RNA sequences generated using a microarray having a base cleavable succinate linker

There is disclosed a microarray having base cleavable succinate linkers. The microarray has a solid surface with known locations, each having reactive hydroxyl groups. The density of the known locations is greater than approximately 100 locations per square centimeter. Amino moieties are attached to the reactive hydroxyl groups. Preferably the attachment is through a phosphorous-oxygen bond between the phosphorous of amino amidite moieties and the oxygen of the hydroxyl groups. Succinate moieties are attached to the amino moieties through amide bonds to form cleavable linkers attached to the microarray. Oligomers may be synthesis in situ onto the cleavable linkers and subsequently cleaved using a cleaving base. The cleaved oligomers are recoverable and include oligonucleotides.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 120 to U.S. patent application Ser. No. 11/229,757, filed Sep. 19, 2005, which is incorporated by reference herein, and to U.S. patent application Ser. No. 10/877,568, filed Jun. 25, 2004, which is incorporated by reference herein and of which claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application Ser. No. 60/482,929, filed Jun. 27, 2003.

TECHNICAL FIELD OF THE INVENTION

This invention provides a plurality of different of double stranded RNA compounds (dsRNA) and single stranded short inhibitory RNA compounds (siRNA) generated on microarrays having cleavable succinate linker moieties attached at known locations, wherein the compounds are synthesized in situ on the cleavable succinate linkers.

BACKGROUND OF THE INVENTION

Microarray preparation methods for synthetic oligomers such as oligonucleotides (oligos) include the following: (1) spotting a solution on a prepared flat surface using spotting robots; (2) in situ synthesis by printing reagents via ink jet or other printing technology and using regular phosphoramidite chemistry; (3) in situ parallel synthesis using electrochemically generated acid for deprotection and using regular phosphoramidite chemistry; (4) maskless photo-generated acid (PGA) controlled in situ synthesis and using regular phosphoramidite chemistry; (5) mask-directed in situ parallel synthesis using photo-cleavage of photolabile protecting groups (PLPG); (6) maskless in situ parallel synthesis using PLPG and digital photolithography; and (7) electric field attraction/repulsion for depositing oligos.

Photolithographic techniques for in situ oligo synthesis are disclosed in Fodor et al. U.S. Pat. No. 5,445,934 and the additional patents claiming priority thereto, all of which are incorporated by reference herein. Electric field attraction/repulsion microarrays are disclosed in Hollis et al. U.S. Pat. No. 5,653,939 and Heller et al. U.S. Pat. No. 5,929,208, both of which are incorporated by reference herein. An electrode microarray for in situ oligo synthesis using electrochemical deblocking is disclosed in Montgomery U.S. Pat. Nos. 6,093,302; 6,280,595, and 6,444,111 (Montgomery I, II, and III respectively), all of which are incorporated by reference herein. Another and materially different electrode array (not a microarray) for in situ oligo synthesis on surfaces separate and apart from electrodes using electrochemical deblocking is disclosed in Southern U.S. Pat. No. 5,667,667, which is incorporated by reference herein. A review of oligo microarray synthesis is provided by: Gao et al., Biopolymers 2004, 73:579.

In addition to other disclosure, U.S. patent application Ser. No. 10/243,367, filed 12 Sep. 2002 (Oleinikov) discloses a process for assembling a polynucleotide from a plurality of oligonucleotides. The claimed process provides a plurality of oligonucleotide sequences that are synthesized in situ or spotted on a microarray device. The plurality of oligonucleotide sequences is attached to a solid or porous surface of the microarray device. The oligonucleotide sequences are cleaved at a cleavable linker site to form a soluble mixture of oligonucleotides. The cleavable linker is a chemical composition having a succinate moiety bound to a nucleotide moiety such that cleavage produces a 3′-hydroxy nucleotide.

