carbon-hydrogen bond by which a photoreactive group can react and immobilize the nucleic acid on the surface. Examples of suitable substrates include polypropylene, polystyrene, poly (vinyl chloride), polycarbonate, poly (methyl methacrylate), parylene and any of a number of organosilanes used to pretreat glass or other inorganic surfaces. But not limited to them. The photoactivatable nucleic acids are printed on the surface in the array and can then be photoactivated by uniform irradiation to immobilize them in a specific pattern on the surface. They can also be applied uniformly to the surface continuously and then be photoactivated by irradiation through a series of masks to immobilize a particular arrangement in a particular area. Thus, with multiple irradiations through various masks and careful washing to remove unbound light-nucleic acid after each light-binding step, multiple applications of specific photo-derivatized nucleic acids prepare arrays of immobilized nucleic acids. Can be used to Photoactivatable nucleic acids can also be uniformly immobilized on a surface by coating and photoimmobilization.
[0080]
Masks are well known in the art and can be provided in the form of a transparent support material that is selectively coated with a layer of opaque material. Generally, a mask is a spatially allocated barrier material that is affixed to a given substrate to block photon irradiation from a portion of the substrate surface over which the barrier material is covered. Such a mask blocks the underlying portions of the substrate from contact with the activating light. Suitable masks are discussed, for example, in US Pat. No. 5,831,070 (November 3, 1998, Pease et al.). The opaque material portions can be removed, leaving the opaque material in the desired precise pattern on the substrate surface. The mask is imaged on, or in direct contact with, the master array support surface. "Openings" in the mask correspond to the portions on the substrate where it is desired to bind the desired portions, such as nucleic acids and microbeads. In some embodiments, the mask can be repositioned for multiple exposures. Alternatively, a multi-mask can be used.
[0081]
Multiligand conjugate
The invention further includes multiligand conjugates that contain multiple active (eg, binding or polymerizable) regions. The multi-ligand conjugate includes a first ligand binding region, a second ligand binding region, and a core having a third ligand bound thereto. The first and second ligand binding regions are preferably nucleic acids, while the third ligand is preferably a non-nucleic acid moiety. Preferably, the third ligand comprises a binding ligand or a polymerizable group. Alternatively, a multi-ligand conjugate comprises a second ligand binding region, and a core having a third ligand attached thereto. In this embodiment, the second ligand binding region is complementary to both the address ligand and the target ligand to be detected in the sample. Multiligand conjugates carry conjugates that can use the template set by the master array, creating one or more replication assay arrays as described herein.
[0082]
The core of the multi-ligand conjugate provides a first and second ligand binding region, as well as a surface for binding of a third ligand. The core comprises suspended particles or soluble molecules, as described herein. Suitable suspended particles include microbeads. Alternatively, the core comprises a molecular moiety that is preferably soluble. As used herein, when the core of the multiligand conjugate is provided in the form of microbeads, the microbeads are referred to as "transfer microbeads".
[0083]
In one embodiment, the core is provided in the form of transfer microbeads. Microbeads useful in preparing multiligand conjugates of the invention can take the same diversity as described above for "ligated microbeads." Preferred beads desirably have a greater specific gravity than that of water and have a spherical, uniform size. Also, preferred beads have a surface that readily binds the chemical composition, or at least the organic reagent. For example, a uniform silica sphere between about 0.5 microns to about 20 microns, preferably between about 1 micron to about 10 microns, and more preferably between about 1 micron to about 5 microns (μ) in diameter, will bind the reagent to It can be reacted with an organosiloxane reagent containing a thermochemically reactive group (eg, an epoxy group) to attach to the corresponding group on the ligand.
[0084]
Transfer microbeads suitable for use in the present invention can be bound to a first ligand binding region, a second ligand binding region, and a third ligand, as described herein. Any three-dimensional structure that is Preferably, the transfer microbeads have a specific gravity greater than water to ensure that the beads are close to the surface of the master array for hybridization between the address oligonucleotide and the first binding region of the multiligand conjugate. Make it possible. Transfer microbeads can be provided in any suitable size, and nanobeads as well as microbeads are contemplated in the present invention.
[0085]
As contemplated in the present invention, the beads can be made from virtually any insoluble or solid material. For example, beads can be silica gel, glass, nylon, Wang resin, Merrifield resin, Sephadex, Sepharose, cellulose, magnetic beads, Dynabeads, metal surfaces (eg, steel, gold, silver, aluminum , Silicon and copper), plastic materials (eg, polyethylene, polypropylene, polyamide, polyester, and vinylidene difluoride (PVDF)), and the like, and combinations thereof. Examples of suitable microbeads are described, for example, in US Pat. No. 5,900,481 (May 4, 1999, Lough et al.). Other examples of suitable microbeads are commercially available, for example, from Interface Dynamics (Portland, Oreg.).
[0086]
In another embodiment, the core is provided in the form of a molecular core. Preferably, the molecular core is soluble. Suitable molecular cores include multifunctional polymers or multifunctional monomer molecules. Examples of multifunctional polymers include linear or branched polymers or dendrimers. Examples of suitable multifunctional monomer molecules include cyanuric chloride, homocysteine, cysteine, lysine, aspartic acid, glutamic acid, and serine.
[0087]
In one embodiment, a multi-ligand conjugate of the invention further comprises a first ligand binding region. In a preferred embodiment, the first ligand binding region comprises a nucleic acid, preferably an oligonucleotide. Oligonucleotides are selected for the first binding region by preparing a sequence complementary to the irregular sequence containing the address ligand of the master array. Once the sequence of the address ligand is known, the sequence of the first ligand binding region may first determine the complementary nucleic acid sequence to the address oligonucleotide using standard Watson-Crick base pairing rules, and then It is prepared by synthesizing a defined nucleic acid sequence. This preparation can be accomplished using any suitable method known in the art, for example, by solid phase DNA synthesis. Once the oligonucleotides for the first binding region have been synthesized, the ends of each oligonucleotide can be modified, if necessary, to contain an alkylamine group on the spacer.
[0088]
The length of the oligonucleotide is optimized for the desired hybridization strength and kinetics. Usually, the length of the first ligand binding region is in the range of 20-50 nucleotides. Preferably, the length of the first ligand binding region is selected based on the length of the address ligand on the master array. In a preferred embodiment, the sequences of the first binding region are either relative to each other or to any known natural gene sequence and / or gene fragment that has a significant probability of being present in the sample to be tested. Are not complementary. As a result, the first binding region oligonucleotide sequences will only hybridize to their respective complementary address oligonucleotide sequences immobilized on the master array support surface. This results in the first ligand binding region responsible for placing the multiligand conjugate in a spatially defined location on the surface of the assay array support, and the intersection between the target nucleic acid sequence selected in the test sample. Avoid reactivity (eg, cross-hybridization).
[0089]
In a preferred embodiment, the second ligand binding region of the multi-ligand conjugate also comprises a nucleic acid, preferably an oligonucleotide. Oligonucleotides are selected for the second binding region by first selecting the target ligand (eg, gene and / or gene fragment) to be detected by the assay array constructed according to the invention. The sequences of these genes and / or gene fragments to be detected can be known or can be determined using techniques well known in the art (eg, the Sanger dideoxy method, PCR sequencing, etc.). It is possible. Once the sequence of the target nucleic acid is known, the sequence of the second ligand binding region can be determined by first determining the complementary nucleic acid sequence to the target nucleic acid using standard Watson-Crick base pairing rules, and then determining the determined nucleic acid sequence. Prepared by synthesizing the sequence. This preparation can be accomplished using any suitable method known in the art, for example, by solid phase DNA synthesis. Once the second binding region has been selected, the termini of each oligonucleotide can be modified, if necessary, to contain an alkylamine group on the spacer.
[0090]
As with the first ligand binding region, the length of the oligonucleotide for the second ligand binding region preferably ranges from 10 to 100 nucleotides, more preferably from 20 to 100, for the desired strength and kinetics of hybridization. Optimized within the 50 nucleotide range. Preferably, the sequence of the second binding region is selected from the sample while avoiding cross-reactivity, such as cross-hybridization with either the address ligand on the master array or the first ligand binding region of the multi-ligand conjugate. Selected to provide sufficient specificity for a particular target ligand therein.
[0091]
In an alternative embodiment, the second ligand binding region is selected to be complementary to the address ligand of the master array and the target ligand to be detected in the sample. In this embodiment, the sequence of the second ligand binding region is determined by selecting the target ligand (eg, gene and / or gene fragment) to be detected by the assay array constructed according to the invention. The sequence of the second ligand binding region is prepared by determining a complementary nucleic acid sequence to the target nucleic acid using Watson-Crick base pairing rules, as described above, and then synthesizing the determined nucleic acid sequence. Is done. Next, the address ligand in this embodiment is assembled to include a sequence that is substantially complementary to the target ligand. With this embodiment, there is no need to provide a multi-ligand conjugate with the first ligand binding region since the second ligand binding region hybridizes with the address ligand of the master array.
[0092]
A multiligand conjugate is complementary to, and therefore hybridizes to, any desired target nucleic acid found in a biological sample and can be assembled to include oligonucleotides having a known or determinable sequence. In one embodiment, the various second ligand binding regions of the multiligand conjugates of the invention can include various oligonucleotides that are complementary to various target nucleic acids found in a biological sample.
[0093]
In an alternative embodiment, the second binding region of the multiligand conjugate is assembled such that the resulting assay array maps a particular series of target nucleic acids of the naturally occurring gene. One example of this particular embodiment would be a set of multiligand conjugates containing a second binding region complementary to 50-60 known target sequences of cystic fibrosis. Thus, the resulting assay array will be assembled to detect the presence of sequences known or suspected to bind only to this obstacle.
[0094]
The third ligand of the multiligand conjugate is a molecule that transports the multiligand conjugate to an assay array prepared according to the present invention. In one embodiment, once the multi-ligand conjugate has hybridized to the master array at the desired address location, the third ligand retains the positional arrangement of the nucleic acids set by the master array. Allows the multiligand conjugate to be transferred to the assay array. Preferably, the third ligand is provided as a binding ligand or a polymerizable group.
[0095]
In one embodiment, the third ligand is a binding ligand. As used herein, "binding ligand" refers to any ligand that can bind to a binding partner. Preferably, the binding ligand is a member of a specific binding pair capable of binding a complementary binding moiety. As contemplated in the present invention, a binding ligand is a biological or synthetic molecule that has a high affinity for another molecule or macromolecule through covalent or non-covalent bonds. Preferably, the binding ligand is a functional chemical group (primary amine, sulfhydryl group, primary amine, sulfhydryl group, etc.) which allows it to react covalently or non-covalently to common functional groups. Or aldehyde groups), epitopes (antibodies), haptens or ligands (either naturally or by modification). For example, where the third ligand comprises avidin or streptavidin, the multiligand conjugate can bind to a surface that is coated with a biotin derivative such as biotin or imino-biotin. It is understood that the binding members can be reversed, for example, biotin-coated beads can be bound to a streptavidin-coated solid support. Other specific binding pairs contemplated for use in the present invention include haptens (digoxigen, 2,4-dichlorophenoxyacetic acid, trinitrophenyl) and high affinity antibodies, hormone-receptors, enzymes-inhibitors Agents and antibodies-antigens.
[0096]
In one embodiment, the third ligand is provided in the form of a biotin-poly (alkylene oxide) amine derivative. Such derivatives are prepared, for example, by reacting the N-oxysuccinimide ester of biotin with a poly (ethylene oxide) diamine of approximately the same molecular length as the oligonucleotide derivative to be placed on the same core or microbead. can do.
[0097]
The third ligand can be directly or indirectly through the use of a spacer in a manner that allows the ligand to be free of steric hindrance (eg, steric hindrance caused by proximity of the ligand to the transfer microbeads) It can be attached to the core of the multiligand conjugate or to transfer microbeads. In other words, the use of a spacer to bind the third ligand is such that the ligand is at a sufficient distance from the surface of the core or transfer microbeads and that it performs its intended function, e.g., at the surface of an assay array. Ensure that it can polymerize or bind to the corresponding binding partner contained above.
[0098]
As used herein, a "spacer" for binding a third ligand to the core allows for binding of the third ligand to the core in a spatial relationship that does not interfere with the function or reactivity of the third ligand. Is a molecule. Suitable spacers include polyethylene glycol and water-soluble bi- or multi-functional monomers such as acrylamide, vinylpyrrolidone, and carbohydrates. Preferably, such water-soluble bi- or multi-functional monomers include oligomers or polymers. In a preferred embodiment, the spacer comprises, for example, a hydrophilic spacer of sufficient length to allow the binding region to be sterically unhindered, such as a polyethylene glycol spacer.
[0099]
In one embodiment, a multi-ligand conjugate of the invention further comprises a polymerizable group. As used herein, a "polymerizable group" refers to a molecule that is capable of undergoing, under suitable conditions, a chemical reaction in which relatively simple molecules combine to form a macromolecule or chain-like molecule. The third ligand of the multi-ligand conjugate is provided in the form of a polymerizable group, for example, to allow the conjugate to be formed into a stable film while in place on the master array. can do.
[0100]
Examples of suitable polymerizable groups include free radical products such as vinyl, acrylic, maleic, itacone, unsaturated fatty acids, and other ethylenically and acetylenically unsaturated hydrocarbon groups. In a preferred embodiment, the polymerizable groups are adapted to perform addition polymerization in the presence of additional monomers and other reagents (eg, initiators) to form a stable film. The polymerization can be carried out via any suitable reaction, including aqueous free radical solution polymerization, exposure to heat, high energy irradiation, ultrasound, ultraviolet irradiation, and ionic polymerization catalysts to produce water soluble or swellable polymers. it can. Further, the polymerization of the groups can be carried out via base catalyzed hydrogen transfer polymerization. Given the present description, those skilled in the art will recognize that such groups can be used to copolymerize ligands with other monomers and then with their corresponding conjugates to form a complete polymeric structure, for example, an assay array support surface It will be appreciated that it can be used to take the form of a film backing that can be transported to or into it.
[0101]
In this embodiment, once the address oligonucleotides of the master array have each hybridized to their respective first binding region of the multi-ligand conjugate, the polymerizable groups are bound to the reagents and the skill of the artisan. The reaction can be performed using the conditions in the above to enable formation of a polymer film. The polymerizable groups are copolymerized on the film backing while substantially maintaining their respective positions determined by the master array. The film exists so that it is sufficiently stable (eg, self-supporting) to maintain the spatially defined position of each “address” or is optionally stabilized (eg, gelled or , Another layer is laminated or otherwise solidified). At the same time or thereafter, the polymeric backing can be transferred to or otherwise incorporated into the assay array surface in a manner that maintains the desired orientation of the conjugate.
[0102]
According to the present invention, the individual ligands of a multi-ligand conjugate may be combined in any suitable manner and / or order, for example, individually or together (eg, in a linear array and at a single position or multiple positions). It can be attached to an atom or a molecule. For example, in one embodiment, the ligand is simultaneously bound to the core, eg, under similar reaction conditions. Optionally, the ligand serving as the third ligand is bound to the core after hybridization between the address oligonucleotide of the master array and the complementary oligonucleotide provided by the multiligand conjugate. In this embodiment, the third ligand is present as a separate reagent, however, the binding of the third ligand can occur under the same or similar reaction conditions as the first and second binding regions. In yet another embodiment, each ligand is bound to the core under separate conditions in a separate reaction.
[0103]
The sequence of binding of these ligands to the core of the multiligand conjugate is not considered critical to the invention, and individual active (eg, binding) regions bind their specific complement. They can be bonded to the core or to each other in any order, as long as it is available for that. In a particularly preferred embodiment, each individual binding region is individually attached to the core at a plurality of locations on the surface.
[0104]
In one embodiment, an epoxy-glass surface is formed on the surface of the transfer microbeads that subsequently react with the first and second ligand binding regions, and the third ligand. In this embodiment, the glycidoxypropyltriethoxy siloxane is prepared according to published reaction conditions (see "Grafting Rates of Amine-Functionalized Polystyrenes on Epoxidized Silica Surfaces," Macromolecules, 33: 1105-33, 1105-1305). ) Reacts with the glass microbeads below to provide an epoxy-glass surface. The three binding regions of the invention, each terminated with an alkylamine group on the spacer, can readily bind to epoxy groups on the surface of the bead.
[0105]
The first and second oligonucleotide binding regions can be attached to the transfer microbeads in any suitable manner, for example, an alkylamine-terminated nucleic acid can be synthesized and attached thermochemically. As described above, the first binding region comprises an oligonucleotide that is complementary to the address oligonucleotide immobilized on the master array support surface, while the second binding region It is complementary to a selected target nucleic acid suspected or known to be present in the sample to be sought. As mentioned above, the sequences of the first ligand binding region are preferably relative to each other or to any known natural gene sequence and / or gene fragment that has a significant probability of being present in a biological sample. Not complementary.
[0106]
In one embodiment, the first and second ligand binding regions are provided in the form of an alkylamine nucleic acid derivative, and the third ligand is provided in the form of biotin hydrazide. The core is provided as microbeads provided with a photoreactive poly-N-oxysuccinimide (polyNOS) or epoxide surface. The alkylamine derivative and biotin hydrazide react with the surface of the core to allow binding of a binding ligand and a third ligand. Alternatively, 5'-OH oligonucleotides and allylamine oligonucleotide derivatives are used as first and second ligand binding regions, and the third ligand is biotin hydrazide (binding) with triazine trichloride as the soluble nucleus. (Ligands) or allylamine (polymerizable groups, for in-situ formation of patterned films).
[0107]
In an alternative embodiment, the third ligand is provided in solution rather than being bound to the core of the multi-ligand conjugate. In this embodiment, the third ligand is incorporated after hybridization of the address oligonucleotide of the master array with the first binding region of its complementary multiligand conjugate. Thus, in this embodiment, the third ligand is present in the complex formed on the master array after binding of the multiligand conjugate and before the assay array is brought close to the binding distance with the master array. Be incorporated. Preferably, the addition of the third ligand does not require different reaction conditions than that of the first and second binding regions; rather, the addition of the third ligand can be performed under the same conditions.