The succinate moiety disclosed in Oleinkov as a cleavable linker is bound to the solid or porous surface through an ester linkage by reacting the succinate moieties with the solid or porous surface. In general, formation of an ester linkage to an organic hydroxyl on a solid surface using a succinate is relatively difficult and results in relatively low yield. Additionally, the reaction conditions require a relatively long period of time at relatively high temperature. Increasing yield would increase oligonucleotide density and provide more efficient production of oligonucleotides on a microarray. The present invention addresses the issue of low yield and hence low oligonucleotide density at a location on a microarray by providing a more reactive solid or porous surface for attachment of a succinate moiety. This approach to oligonucleotide synthesis is disclosed herein for the generation of RNA for use in treatment of diseases through RNA interference.

The present invention provides pools of RNA compounds useful for treating virally infected individuals. The RNA interference process (RNAi) is triggered by a dose of double stranded RNA (dsRNA) sufficient to maintain interference of the target gene expression for several days. RNAi for viral treatment is designed to target specific viral gene expression in a host infected cell. When infected, a cell has incorporated the viral genome, and the viral genes become expressed (i.e., form mRNAs) to build new virons or viral particles that leave the infected cell and spread to infect other cells. Through this process, the viral infection is augmented in an infected individual by having more cells become infected.

RNAi is triggered or enabled by using dsRNA. RNAi causes target gene silencing by targeting specific mRNA transcripts for degradation within an infected cell. Short dsRNA of 18-23 nucleotides in length (siRNAs) were sufficient to initiate RNAi in mammalian cells (Elbashir et al., Nature 411:494-498, 2001).

RNAi is a gene silencing or an antagonist approach to drug targeting. Within cells, dsRNA becomes incorporated into a protein-RNA effecter nuclease complex that can recognize and destroy a specific mRNA target or the expression of the targeted gene within a cell. The effecter nuclease is known as the RNA-induced silencing complex (RISC). RISC becomes activated by an ATP-dependent mechanism that involves unwinding the siRNA molecule introduced into the cell. This primes the specific RISC to recognize and cleave target mRNA containing a sequence complementary to the “guide” strand of the dsRNA or siRNA. Moreover, the RISC complex may be recycled to enable multiple rounds of mRNA degradation.

RNAi depends on normal mammalian cellular mechanisms such as the RNase III DCR-1 and Dicer (Knight and Bass, Science 293:2269-2271, 2001; Grishok et al., Cell 106:23-34, 2001; and Hutvagner et al., Science 293:834-838, 2001).

RNAi is performed experimentally by introducing synthetic siRNA's into cells (Elbashir et al., Nature 411:494-498, 2001; and Elbasjir et al., Methods 26:199-213, 2002). A single siRNA dose in vitro can maintain gene silencing up to 4 days. Drug delivery of single siRNA doses has been accomplished in cell culture with liposome packaging or in vivo with a high blood pressure IV-injection of naked siRNA molecules (McCaffrey et al., Nature 418:38-39, 2002; Lewis et al., Nature Genetics 32:107-108, 2002; and Song et al., Nature Med. 9:347-351, 2003).

RNAi has taken over the strategies previously used by antisense techniques. Antisense techniques had problems with target sequence accessibility in the mRNA. Antisense also had problems with nonspecific side effects in clinical trials, particularly when chemically modified synthetic antisense oligonucleotides with modified backbones were used. For example, when phosphorothiate-oligonucleotides were used and were designed to confer nuclease resistance, there was unanticipated binding to cellular proteins. This caused nonspecific cellular disruptive effects. Another problem was one of mRNA cleavage fragments produced by treatment with antisense oligonucleotides. Antisense oligonucleotides induce cleavage of the target mRNA by RNase H. Thus, stable 3′ mRNA cleavage products accumulated within cells (Thoma et al., Mol. Cell. 8:865-872, 2001; and Steiger and Decker, Mol. Cell. 8:732-733, 2001). Sometimes, the 3′ mRNA fragment is translated to produce N-terminally truncated protein products (Panchole et al., J. Virol. 77:382-390, 2003). The resulting N-truncated protein products accumulate within cells and can have effects if they lack N-terminal regulatory domains. Therefore, there is a need in the art to avoid the generation of stable 3′ mRNA cleavage products that can be addressed by employing pools of different siRNA molecules.