[0108]
In a preferred embodiment, the third ligand is attached to the multiligand conjugate using photoreaction chemistry. In this embodiment, the surface of the microbeads is pretreated with, for example, an organosiloxane to prepare a surface for reaction with a photoreactive compound. In this embodiment, the third ligand is attached to a hydrophilic spacer which is then attached to a photoreactive group. The photoreactive group allows for the formation of a bond between the spacer (attached to the binding ligand) and the core of the multiligand conjugate.
[0109]
The third ligand is preferably covalently attached to the core, whether or not the core contains suspended particles or soluble molecules. In one embodiment, the third ligand derivative comprises a reactive group selected to form a covalent bond with a target group found or located on the core of the multi-ligand conjugate. For example, the core can contain an epoxy or active ester group, and the third ligand can contain an amine or hydrazide group; these two classes of reactive groups are aqueous and slightly alkaline. React together to form a covalent bond when placed together. If the core comprises, for example, suspendable particles, the biotin-poly (alkylene oxide) amine derivative will react with a soluble copolymer containing photoreactive groups and N-oxysuccinimide (NOS) ester groups ( As would be the other two ligands or amine-containing derivatives of the ligand binding region). The multi-ligand conjugate will be photochemically linked to the core particle.
[0110]
In an alternative embodiment, where the present invention involves the use of porous linked microbeads immobilized on the surface of a master array and covalently linked to address oligonucleotides, as the core of a multiligand conjugate No transfer microbeads are required. In this embodiment, the porous linking microbeads are immobilized on the surface of a master array, and each porous linking microbead can have multiple address oligonucleotides attached to its surface. According to this embodiment, the multiligand conjugate comprises a first binding region complementary to the address oligonucleotide, a second binding region complementary to the target ligand found in the biological sample, and a second binding region comprising the binding ligand. Includes triligands. Multiligand conjugates preferably do not include transfer microbeads, but rather a chemical (eg, molecular) core.
[0111]
Multiple multiligand conjugates are applied to the master array and cultured under conditions suitable to allow hybridization of the master array address sequence to its complementary oligonucleotide contained within the multiligand conjugate. Is done. One skilled in the art can determine suitable conditions for temperature, salt concentration, and buffer composition for hybridization of the address oligonucleotide to the first binding region. For a discussion of hybridization conditions and methods for applying them, see pages 3-15, 62-66, 73-112, Nucleic Acid Hybridization: A Practical Approach, Hames and Higgins, eds. (1985). The disclosure is incorporated herein by reference.
[0112]
Assay array support
The assay array support of the present invention comprises a support surface that is preferably coated with a binding site for a third ligand of a multi-ligand conjugate. The assay array support provides a support for the replicated array to be assembled using the master array.
[0113]
The assay array support is made from any suitable material to provide an optimal combination of properties such as dimensional stability, shape, and smoothness. Suitable materials for use in assembling the assay array include those described as being useful for the master array itself, and preferably include silicon and glass. In a particularly preferred embodiment, the assay support is provided in planar dimensions between about 0.5 cm to about 7.5 cm long, and between about 0.5 cm to about 7.5 cm, preferably between about 1 cm to about 2 cm wide. . Preferably, the assay array support is sized to accommodate the master array to provide a direct spatial relationship.
[0114]
In yet another embodiment, multiple arrays can be replicated on a single assay array support. In this embodiment, one or more master arrays are used to create individual replicate arrays at defined surfaces on the assay array support. The resulting assay array contains multiple probe arrays on a single support surface, and the composition of each array can be tailored for a particular application as desired.
[0115]
As used herein, "binding site" refers to the portion on the surface of an assay array that can bind a third ligand of a multi-ligand conjugate. Preferably, the binding site comprises a member of a specific binding pair capable of binding in a covalent or non-covalent manner a complementary binding moiety, eg, a third ligand of a multi-ligand conjugate. Including. Suitable specific binding pairs are described herein with respect to the third ligand of the multi-ligand conjugate. One of skill in the art can select a suitable binding site based on the composition of the multiligand conjugate and the intended use of the assay array.
[0116]
The surfaces of the individual assay array supports can be pre-treated to facilitate binding of the oriented conjugate layer. In a preferred embodiment, the assay array is provided in the form of a silicon chip that is cleaned, polished, and treated to provide a smooth silicon oxide surface. Next, the silicon oxide surface can be treated with an organosiloxane reagent (as described herein) to provide a smooth photoactivatable surface.
[0117]
In one embodiment, an assay array support of the invention can be coated with a binding partner molecule available to form a bond with a third ligand of a multiligand conjugate. Assay array support surfaces can be prepared by coating the surface with a photoactivatable compound (eg, a photoreactive siloxane reagent) to render the surface photoreactive. Thereafter, the assay array support surface can be exposed to a solution containing a binding partner molecule (eg, avidin), which solution is used to allow the formation of a bond between the binding partner and the surface of the assay array. Irradiated. Optionally, the surface of the assay array is found in the test sample before and / or after exposure to the binding partner, for example, protected with a coating (eg, a biotin triblock copolymer) using a surfactant. Prevents nonspecific binding of molecules to the surface of the assay array support.
[0118]
In a preferred embodiment, the binding partner is provided in the form of a monolayer on the surface of the assay array support. Optionally, the binding partner can be provided in the form of a three-dimensional layer, eg, avidin-hydrogel composite up to a sufficient thickness, eg, up to about 20 microns (μ) thick. The thickness of the three-dimensional layer depends on factors such as the molecular weight of the polymer used, the swelling of the polymer (eg, hydrophilicity), and the amount of applied material (eg, concentration and volume). Suitable thicknesses are from about 0.1μ to about 20μ, preferably from about 0.5μ to about 10μ, more preferably from about 0.5μ to about 5μ. Such structures are, for example, wetted with a solution of avidin plus a photoreactive hydrogel (eg, a copolymer of benzoylbenzamidopropyl methacrylamide and vinylpyrrolidone or acrylamide) on a photoreactive assay array surface. It can be obtained by irradiation in between.
[0119]
In another preferred embodiment, the binding partner is provided by a thin film of a photopolymer applied to a dimensionally stable assay array support surface. High surface density (for example, 0.1 pmol / mm ² A) self-assembled monolayer of a protective surfactant containing a high affinity ligand group (e.g., biotin) is applied to the photoreactive surface. Next, the monolayer surfactant film is optically bonded to the surface and any unbound surfactant is rinsed away. The ligand-bearing surface protected by the membrane is saturated with a high affinity binding partner molecule (eg, x-avidin), and any excess binding partner is rinsed away. Suitable high affinity binding partner molecules include, for example, x-avidin membranes, or high affinity antibodies (eg, anti-digoxigenin).
[0120]
Optionally, the system further comprises dissociating, for example, complementary and hybridized oligonucleotides (eg, the address oligonucleotide of the master array and its complementary oligonucleotide carried by the multiligand conjugate). Other components are also included, including reagents or other mechanisms for use in. For example, suitable mechanisms for dissociating hybridized DNA include techniques or reagents that alter the temperature, pH, or salt concentration of the system. One skilled in the art would be able to dissociate the hybridized DNA and apply such a mechanism to achieve the invention herein.
[0121]
Optionally, the system further comprises a commercially available scanner (eg, a confocal fluorescence microscope commercially available from Molecular Dynamics) to detect hybridization of a nucleic acid target to a particular address. included.
[0122]
Method
In another aspect, the invention provides a method for reproducibly preparing a nucleic acid array, comprising:
a) providing a master array having a support surface;
b) immobilizing a plurality of address ligands on a master array support surface in the form of a patterned array;
c) determining the pattern of the immobilized address oligonucleotide;
d) the first ligand binding of at least one molecule, wherein each multi-ligand conjugate comprises: (i) a core; (ii) a ligand selected to bind to the address ligand of the master array in a complementary manner. Region (iii) a second ligand binding region of at least one molecule comprising a ligand selected to bind to a specific target ligand in a complementary manner, and (iv) a third ligand of at least one molecule Providing a plurality of multi-ligand conjugates, wherein the first ligand binding region, the second ligand binding region, and the third ligand are bound to a core;
e) contacting the multiligand conjugate with the master array support surface under conditions suitable to allow each address ligand to bind its complementary first ligand binding region of the multiligand conjugate Allowed to incubate;
f) providing an assay array support surface;
g) combining the bound multiligand conjugate with the assay support surface under conditions suitable to allow the conjugates to be transferred to the assay support in a pattern corresponding to their pattern on the master array. Contact; and
h) dissociating the first ligand binding region from the master array support in a manner that allows the conjugate to remain on the assay support surface in a desired pattern;
Including.
[0123]
In another aspect, the invention provides a method for preparing a nucleic acid array to be replicable, comprising:
a) providing a master array having a support surface;
b) immobilizing a plurality of address ligands on a master array support surface in the form of a patterned array;
c) determining the pattern of the immobilized address oligonucleotide;
d) at least one molecule of each multiligand conjugate comprising: (i) a core; (ii) a ligand selected to bind to a specific address ligand and a specific target ligand in a complementary manner. Providing a plurality of multi-ligand conjugates comprising a second ligand binding region and (iii) a third ligand of at least one molecule, wherein the second ligand binding region and the third ligand are bound to a core. And;
e) contacting the multiligand conjugate with the master array support surface under conditions suitable to allow each address ligand to bind its complementary second ligand binding region of the multiligand conjugate. Allowed to incubate;
f) providing an assay array support surface;
g) combining the bound multiligand conjugate with the assay support surface under conditions suitable to allow the conjugates to be transferred to the assay support in a pattern corresponding to their pattern on the master array. Contact; and
h) dissociating the second ligand binding region from the master array support in a manner that allows the conjugate to remain on the assay support surface in a desired pattern;
Including stages.
[0124]
Optionally, the method further comprises using the assay array support surface produced in step h) to prepare a second assay array. The second set of multiligand conjugates is used to generate a second assay array that is a duplicate of the original master array. A second set of multi-ligand conjugates can be assembled according to the teachings herein. In this embodiment, the nucleic acid sequence of the second assay array is substantially identical to the nucleic acid sequence of the original master array.
[0125]
In a preferred embodiment, the immobilization of the address ligand on the surface of the master array support is irregular, and the location of each address is determined after the address ligand is immobilized on the surface. Preferably, the address ligand, the first ligand binding region, and the second ligand binding region are nucleic acid sequences, and the third ligand comprises a binding ligand or a polymerizable group. In use, the cores of a multi-ligand conjugate carrying three or more ligands can be mixed in equal numbers and suspended in aqueous buffer. This suspension contains an excess of each transfer microbead carrying an oligonucleotide sequence that is complementary to the characteristic oligonucleotide sequence of the target nucleic acid expected to be detected in the sample (the microarray on the master array). (Beyond the hybridization capacity of the beads).
[0126]
The master array surface can be flooded with this suspension of the selected mixture of multiligand conjugates, and solid state hybridization can be allowed to proceed to near equilibrium. The excess multiligand conjugate is then recovered from the hybridized master array by rinsing and the bound conjugate is copolymerized to form a stable backing on the assay Either the triligand contains a polymerizable group) or can bind to the corresponding binding partner (if the third ligand contains a binding ligand). Next, the master array is available for iterative replication.
[0127]
The master array can be used repeatedly to then prepare a corresponding assay array that can be packaged, stored, and transported, for example, in wet or dry conditions. Once unpacked, the assay array is ready to use in routine hybridization assay protocols with a scanner. Further, the present invention provides a versatile master array-the master array may be used to create a substantially identical array or to modify the function of the array (e.g., when a user attempts to detect in a sample). To alter the oligonucleotide sequence that is complementary to the characteristic oligonucleotide sequence of the target nucleic acid, so that the target nucleic acid can be altered).
[0128]
Those skilled in the art, given the present description, will understand how commercially available microarrays can themselves be used as the master array of the present invention and replicated one or more times to form a corresponding assay array. Will do. In this case, the individual nucleic acids of the commercially available array, regardless of their original specificity, each may be considered simply as an address sequence and are described herein to provide an assay array of any desired specificity. Can be used in the manner described in. The user can determine the sequence of each address and assemble the first binding ligand of the multi-ligand conjugate using the teachings herein.
[0129]
In a still further aspect, particularly where the third ligand is provided in the form of a binding ligand, the present invention provides a sandwich array comprising:
a) a master array comprising a support surface having a plurality of address ligands immobilized thereon;
b) a molecule of the first ligand binding region wherein each multiligand conjugate comprises (i) a ligand selected such that each molecule binds in a complementary manner to a specific address ligand of the master array; ii) a molecule of the second ligand binding region comprising a ligand wherein each molecule is selected to bind to a characteristic target ligand in a complementary manner; and (iii) a third ligand of at least one molecule. A plurality of multiligand conjugates comprising a core attached thereto, wherein the first ligand binding region is bound to a corresponding address ligand immobilized on a master array; and
c) An assay array support comprising a support surface wherein the binding sites of the assay array are covered by binding sites for a third ligand which is bound to the corresponding binding ligand of the multiligand conjugate.
[0130]
Optionally, the sandwich array further comprises connecting microbeads immobilized on the surface of the master array, to which the connecting microbeads bind a plurality of address oligonucleotides.
[0131]
kit
In yet another alternative embodiment, the invention provides a corresponding kit comprising one or more components for reproducibly preparing a nucleic acid array, wherein the kit components are selected from the group consisting of:
a) a master array comprising a support surface having a plurality of "address" ligands immobilized thereon, wherein the ligands take the form of a patterned array;
b) a molecule of the first ligand binding region wherein each multiligand conjugate comprises (i) a ligand selected such that each molecule binds in a complementary manner to a specific address ligand of the master array; ii) binding a molecule of the second ligand binding region comprising a ligand selected such that each molecule binds to a characteristic target ligand in a complementary manner, and (iii) at least one molecule of a third ligand. A plurality of multi-ligand conjugates comprising a modified core;
c) An assay array support comprising a support surface for a replicated array covered by a binding site for a third ligand.
[0132]
Optionally, the kits of the present invention further comprise reagents for performing the assays described herein, such as suitable buffers to provide appropriate pH levels, and salt concentrates for hybridization. Including. The kit further includes an array reader for determining the location of the address oligonucleotide on the master array and the presence of the hybridizing target nucleic acid on the assay array.
[0133]
Reusable nucleic acid arrays
In yet another alternative embodiment, the present invention provides methods and systems for directly preparing replicable arrays in the form of reusable nucleic acid arrays. In this embodiment, the surface to which the nucleic acids are immobilized is provided by a fiber optic array and the master array support surface is provided by the distal ends of the fibers themselves. Optionally and preferably, micro-wells are etched into the distal end of the optical fiber.
[0134]
In such embodiments, conventional fiber optic bundles can be adapted for use as specific binding array biosensor systems for detecting a variety of target nucleic acids in a sample. Commercial sources provide optical imaging fiber bundles, including thousands (e.g., 3,000-100,000) of hexagonally-closed, individually coated optical fibers. Each fiber in the array transmits its own isolated optical signal from one end of the fiber to the other. An individual specific binding ligand can be attached to one end of each fiber and the other end is functionally connected to an optical signal analyzer (eg, a CCD camera).
[0135]
The assay end of the fiber bundle (containing the specific binding ligand) is exposed to a sample solution containing the unknown target and other necessary reagents, if any, of the optical assay system. The optical signal pattern is recorded and analyzed at the other end of the bundle using an image analysis system.
[0136]
Applicants have discovered that the complementary oligonucleotide binding region of the target sequence can be ligated to the light-emitting device in any suitable manner, for example, either directly or reversibly or irreversibly by a linking molecule or microbead. It has been found that it can bind to the surface of an assay array. Next, the ultimate position of the oligonucleotide binding region is determined (eg, by light means and assays), preferably controlled (eg, by use of reusable address regions), and optionally on a flat surface. Can be replicated (eg, by use of a reversible linker).
[0137]
In one preferred embodiment, the system comprises a plurality of multiligand conjugates, each multiligand conjugate comprising: a) a first oligonucleotide binding region adapted to bind to a corresponding address ligand; and b. A) a second oligonucleotide binding region molecule that is complementary to a characteristic oligonucleotide sequence (eg, a gene and / or gene fragment) of the target nucleic acid. The address ligand is immobilized on the distal end of the master array, either directly or through the use of linkers or microbeads. The multiligand conjugate then carries a known analyte-specific ligand on each addressed fiber. The set of analyte specific ligands on the fibers in the bundle can be arbitrarily changed.
[0138]
In another embodiment, attachment of the oligonucleotide binding region is achieved using microbeads. In this embodiment, the microbead is prepared such that it is bound to the oligonucleotide binding region. Next, the microbeads and the binding region are bound to the assay array support surface. Binding of the microbeads can be accomplished using any suitable binding pair, such as, for example, avidin-biotin. In an alternative embodiment, a photoreactive reagent can be used to bind the microbeads to the surface of an assay array support. Attachment of the address oligonucleotide to the microbeads, and attachment of the microbeads to the surface of the master array can be accomplished using any suitable method described herein.
[0139]
Optionally, the system further includes an image analyzer. In this embodiment, the fiber bundle is connected at one end to an optical sensor that collects signals from these multiple sensors (eg, fibers) and processes the information.
[0140]
In such a method, the present invention provides a reusable nucleic acid array that allows a user to modify the function of the nucleic acid array for each particular application. For example, linkers or microbeads can be coated with iminobiotin, and the master array support surface can be coated with biotin. Thus, the linker or microbead can reversibly bind to the surface of the assay array support. The linker or microbeads can be dissociated by changing the pH such that the lower pH will cause the linker or microbeads to dissociate from the assay array support surface.
[0141]
In one embodiment, the microwells can be etched into the distal end of each optical fiber using techniques known in the art. For example, a wet chemical etching procedure can be used to selectively etch individual fiber cores. This technique takes advantage of the difference in etch rates between the core and fiber cladding materials. By controlling the etch time, one skilled in the art will understand how to obtain densely ordered, well-shaped microwell arrays and well volumes. The well construction is determined by the preformed putative fiber. Since each microwell is obtained at the end of its own optical fiber, each well can serve as an individual sensor. Preferably, the diameter of the etched microwells is slightly larger than that of the individual connecting microbeads used for the connection there.