The consequences of a viral infection depend upon a number of factors, both viral and host dependent. These factors, which affect pathogenesis, include the number of infecting viral particles and their path to susceptible cells, the speed of viral multiplication and spread, the effect of the virus on cell functions, the host's secondary responses to the cellular injury, and the immunologic and non-specific defenses of the host. In general, the effects of viral infection include acute and chronic clinical diseases, asymptomatic infections, induction of various cancers, and chronic progressive neurological disorders. Viruses are potent infectious pathogenic agents because virons produced in one cell can invade other cells and thus cause a spreading infection. Viruses cause important functional alterations of the invaded cells, which can result in cellular death.

Therapeutic studies during the last ten years have identified promising drugs with antiviral effects. Although effective in some patients, such agents have been shown frequently to result in only a transient response or to have significant toxicity. Accordingly, there is a continuing need for methods and therapeutic agents to stop viral replication and prevent the spread of the virus to additional cells.

Another important limitation of antiviral therapy is the emergence of resistant mutants. Accordingly, there is also need in the art for therapies that do not quickly become obsolete due to rapidly developing viral resistance. The present invention was made to address the issue of viral therapy resistance.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a process for forming a microarray having cleavable succinate linkers comprising:

(a) providing a solid surface having free hydroxyl groups at known locations, wherein the density of the known locations is greater than approximately 100 locations per square centimeter;

(b) bonding a linker moiety to the hydroxyl groups, wherein the linker moiety comprises free amine group and a hydroxyl bonding group; and

(c) bonding a succinate-containing moiety having free carboxyl groups to the free amine groups to form cleavable linkers attached to the known locations, wherein the succinate-containing moieties comprise a sugar having both a nucleotide base group and a succinate group bonded to the sugar, wherein the cleavable linkers have a base-labile cleaving site on the succinate group and a reactable hydroxyl group on the sugar group.

Preferably, the sugar moiety has one or a plurality of free hydroxyl groups. Preferably, the process further comprises (d) synthesizing oligomers, such as oligonucleotides, attached to free hydroxyl groups of the sugar moiety. Most preferably, the process further comprises (e) cleaving oligomers at the base-labile cleaving site from the known location using a cleaving base, whereby the oligomers are recoverable.

Preferably, the sugar group is ribose and the nucleotide base group is selected from the group consisting of adenine, guanine, cytosine, and uracyl, or the sugar group is deoxyribose and the base group is selected from the group consisting of adenine, guanine, cytosine, and thymine.

Preferably, the oligomers are selected from the group consisting of DNA, RNA, and polypeptide, and combinations thereof. Preferably, the cleaving base is selected from the group consisting of ammonium hydroxide, electrochemically generated base, sodium hydroxide, potassium hydroxide, methylamine, and ethylamine and combinations thereof, whereby the oligomers comprising DNA and RNA have a 3′ hydroxyl after cleaving from the solid surface.

Preferably, the solid surface comprises an array of electrodes, and each of the known locations are associated with an electrode, wherein each electrode is electronically addressable. More preferably, the known locations are on the same surface as the array of electrodes, on an opposing surface to the electrodes, or on an overlayer over the electrodes.