[0142]
For example, in one embodiment, a solution containing a plurality of microbeads having one or more attached oligonucleotide binding regions is added to the surface of a fiber optic bundle. Preferably, the oligonucleotide binding region is complementary to the target nucleic acid to be detected and / or quantified. The individual connected microbeads settle randomly in each microwell as the solution is left to evaporate from the surface of the fiber optic bundle. Optionally, and when a photoreactive compound is used in connection with this embodiment, the optical fibers are illuminated to allow the linked microbeads to optically couple to the surface of each optical fiber. Optionally, the excess microbeads are then washed from the surface of the fiber bundle.
[0143]
For example, immobilization of a bead or oligonucleotide binding region on the distal end of a fiber can be achieved in a microwell using a photoactivatable compound to form a bond with the well surface. it can. In one embodiment, the well surface of the optical fiber is coated with a photoreactive siloxane reagent, and then a solution containing free (ie, unbound) oligonucleotide binding regions is applied to the well surface. The solution is then irradiated, activating the photoreactive groups, resulting in the formation of a bond between the oligonucleotide binding region and the well surface. Irradiation is achieved by passing the activating light through the fiber and into the well.
[0144]
As described above, each individual fiber of the fiber bundle can be etched at one end to provide a concave container for the microbeads. Photoreactive organosiloxane reagents are prepared by combining a commercially available organosiloxane reagent such as aminopropyldimethyl methoxy siloxane with a thermochemically reactive photoreagent such as benzoyl benzoic acid acid chloride. Can be. The photoreactive siloxane reagent reacts with the concave silicon oxide surface of the etched optical fiber in the image analyzer bundle. In an alternative embodiment, each microwell is coated with x-avidin to create a surface that reacts with biotin. In this embodiment, the microbeads are coated with biotin or an iminobiotin derivative. As described herein, any specific binding pair is suitable for such binding.
[0145]
Preferably, the connecting microbeads are spherical and have a diameter approximately equal (or slightly smaller) than that of the individual optical fibers. The linked microbeads of the present invention are made from any suitable material, including, for example, glass, polystyrene, polyvinylbenzophenone, photopolysiloxane coated glass. Preferably, the microbeads have a greater specific gravity than that of water and have a chemical composition that is amenable to binding with organic reagents.
[0146]
Oligonucleotide derivatives can be synthesized by published and commercially used methods, as described in more detail herein. The length of the oligonucleotide sequence is optimized for the desired strength and rate of hybridization (usually in the range of 20-50 nucleotides). The oligonucleotide sequence is complementary to the selected target nucleic acid.
[0147]
"Address oligonucleotide" derivatives can be attached to epoxy activated linked microbeads for the end of an optical fiber. In a preferred embodiment, for example, uniform silica spheres of approximately fiber diameter are commercially available containing thermochemically reactive groups (eg, epoxy groups) useful for binding to amine groups of oligonucleotide derivatives in the present invention. Reacts with organosiloxane reagents. Glycidoxypropyltriethoxy siloxane reacts with glass microbeads under published reaction conditions to provide an epoxy glass surface. The address oligonucleotide derivative of the present invention is terminated by an alkylamine group on the spacer, and easily binds to an epoxy group on the surface of the linked microbead.
[0148]
The bundle of etched photoreactive optical fibers is exposed to a slurry of ligated microbeads (in sufficient number to provide at least one type of address oligonucleotide per fiber), with each microbead on its surface. It has an immobilized address oligonucleotide sequence. Preferably, one oligonucleotide binding region is attached to each fiber optic well. In this embodiment, the master array is reusable. This slurry consists of approximately the same number of beads as each of the desired address oligonucleotide sequences. The number of beads providing the oligonucleotide sequence is preferably sufficient to provide for the redundancy (at least three-fold) of each target nucleic acid specific site. As the solvent (preferably aqueous) evaporates from the slurry pool on the photoreactive surface in the dark or in dim light, the uniformly sized beads remain in the concave pocket at the ends in the ordered arrangement of fibers. The linked microbeads, which provide the oligonucleotide sequence, are then "immobilized" by irradiation in the dry state to allow photoreactive groups on the fiber optic surface and organic groups on the address oligonucleotide beads (often oligos). Nucleotides). Excess microbeads are rinsed from the system.
[0149]
The resulting fiber optic bundle has a stable, ordered arrangement of oligonucleotides complementary to the target nucleic acid sequence on one end of the fiber, and the positions of the oligonucleotide sequences are then described in detail herein. As such, it can be determined by conventional fluorescent hybridization methods involving sequential addition of fluorescently labeled complementary sequences to the array, with rinsing and reading of the array between each addition.
[0150]
In one preferred embodiment, the complementary sequence is labeled with a fluorophore. Fluorescently labeled oligonucleotides complementary to each sequence are synthesized, each containing several (eg, 1 in 10) different fluorescent compounds that can be distinguished from each other. Thus, each oligonucleotide has a known sequence and a known identifiable label. Several oligonucleotides, each having a different known sequence, each complementary to the oligonucleotide sequence on the surface, and one of a similar number of identifiable fluorophores, were simultaneously applied to the surface and at 55 ° C. Hybridize for minutes.
[0151]
Next, the surface is rinsed and analyzed to identify the location of each sequence. Next, another set of sequences is hybridized to the surface and scanned to identify a second set of sequences. The process is continued until all locations have been identified, after which the master array is peeled off by immersion in boiling water for 2 minutes. Then, it is ready to be used to detect the presence of the target nucleic acid in the sample.
[0152]
In use, the master array is exposed to a test solution containing a plurality of target nucleic acid sequences (eg, genes and / or gene fragments). The test solution is labeled via a fluorescent label, allowing visualization of the results. The test solution is cultured under conditions that allow the oligonucleotide binding region to hybridize to the target nucleic acid contained within the sample. Next, the unbound solution is rinsed from the master array. The hybridized nucleic acid sequences are visualized using the CCD camera of the array system described above.
[0153]
The assay array was washed with phosphate buffered saline containing 0.05% Tween 20 (PBS / Tween), then 4XSCC (0.6M NaCl, 0.06M citrate, pH 7.0), 0.1%. Blocked for 30 minutes at 55 ° C with a hybridization buffer consisting of 1% (w / v) lauroyl sarcosine and 0.02% (w / v) sodium dodecyl sulfate. A sample containing DNA having a sequence complementary to one of the oligonucleotide probes immobilized on the array was applied to the support surface of the fiber and allowed to stand at 55 ° C. for 1 hour in a sealed hybridization chamber. Cultured. Next, the support surface is washed with 2 × SSC containing 0.1% SDS (sodium dodecyl sulfate) at 55 ° C. for 5 minutes, followed by a fluorescently labeled detection probe that is complementary to another sequence on the target DNA. 50 fmol is applied to the support surface and incubated at 55 ° C. for 1 hour. Next, the support surface is washed with 2 × SSC containing 0.1% SDS at 55 ° C. for 5 minutes, followed by 0.1 × SSC, then dried and the fluorescent pattern is analyzed by image analysis equipment. Be recorded.
[0154]
Those skilled in the art will appreciate that the present invention can provide a variety of oligonucleotide sequences integrated with a particular fiber bundle. In one embodiment, for example, the fiber wells are individually coated with a specific binder. In another embodiment, the fiber wells are simultaneously coated with a common binder (eg, iminobiotin). In this embodiment, the fiber wells are loaded from a mixture of specific binder microbeads, each containing a complement to a common binder (eg, x-avidin).
[0155]
Optionally, the system further includes an image analysis system, for example, a CCD camera with associated software. The resulting assay array can be used in a conventional manner, e.g., the assay array can be read by a standard array reader, while the master array can be reused to re-use some or all of the steps described above. By repeating the above, more assay arrays can be prepared.
[0156]
It will be apparent to those skilled in the art that the present invention can be applied to any molecular species involved in molecular recognition. Discussion has provided details regarding the nucleic acid applications of the present invention, while the present invention is not so limited.
[0157]
All patents, patent applications, provisional applications, and publications referenced or cited herein are incorporated by reference in their entirety to the extent they do not conflict with the express teachings herein.
[0158]
The following is an example illustrating a procedure for performing the present invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise specified.
[0159]
Example
Example 1
Preparation of 4-benzoylbenzoyl chloride (BBA-Cl) (Compound I)
1.0 kg (4.42 mol) of 4-benzoylbenzoic acid (BBA) is added to a dry 5 liter Morton flask equipped with a reflux condenser and a top stirrer, followed by 645 ml (8.84 mol) of thionyl chloride. ) And 725 ml of toluene were added. Then 3.5 ml of dimethylformamide were added and the mixture was heated at reflux for 4 hours. After cooling, the solvent was removed under reduced pressure and residual thionyl chloride was removed by three evaporations using 3 × 500 ml of toluene. The product was recrystallized from 1: 4 toluene: hexane to give 988 g (91% yield) after drying in a vacuum oven. The product melting point was 92-94 ° C. 80MHz ( ¹ HNMR (CDCl ₃ Nuclear magnetic resonance (NMR) analysis under)) was consistent with the expected product: aromatic protons 7.20-8.25 (m, 9H). All chemical shift values are ppm downfield from tetramethylsilane internal standard. The final compound was saved for use in preparing monomers used in the synthesis of photoactivatable polymers, for example, as described in Example 3.