Preferably, the amino moiety is selected from the group consisting of aminopropyltrimethoxysilane, aminopropyltriethoxysilane, aminopropylmethyldiethoxysilane, aminopropylmethyldiethoxysilane hydrozylate, m-aminophenyltrimethoxysilane, phenylaminopropyltrimethoxysilane, 1,1,2,4-tetramethyl-1-sila-2-azacyclopentane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltriethoxysilane, aminoethylaminopropylmethyldimethoxysilane, aminoethylaminopropyltrimethoxysilane hydrolyzate, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane hydrolyzate, trimethoxysilylpropyldiethylenetriamine, vinylbenzylethylenediaminepropyltrimethoxysilane monohydrochloride, vinylbenzylethylenediaminepropyltrimethoxysilane, benzylethylenediaminepropyltrimethoxysilane monohydrochloride, benzylethylenediaminepropyltrimethoxysilane, and allylethylenediaminepropyltrimethoxysilane monohydrochloride, and combinations thereof.

More preferably, the amino moieties are an amino amidite moiety selected from the group consisting of 3-(trifluoroacetylamino)propyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 12-(4-monomethoxytritylamino)dodecyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and 6-(trifluoroacetylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and combinations thereof.

Preferably, the succinate moieties are selected from a salt of a chemical selected from the group consisting of 5′dimethoxytrityl-N-benzoyl-2′-deoxycytidine-3′-O-succinate, 5′dimethyoxytrityl-N-isobutyryl-2′-deoxyguanosine-3′-O-succinate, 5′-dimethoxytrityl-thymidine-3′-O-succinate, and 5′-dimethoxytrityl-N-benzoyl-2′-deoxyadenosine-3′-O-succinate, and combinations thereof. Preferably the salt is a pyridium salt of the succinate moieties.

Preferably, the linker moiety comprises hydroxyl binding groups, free amine groups and a spacer moiety and further comprising amino amidite moieties are bound to the spacer moieties. Preferably, the spacer moiety is an oligomer selected from the group consisting of DNA, RNA, polyethylene glycol, and polypeptides, and combinations thereof. Preferably, the spacer moiety oligomer is from approximately 1 to 35 mers or units in length.

Preferably, the oligomers are synthesized in situ using electrochemical synthesis techniques. Optionally, the oligomers are synthesized in situ by a method selected from the group consisting of (a) printing reagents via ink jet or other printing technology and using regular phosphoramidite chemistry, (b) maskless photo-generated acid controlled synthesis and using regular phosphoramidite chemistry, (c) mask-directed parallel synthesis using photo-cleavage of photolabile protecting groups, and (d) maskless parallel synthesis using photo-cleavage of photolabile protecting groups and digital photolithography.

Preferably, a porous reaction layer is attached to the known locations and provides the hydroxyl groups, wherein the porous reaction layer comprises a chemical species or mixture of chemical specie, wherein the chemical species is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, polyethylene glycol, polyethylene glycol derivative, N-hydroxysuccinimide, formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, and combinations thereof, wherein formula I is

formula II is

formula III is HOR4(OR5)mR7 formula IV is

formula V is

formula VI is

and
formula VII is

wherein in each formula III is an integer from 1 to 4; R1, R2, R7, and R8 are independently selected from the group consisting of hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl, acyloxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino, tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato, isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato, selenocyanato, carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide, sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate, hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino, hydrothiol, tetrazolyl; thiocarbamoyl, thiocarbazono, thiocarbodiazono, thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato, thiosemicarbazido, thiosulfino, thiosulfo, thioureido, triazano, triazeno, triazinyl, trithiosulfo, sulfinimidic acid, sulfonimidic acid, sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid, sulfonohydroximic acid, and phosphoric acid ester; R3 is selected from the group consisting of heteroatom group, carbonyl, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; R4 and R5 are independently selected from the group consisting of methylene, ethylene, propylene, butylene, pentylene, and hexylene; R6 forming a ring structure with two carbons of succinimide and is selected from the group consisting of substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; and R7 is selected from the group consisting of amino and hydroxyl.

Preferably, the monosaccharide is selected from the group consisting of allose, altrose, arabinose, deoxyribose, erythrose, fructose, galactose, glucose, gulose, idose, lyxose, mannose, psicose, L-rhamnose, ribose, ribulose, sedoheptulose, D-sorbitol, sorbose, sylulose, tagatose, talose, threose, xylulose, and xylose. Preferably, the disaccharide is selected from the group consisting of amylose, cellobiose, lactose, maltose, melibiose, palatinose, sucrose, and trehalose Preferably, the triaccharide is selected from the group consisting of raffinose and melezitose.

Preferably, the polyethylene glycol derivative is selected from the group consisting of diethylene glycol, tetraethylene glycol, polyethylene glycol having primary amino groups, 2-(2-aminoethoxy)ethanol, ethanol amine, di(ethylene glycol) mono allyl ether, di(ethylene glycol) mono tosylate, tri(ethylene glycol) mono allyl ether, tri(ethylene glycol) mono tosylate, tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol) mono trityl ether, tri(ethylene glycol) mono chloro mono methyl ether, tri(ethylene glycol) mono tosyl mono allyl ether, tri(ethylene glycol) mono allyl mono methyl ether, tetra(ethlyne glycol) mono allyl ether, tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) mono tosyl mono allyl ether, tetra(ethylene glycol) mono tosylate, tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) mono trityl ether, tetra(ethylene glycol) mono 1-hexenyl ether, tetra(ethylene glycol)mono 1-heptenyl ether, tetra(ethylene glycol) mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether, tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol) mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether, penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethylene glycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allyl ether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol) mono benzyl ether, hexa(ethylene glycol) mono trityl ether, hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono 1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether, hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono 1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono 1-undecenyl ether, hepta(ethylene glycol) mono allyl ether, hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) mono tosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methyl ether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol) mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether, undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) mono allyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methyl ether, undeca(ethylene glycol) mono allyl ether, octadeca(ethylene glycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol), dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethylene glycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol) allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether, benzophenone-4-hexa(ethylene glycol) octenyl ether, benzophenone-4-hexa(ethylene glycol) decenyl ether, benzophenone-4-hexa(ethylene glycol) undecenyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and 4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether. Preferably, the polyethylene glycol has a molecular weight of approximately 1,000 to 20,000.

The present invention further provides a microarray having base cleavable succinate linkers comprising:

(a) a solid surface having known locations and reactive hydroxyl groups, wherein the known locations have a density greater than approximately 100 per square centimeter;

(b) a plurality of reactive amino amidite moieties bonded to the reactive hydroxyl groups on the solid surface, wherein the reactive amino moieties comprise an amine group and a hydroxyl bonding group, wherein the hydroxyl bonding group is bonded to the reactive hydroxyl groups at the known locations; and

(c) a plurality of reactive succinate moieties bonded to the amine groups, wherein the reactive succinate moieties comprise a sugar group bonded to the succinate group and to a base group bonded.

Preferably, the microarray further comprises (d) oligomers bonded onto the reactable hydroxyl groups. Preferably, the sugar group is ribose and the base group is selected from the group consisting of adenine, guanine, cytosine, and uracyl, or the sugar group is deoxyribose and the base group is selected from the group consisting of adenine, guanine, cytosine, and thymine.

Preferably, the oligomers are selected from the group consisting of DNA, RNA, and polypeptide, and combinations thereof.

Preferably, the solid surface has a plurality of electrodes, each at a known location, wherein the electrodes are electronically addressable. More preferably, the known locations are on the same surface as the plurality of electrodes, on an opposing surface to the electrodes, or on an overlayer over the electrodes. Optionally, the solid surface is glass and the reacted amino moieties are an amino silane coupling agent selected from the group consisting of aminopropyltrimethoxysilane, aminopropyltriethoxysilane, aminopropylmethyldiethoxysilane, aminopropylmethyldiethoxysilane hydrozylate, m-aminophenyltrimethoxysilane, phenylaminopropyltrimethoxysilane, 1,1,2,4-tetramethyl-1-sila-2-azacyclopentane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltriethoxysilane, aminoethylaminopropylmethyldimethoxysilane, aminoethylaminopropyltrimethoxysilane hydrolyzate, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane hydrolyzate, trimethoxysilylpropyldiethylenetriamine, vinylbenzylethylenediaminepropyltrimethoxysilane monohydrochloride, vinylbenzylethylenediaminepropyltrimethoxysilane, benzylethylenediaminepropyltrimethoxysilane monohydrochloride, benzylethylenediaminepropyltrimethoxysilane, and allylethylenediaminepropyltrimethoxysilane monohydrochloride and combinations thereof.