[0160]
Example 2
Preparation of N- (3-aminopropyl) methacrylamide hydrochloride (APMA) (Compound II)
CH ₂ Cl ₂ A solution of 1910 g (25.77 mol) of 1,3 diaminopropane in 1000 ml was added to a 12 liter Morton flask and cooled on an ice bath. Next, 1000 g (5.15 mol) of t-butylphenyl carbonate, CH ₂ Cl ₂ 250 ml of the solution were added dropwise at a rate to keep the reaction temperature below 15 ° C. After the addition, the mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture is CH ₂ Cl ₂ Dilute with 900 ml and 500 g of ice, followed by slow addition of 2500 ml of 2.2N NaOH. After testing to confirm that the solution was basic, the product was transferred to a separatory funnel and the organic layer was removed and set aside as extract # 1. Next, the aqueous fraction was CH 2 ₂ Cl ₂ Extracted with 3 × 1250 ml and stored each extract as a separate fraction. The four organic extracts were washed continuously with a single 1250 ml portion of 0.6N NaOH starting from fraction # 1 and passing through fraction # 4. This washing procedure was repeated a second time with 1250 ml portions of fresh 0.6N NaOH. Next, the organic extracts were mixed and mixed with Na ₂ SO ₄ Dried on. Filtration and evaporation to constant weight gave 825 g of N-mono-t-butoxycarbonyl-1,3-diaminopropane which was used without further purification.
[0161]
CHCl ₃ A 806 g (5.23 mol) solution of methacrylic anhydride in 1020 ml was placed in a 12 liter Morton flask equipped with a top stirrer and cooled on an ice bath. 60 mg of phenothiazine is added as inhibitor followed by CHCl ₃ 825 g of N-mono-t-butoxycarbonyl-1,3-diaminopropane in 825 ml were added dropwise. The addition rate was adjusted so that the reaction temperature was always kept below 10 ° C. After the addition was complete, the ice bath was removed and the mixture was allowed to stir overnight. The product was diluted with 2400 ml of water and transferred to a separatory funnel. After thorough mixing, the aqueous layer was removed, and the organic layer was washed with 2400 ml of 2N NaOH, confirming that the aqueous layer was basic. Next, the organic layer was washed with Na ₂ SO ₄ Dry over and filter to remove desiccant. CHCl ₃ Some of the solvent was removed under reduced pressure until the combined weight of product and solvent was approximately 3000 g. Next, the stirred CHCl ₃ The expected product was precipitated by the slow addition of 11.0 liter of hexane into the solution. The product is isolated by filtration and the solid is dried in 900 ml hexane and CHCl ₃ Rinse twice with 150 ml. Thorough drying of the solid provided N- [N '-(t-butyloxycarbonyl) -3-aminopropyl] -methacrylamide with a melting point of 85.8 ° C by DSC. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (CDCl ₃ ) Amide NH's 6.30-6.80, 4.55-5.10 (m, 2H), vinyl proton 5.65, 5.20 (m, 2H), N adjacent methylene 2.90-3. 45 (m, 4H), methyl 1.95 (m, 3H), residual methylene 1.50-1.90 (m, 2H), and t-butyl 1.40 (s, 9H).
[0162]
A 3-neck, 2 liter round bottom flask was equipped with a top stirrer and gas spray tube. 700 ml of methanol was added to the flask and cooled on an ice bath. With stirring, HCl gas was bubbled through the solvent at a rate of approximately 5 liters / minute for a total of 40 minutes. The molarity of the final HCl / MeOH solution was determined to be 8.5 M by titration with 1 N NaOH using phenolphthalein as indicator. 900 g (3.71 mol) of N- [N '-(t-butyloxycarbonyl) -3-aminopropyl] -methacrylamide are added to a 5 liter Morton flask equipped with a top stirrer and gas outlet adapter, followed by Then, 1150 ml of a methanol solvent was added. Some solids remained in the flask with this volume of solvent. 30 mg of phenothiazine was added as inhibitor, followed by 655 ml (5.57 mol) of an 8.5 M HCl / MeOH solution. The solid dissolved slowly with outgassing, but the reaction was not exothermic. The mixture was stirred at room temperature overnight to ensure complete reaction. Next, any solids were removed by filtration and an additional 30 mg of phenothiazine was added. The solvent was then removed under reduced pressure and the resulting solid residue was azeotropically distilled with 3 × 1000 ml by evaporation under reduced pressure. Finally, the product was dissolved in 2000 ml of refluxing isopropanol and 4000 ml of ethyl acetate were added slowly with stirring. The mixture was allowed to cool slowly and stored at 4 ° C. overnight. Compound II was isolated by filtration and dried to constant weight to give a yield of 630 g with a melting point of 124.7 ° C. by DSC. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (D ₂ O) Vinyl proton 5.60, 5.30 (m, 2H), methylene N adjacent methylene 3.30 (t, 2H), amine N adjacent methylene 2.95 (t, 2H), methyl 1.90 (m , 3H), and residual methylene 1.65-2.10 (m, 2H). The final compound was saved for use in preparing the monomer used in the synthesis of the photoactivatable polymer, for example, as described in Example 3.
[0163]
Example 3
Preparation of N- [3- (4-benzoylbenzamido) propyl] methacrylamide (BBA-AMPA) (Compound III)
120 g (0.672 mol) of compound II prepared by the general method described in Example 2 was added to a dry 2-liter 3-neck round bottom flask equipped with a top stirrer. 23-25 mg of phenothiazine was added as an inhibitor, followed by 800 ml of chloroform. The suspension was cooled below 10 ° C. on an ice bath and 172.5 g (0.705 mol) of compound I prepared by the general method described in Example 1 were added as a solid. Next, 207 ml (1.485 mol) of triethylamine in 50 ml of chloroform was added dropwise over a period of 1 to 1.5 hours. The ice bath was removed and stirring at room temperature was continued for 2.5 hours. The product was then washed twice with 600 ml of 0.3N NaCl and 300 ml of 0.07N HCl. After drying over sodium sulfate, the chloroform was removed under reduced pressure and the product was recrystallized twice from 4: 1 toluene: chloroform using 23-25 mg of phenothiazine in each recrystallization to prevent polymerization. . The general yield of compound III was 90%, mp 147-151 ° C. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (CDCl ₃ ) Aromatic proton 7.20-7.95 (m, 9H), amide NH 6.55 (t, 1H wide), vinyl proton 5.65, 5.25 (m, 2H), adjacent to amide N's Methylene 3.20-3.60 (m, 4H), methyl 1.95 (s, 3H), and residual methylene 1.50-2.00 (m, 2H). The final compound was stored for use in the synthesis of the photoactivatable polymer, for example, as described in Example 6.
[0164]
Example 4
Preparation of N-succinimidyl 6-maleimidohexanoate (MAL-EAC-NOS) (Compound VI)
The functionalized monomer was prepared in the following manner and used as described in Example 6 to introduce an activated ester group on the polymer backbone. 100 g (0.762 mol) of 6-hexanoic acid were dissolved in 300 ml of acetic acid in a three-necked 3 liter flask equipped with a top stirrer and a drying tube. 78.5 g (0.801 mol) of maleic anhydride were dissolved in 200 ml of acetic acid and added to the 6-aminohexanoic acid solution. The mixture was stirred for 1 hour while heating on a boiling water bath, resulting in the formation of a white solid. After cooling overnight at room temperature, the solid was collected by filtration and rinsed with 2 × 50 ml of hexane. After drying, the general yield of (Z) -4-oxo-5-aza-2-undecenoic acid was 158-165 g (90-95%) and the melting point was 160-165 ° C. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (DMSO-d ₆ ) Amide proton 8.65 to 9.05 (m, 1H), vinyl proton 6.10, 6.30 (d, 2H), methylene 2.85 to 3.25 (m, 2H) adjacent to nitrogen, carbonyl Neighboring methylene 2.15 (t, 2H), and residual methylene 1.00 to 1.75 (m, 6H).
[0165]
(Z) -4-oxo-5-aza-2-undecenoic acid 150.0 g (0.654 mol), acetic anhydride 68 ml (73.5 g, 0.721 mol), and phenothiazine 500 mg, equipped with a top stirrer Added to a 2 liter, 3 neck round bottom flask. 91 ml (0.653 mol) of triethylamine and 600 ml of tetrahydrofuran (THF) were added and the mixture was heated to reflux with stirring. After a total of 4 hours of reflux, the dark mixture was cooled to below 60 ° C. and poured into 250 ml of 12N HCl in 3 liters of water. The mixture was stirred at room temperature for 3 hours, then filtered through a filter pad (Celite 545, JT Baker, Jackson, TN) to remove solids. The filtrate was extracted with 4 × 500 ml of chloroform and the combined extracts were dried over sodium sulfate. After addition of 15 mg of phenothiazine to prevent polymerization, the solvent was removed under reduced pressure. The 6-maleimidohexanoic acid was recrystallized from 2: 1 hexane: chloroform to give a general yield of 76-83 (55-60%) having a melting point of 81-85 ° C. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (CDCl ₃ ) Maleimide proton 6.55 (s, 2H), methylene adjacent 3.40 (t, 2H), carbonyl adjacent 2.30 (t, 2H), and residual methylene 1.05-1.85 (m, 6H).