Preferably, the reacted amino moieties are made from an amino amidite selected from the group consisting of 3-(trifluoroacetylamino)propyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 12-(4-monomethoxytritylamino)dodecyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and 6-(trifluoroacetylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and combinations thereof.

Preferably, the reacted succinate moieties are selected from a salt of a chemical selected from the group consisting of 5′dimethoxytrityl-N-benzoyl-2′-deoxycytidine-3′-O-succinate, 5′ dimethyoxytrityl-N-isobutyryl-2′-deoxyguanosine-3′-O-succinate, 5′-dimethoxytrityl-thymidine-3′-O-succinate, and 5′-dimethoxytrityl-N-benzoyl-2′-deoxyadenosine-3′-O-succinate, and combinations thereof. Preferably, the salt is a pyridium salt of the succinate moieties.

Optionally, a spacer having reactive hydroxyl groups is bound to the reacted hydroxyl groups, wherein the amino amidite is bound to the reactive hydroxyl groups of the spacer. Preferably, the spacer is selected from the group consisting of DNA, RNA, polyethylene glycol, and polypeptides, and combinations thereof. Preferably, the spacer is from approximately 1 to 35 mers.

Preferably, the oligonucleotides are synthesized in situ using electrochemical synthesis. Optionally, the oligonucleotides are synthesized in situ by a method selected from the group consisting of (a) printing reagents via ink jet or other printing technology and using regular phosphoramidite chemistry, (b) maskless photo-generated acid controlled synthesis and using regular phosphoramidite chemistry, (c) mask-directed parallel synthesis using photo-cleavage of photolabile protecting groups, and (d) maskless parallel synthesis using photo-cleavage of photolabile protecting groups and digital photolithography.

Preferably, a porous reaction layer attached to the locations provides the reacted hydroxyl groups, wherein the porous reaction layer comprises a chemical species or mixture of chemical specie, wherein the chemical species is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, polyethylene glycol, polyethylene glycol derivative, N-hydroxysuccinimide, formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, and combinations thereof, wherein formula I is

formula II is

formula III is HOR4(OR5)mR7 formula IV is

formula V is

formula VI is

and formula VII is

wherein in each formula m is an integer from 1 to 4; R1, R2, R7, and R8 are independently selected from the group consisting of hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl, acyloxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino, tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato, isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato, selenocyanato, carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide, sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate, hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino, hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono, thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato, thiosemicarbazido, thiosulfino, thiosulfo, thioureido, triazano, triazeno, triazinyl, trithiosulfo, sulfinimidic acid, sulfonimidic acid, sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid, sulfonohydroximic acid, and phosphoric acid ester; R3 is selected from the group consisting of heteroatom group, carbonyl, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; R4 and R5 are independently selected from the group consisting of methylene, ethylene, propylene, butylene, pentylene, and hexylene; R6 forming a ring structure with two carbons of succinimide and is selected from the group consisting of substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; and R7 is selected from the group consisting of amino and hydroxyl.

Preferably, the monosaccharide is selected from the group consisting of allose, altrose, arabinose, deoxyribose, erythrose, fructose, galactose, glucose, gulose, idose, lyxose, mannose, psicose, L-rhamnose, ribose, ribulose, sedoheptulose, D-sorbitol, sorbose, sylulose, tagatose, talose, threose, xylulose, and xylose. Preferably, the disaccharide is selected from the group consisting of amylose, cellobiose, lactose, maltose, melibiose, palatinose, sucrose, and trehalose. Preferably, the trisaccharide is selected from the group consisting of raffinose and melezitose.