[0166]
20.0 g (94.7 mmol) of 6-maleimidohexanoic acid was dissolved in 100 ml of chloroform under an argon atmosphere, followed by addition of 41 ml (0.47 mol) of oxalyl chloride. After stirring for 2 hours at room temperature, the solvent was removed under reduced pressure with 4 × 25 ml of additional chloroform used to remove the last excess oxalyl chloride. The acid chloride was dissolved in 100 ml of chloroform, followed by addition of 12 g (0.104 mol) of N-hydroxysuccinimide and 16 ml (0.114 mol) of triethylamine. After stirring overnight at room temperature, the product was washed with 4 × 100 ml of water and dried over sodium sulfate. Removal of the solvent gave 24 g (82%) of the product which was used without further purification. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (CDCl ₃ ) Maleimide proton 6.60 (s, 2H), nitrogen adjacent methylene 3.45 (t, 2H), succinimidyl proton 2.80 (s, 4H), carbonyl adjacent methylene 2.55 (t, 2H), and Residual methylene 1.15-2.00 (m, 6H). The final compound was stored for use in the synthesis of the photoactivatable polymer, for example, as described in Example 6.
[0167]
Example 5
Preparation of functionalized monomer: N-succinimidyl 6-methacrylamide hexanoate (MA-EAC-NOS) (compound V)
Functionalized monomers were prepared in the following manner and used to introduce activated ester groups on the polymer backbone.
[0168]
4.00 g (30.5 mmol) of 6-aminocaproic acid was placed in a dry round bottom flask equipped with a drying tube. Next, 5.16 g (33.5 mmol) of methacrylic anhydride were added and the mixture was stirred at room temperature for 4 hours. The thick oil obtained was triturated three times with hexane, the residual oil was dissolved in chloroform and subsequently dried over sodium sulfate.
[0169]
After filtration and evaporation, a portion of the product was purified by silica gel flash chromatography using 10% methanol in a chloroform solvent system. The appropriate fractions were combined, 1 mg of phenothiazine was added and the solvent was removed under reduced pressure. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (CDCl ₃ ) Carboxylic acid protons 7.80-8.20 (b, 1H), amide protons 5.80-6.25 (b, 1H), vinyl protons 5.20 and 5.50 (m, 2H), nitrogen Adjacent methylenes 3.00 to 3.45 (m, 2H), carbonyl adjacent methylenes 2.30 (t, 2H), methyl group 1.95 (m, 3H), and residual methylene 1.10 to 1.90 (m , 6H).
[0170]
3.03 g (15.2 mmol) of 6-methacrylamidohexanoic acid was dissolved in 30 ml of dry chloroform, followed by 1.92 g (16.7 mmol) of N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide 6 .26 g (30.4 mmol) were added. The reaction was stirred overnight at room temperature under a dry atmosphere. The solid was then removed by filtration and a portion was purified by silica gel flash chromatography. Non-polar impurities were removed using chloroform solvent, followed by elution of the desired product with 10% tetrahydrofuran in chloroform solvent. The appropriate fractions were pooled, phenothiazine 0.2 mg was added and the solvent was evaporated under reduced pressure. This product, which contained a small amount of 1,3-dicyclohexylurea as an impurity, was used without further purification. Analysis on the NMR spectrometer corresponded to the expected product: ¹ HNMR (CDCl ₃ 5.) Amide protons 5.60-6.10 (b, 1H), vinyl protons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.05-3.40 (m, 2H), succinimidyl -Proton 2.80 (s, 4H), carbonyl adjacent methylene 2.55 (t, 2H), methyl 1.90 (m, 3H), and residual methylene 1.10 to 1.90 (m, 6H). The final compound was saved for use in the synthesis of the photoactivatable polymer described herein.
[0171]
Example 6
Preparation of Copolymer of Acrylamide, BBA-APMA, and MAL-EAC-NOS (Photosensitive PA-Poly NOS)
The photoactivatable copolymer of the present invention was prepared in the following manner. 4.298 g (60.5 mmol) of acrylamide are dissolved in 57.8 ml of tetrahydrofuran, followed by 0.219 g (0.63 mmol) of compound III prepared by the general method described in Example 3, Example 0.483 g (1.57 mmol) of compound IV prepared by the general method described in 4, N, N, N ', N'-tetramethylethylenediamine (TEMED) 0.058 ml (0.39 mmol), And 0.154 g (0.94 mmol) of 2,2'-azobisisobutyronitrile (AIBN) were dissolved. The solution was deoxygenated with a helium atomizer for 3 minutes, followed by an additional 3 minutes with an argon atomizer. Next, the closed vessel was heated at 60 ° C. overnight to complete the polymerization. The solid product is isolated by filtration and the filter cake is washed with THF and CHCl ₃ And rinsed thoroughly. The product was dried in a vacuum oven at 30 ° C. to give 5.34 g of a white solid. NMR analysis (DMSO-d ₆ ) Confirmed the presence of N-oxysuccinimide (NOS) groups at 2.75 ppm and determined that the optical loading was 0.118 mmol BBA / g of polymer. MAL-EAC-NOS constituted 2.5 mol% of polymerizable monomer in this reaction to give compound VI-A.
[0172]
The procedure described above was used to prepare a polymer having 5 mol% of compound IV. 3.849 g (54.1 mmol) of acrylamide are dissolved in 52.9 ml of THF, followed by 0.213 g (0.61 mmol) of compound III prepared by the general method described in Example 3, Example 4 0.938 g (3.04 mmol) of compound IV, 0.053 ml (0.35 mmol) of TEMED and 0.142 g (0.86 mmol) of AIBN were prepared according to the general procedure described in The resulting solid, compound VI-B, when isolated as described above, gave 4.935 g of product with a photogroup loading of 0.101 mmol BBA / g of polymer.
[0173]
The procedure described above was used to prepare a polymer having 10 mol% of compound IV. 3.241 g (45.6 mmol) of acrylamide are dissolved in 46.4 ml of THF, followed by 0.179 g (0.51 mmol) of compound III prepared by the general method described in Example 3, Example 4 1.579 g (5.12 mmol) of compound IV, 0.047 ml (0.31 mmol) of TEMED and 0.126 g (0.77 mmol) of AIBN were prepared according to the general procedure described in The resulting solid, Compound VI-C, when isolated as described above, gave 4.758 g of product having an optical loading of 0.098 mmol BBA / g of polymer.
[0174]
A procedure similar to that described above was used to prepare a polymer having 2.5 mol% of compound IV and 2 mol% of compound III. 16.43 g (231.5 mmol) of acrylamide; 1.70 g (4.85 mmol) of compound III prepared by the general method described in Example 3; prepared by the general method described in Example 4 1.87 g (6.06 mmol) of the prepared compound IV; and THF (222 ml) were stirred in a round bottom flask at room temperature with an argon atomizer. 0.24 ml (2.14 mmol) of TEMED and 0.58 g (3.51 mmol) of AIBN were added to the reaction. The reaction was then refluxed for 4 hours under an atmosphere of argon. The resulting solid, compound VI-D, when isolated as described above, gave 19.4 g of product with an optical loading of 0.23 mmol BBA / g of polymer.
[0175]
Example 7
Synthesis of multiligand conjugate
A multiligand conjugate was prepared as follows. 200 μl aliquot of polystyrene microbeads (1 μm diameter) (obtained from Prolabo, Bangs Labs, Carmel, IN, product number K100) containing 10% by weight microbeads in aqueous suspension Was mixed with a solution of photosensitive PA-polyNOS (prepared as described in Example 6 above) containing 1.0 mg of polymer in 200 μl of deionized water. The solution was mixed at room temperature for 1 hour.
[0176]
After mixing for 1 hour at room temperature, a Dymax lamp (25 mJoules / cm, measured at 335 nm with a 10 nm bandpass filter on an International Light Radiometer). ² ), Irradiate the suspension for 2 minutes with mixing. The microbeads were then washed twice with 2 ml of deionized water by centrifugation at 15,000 rpm for 15 minutes and resuspension in phosphate buffer (PBS, pH 7.5).
[0177]
The washed beads were then combined with two oligonucleotide sequences, each modified at the 3 'end to contain a primary amine, and biocytin (10 μM) in 0.05 M phosphate buffer pH 7.5. Culture with a solution containing the mixture of The first oligonucleotide sequence is the complement to the known sequence of the specific address oligonucleotide provided on the master array. The first oligonucleotide is provided as a 20-mer at 5 μM. The first oligonucleotide is synthesized by first determining the sequence of the specific address oligonucleotide to be used in the present invention. Next, the complement of the address oligonucleotide is determined using Watson-Crick base pairing rules. Once the complementary sequence has been determined, the first oligonucleotide is synthesized using standard nucleic acid synthesis techniques, for example, solid phase synthesis.
[0178]
The second oligonucleotide sequence is an analytical sequence that is complementary to a known sequence of the target nucleic acid suspected of being present in the test sample. The second oligonucleotide is provided as a 30 mer at 10 μM. A second oligonucleotide is synthesized by first determining the sequence of the particular target ligand to be detected using the probe array of the present invention. Complement for the target ligand is determined using Watson-Crick base pairing rules. Once the complementary sequence has been determined, a second oligonucleotide is synthesized using standard nucleic acid synthesis techniques.
[0179]
The microbead suspension is incubated with the oligonucleotide overnight at room temperature. After overnight culture, the beads are washed twice with 1 ml aliquots of phosphate buffer (PBS, pH 7.5) and then stored at 4 ° C. until used. A series of multiligand conjugates, each with a unique address and analytical sequence, are created in this manner to replicate the probe array.
[0180]
Example 8
Synthesis of Separated Epoxidized Monomer (Compound VII)
To 3 ml of chloroform isocyanate ethyl methacrylate (1.0 ml, 7.04 mmol), glycidol (0.50 ml, 7.51 mmol) and triethylamine (50 μl, 0.27 mmol) are added. The reaction was stirred overnight at room temperature. The product was purified on a silica gel column and the structure was confirmed by NMR. The yield was 293 mg (18% yield).
[0181]
Example 9
Preparation of Copolymer of Acrylamide, BBA-APMA and Glycidyl Methacrylate (Photosensitive PA-Polyepoxide) (Compound VIII)
Acrylamide (7.1 g, 99.35 mmol), BBA-APMA (0.414 g, 1.18 mmol) and 2,2′-azobis (2-methylbutyronitrile) (“VAZO67”, 0.262 g, 1 .4 mmol) was dissolved in 108 ml of THF. To this solution, 2.4 ml (17.7 mmol) of glycidyl methacrylate was added. The solution was sparged with helium for 4 minutes and then with argon for 4 minutes, then stoppered tightly and heated at 61 ° C. overnight with mixing. The polymer was collected by filtration, then suspended and mixed in methanol. It was then collected again by filtration, washed with chloroform and then dried at 30 ° C. in a vacuum oven.
[0182]
Example 10
Preparation of microscope slides coated with polyepoxide
Soda lime glass microscope slides (Erie Scientific, Portsmouth, NH) were run for 1 minute each with 1% p-tolyldimethylchlorosilane (T-silane) and N-decyldimethyl in acetone. Silane treatment was performed by immersion in a mixture of chlorosilane (D-silane, United Chemical Technologies, Bristol, PA). After air drying, the slides were cured in an oven at 120 ° C. for 1 hour. Next, the slides were washed with acetone and subsequently immersed in distilled water. Slides were dried in the oven for an additional 5-10 minutes.
[0183]
Compound VIII (Example 9) was sprayed onto silanized slides, which were then measured while wet at 335 nm with a Dymax lamp (International Light Radiometer 10 nm bandpass filter). 25mJoule / cm ² ), Washed with water and dried.
[0184]
Example 11
Preparation of microarray with photosensitive-polyepoxide coated slide
Oligonucleotides, either aminated or non-aminated, were placed on a chipmaker (ChipMaker) 2 microarray spotting pin (Telechem International) using an X, Y, Z motion controller to position the oligonucleotide. Printed on slides at 8 μM in .15M phosphate buffer. Slides were prepared using photo-polyepoxides as in Example 10, photo-sensitive PA-polyNOS by the same procedure, or published methods (US Pat. No. 5,087,522, Brown et al., Methods for Fabricating Microarrays). of Biological Samples, "and references cited therein).
[0185]
Printed epoxide and NOS slides were incubated overnight at room temperature and 75% relative humidity. Printed polylysine slides were processed according to published procedures. The slide was scanned to measure the Cy3 fluorescence of the immobilized capture oligonucleotide and then hybridized overnight at 41 ° C. with a Cy5-labeled oligonucleotide (1 pmol / slide) that was complementary to the capture oligonucleotide. . The slide was scanned with a laser scanner to measure the fluorescence intensity of the hybridized oligonucleotide. Due to the low amount of capture oligonucleotide immobilized by the amine-silane slide, they did not hybridize.
[0186]
Embedded image

[0187]
Embedded image

[0188]
Embedded image

(Where x = 0.1-5 mol%, y = 2-30 mol%, and z = 65-97.9 mol%)
[0189]
While the preferred embodiment of the invention has been described, it should be understood that various changes, adaptations, and modifications can be made therein without departing from the spirit of the invention and the scope of the appended claims. .

Claims (25)

A system for replicating a specific binding ligand probe array, the system comprising:
a) a master array comprising a support surface having a plurality of address ligands immobilized thereon;
b) at least one molecule of the first, wherein each multiligand conjugate comprises: (i) a core; (ii) a ligand selected to bind in a complementary manner to a specific address ligand of the master array. A ligand binding region; (iii) at least one molecule of a second ligand binding region comprising a ligand selected to bind the target ligand in a complementary manner; and (iv) at least one molecule of a third ligand. A plurality of multi-ligand conjugates, wherein the first ligand binding region, the second ligand binding region, and the third ligand are attached to a core; and c) providing a support surface for the replication array. An assay array support, including
including. The system of claim 1, wherein the address ligand and the first and second binding ligands each independently comprise a nucleic acid. 2. The system of claim 1, further comprising a linking agent bound to the address ligand and the master array support surface. 4. The system of claim 3, wherein the linking agent comprises a microbead. The system of claim 1, wherein the first ligand binding region, the second ligand binding region, and the third ligand are independently bound to the multi-ligand conjugate core. The system of claim 1, wherein the third ligand comprises a biotin derivative. The system of claim 1, wherein the third ligand comprises a polymerizable group. The system according to claim 7, wherein the polymerizable group is selected from the group consisting of an acrylic group and a vinyl group. The system of claim 1, wherein the assay array support further comprises a binding site for a third ligand. 10. The system of claim 9, wherein the third ligand comprises a binding ligand and the binding site comprises a binding partner molecule specific for the third ligand. A method for replicating a specific binding ligand probe array, the method comprising:
a) providing a master array including a support surface having a plurality of address ligands immobilized thereon;
b) at least one molecule of the first, wherein each multiligand conjugate comprises: (i) a core; (ii) a ligand selected to bind in a complementary manner to a specific address ligand of the master array. (Iii) at least one molecule of a second ligand-binding region comprising a ligand selected to bind the target ligand in a complementary manner; and (iv) at least one molecule of a third ligand. Providing a plurality of multi-ligand conjugates, wherein the first ligand binding region, the second ligand binding region, and the third ligand are bound to a core;
c) binding the multi-ligand conjugate to the master array by binding the first ligand binding region to a complementary address ligand;
d) providing an assay array support that includes a support surface for the replication array;
e) bringing the assay array support sufficiently close to the master array under conditions suitable to allow the bound multiligand conjugate to bind to the assay array support; and f) supporting the assay array support. Dissociating the bound complementary address ligand and the first ligand binding region under conditions suitable to allow the body to be recovered and utilized;
A method that includes The method according to claim 11, wherein the third ligand comprises a biotin derivative. The method of claim 11, wherein the third ligand comprises a polymerizable group, the method further comprising the step of reacting the third ligand to form a polymer film. 14. The method according to claim 13, wherein the polymerizable group is selected from a vinyl group and an acrylic group. 12. The method of claim 11, wherein the address ligand and the first and second binding ligands each independently comprise a nucleic acid. 12. The method of claim 11, wherein the master array further comprises an address ligand and a linking agent bound to the master array support surface. 17. The method of claim 16, wherein the linking agent comprises a microbead. The method of claim 11, wherein the assay array support further comprises a binding site for a third ligand. 19. The method of claim 18, wherein the third ligand comprises a binding ligand and the binding site comprises a binding partner molecule specific for the third ligand. A system for replicating a specific binding ligand probe array, the system comprising:
a) a master array comprising a support surface having a plurality of address ligands immobilized thereon;
b) at least one molecule of each multiligand conjugate comprising (i) a core; (ii) a ligand selected to bind to a specific target ligand and a specific address ligand in a complementary manner. A plurality of multi-ligand conjugates comprising: a second ligand binding region; and (iii) at least one molecule of a third ligand, wherein the second ligand binding region and the third ligand are attached to a core. And c) an assay array support comprising a support surface for the replication array;
Including the system. 21. The system of claim 20, wherein the address ligand and the first and second binding ligands each independently comprise a nucleic acid. 21. The system of claim 20, wherein the third ligand comprises a biotin derivative. 21. The system of claim 20, wherein the third ligand is selected from the group consisting of a binding ligand and a polymerizable group. 24. The system according to claim 23, wherein the polymerizable groups are selected from vinyl groups and acrylic groups. A system for preparing a replicable array in the form of a reusable array, the system comprising:
a) a master array comprising a plurality of optical fibers, each optical fiber having a support surface located at the distal end of the fiber; and b) such that each oligonucleotide binding region binds a target ligand in a specific manner. A plurality of oligonucleotide binding regions comprising a selected oligonucleotide sequence,
Including the system.