Preferably, the polyethylene glycol derivative is selected from the group consisting of diethylene glycol, tetraethylene glycol, polyethylene glycol having primary amino groups, 2-(2-aminoethoxy)ethanol, ethanol amine, di(ethylene glycol) mono allyl ether, di(ethylene glycol) mono tosylate, tri(ethylene glycol) mono allyl ether, tri(ethylene glycol) mono tosylate, tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol) mono trityl ether, tri(ethylene glycol) mono chloro mono methyl ether, tri(ethylene glycol) mono tosyl mono allyl ether, tri(ethylene glycol) mono allyl mono methyl ether, tetra(ethlyne glycol) mono allyl ether, tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) mono tosyl mono allyl ether, tetra(ethylene glycol) mono tosylate, tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) mono trityl ether, tetra(ethylene glycol) mono 1-hexenyl ether, tetra(ethylene glycol) mono 1-heptenyl ether, tetra(ethylene glycol) mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether, tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol) mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether, penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethylene glycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allyl ether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol) mono benzyl ether, hexa(ethylene glycol) mono trityl ether, hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono 1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether, hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono 1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono 1-undecenyl ether, hepta(ethylene glycol) mono allyl ether, hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) mono tosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methyl ether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol) mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether, undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) mono allyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methyl ether, undeca(ethylene glycol) mono allyl ether, octadeca(ethylene glycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol), dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethylene glycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol) allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether, benzophenone-4-hexa(ethylene glycol) octenyl ether, benzophenone-4-hexa(ethylene glycol) decenyl ether, benzophenone-4-hexa(ethylene glycol) undecenyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and 4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether. Preferably, the polyethylene glycol has a molecular weight of approximately 1,000 to 20,000.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an image of a portion of the microarray after exposure to the fluorescently labeled oligonucleotide. There are four different areas, A, B, C, and D, shown in the figure. In areas A, B, and C, oligonucleotides were synthesized with and without a cleavable linker. As can be seen in the figure, the microarray locations having the cleavable linker between the oligonucleotide and the microarray are dark, indicating little or no hybridizable oligonucleotide remained after cleaving. In contrast, those locations that did not have the cleavable linker between the oligonucleotide and the microarray are brighter, which indicates that the oligonucleotide remained on the microarray. In area D, some electrodes had cleavable linker while others did not; however, no oligonucleotides were synthesized so that the entire area appears dark.

FIGS. 2A and 2B are a schematics showing the construction of the microarray of the present invention.

FIG. 3 provides exemplary compounds used to construct the microarray of the present invention.

FIG. 4 provides an image of the results from gel electrophoresis of DNA strands that were amplified by PCR. The image shows recovery of the three different DNA strands from a microarray after cleaving the strands from a cleavable linker. The DNA strands were synthesized in situ using electrochemical synthesis on the cleavable linker attached to the microarray.

 DETAILED DESCRIPTION OF THE INVENTION Definitions

Generally, nomenclature for chemical groups as used herein follows the recommendations of “The International Union for Pure and Applied Chemistry”, Principles of Chemical Nomenclature: a Guide to IUPAC Recommendations, Leigh, G. J.; Favre, H. A. and Metanomski, W. V., Blackwell Science, 1998, the disclosure of which is incorporated by reference herein. Formation of substituted structures is limited by atom valence requirements.

“Oligomer” means a molecule of intermediate relative molecular mass, the structure of which essentially comprises a small plurality of units derived, actually or conceptually, from molecules of lower relative molecular mass. A molecule is regarded as having an intermediate relative molecular mass if it has properties which do vary significantly with the removal of one or a few of the units. If a part or the whole of the molecule has an intermediate relative molecular mass and essentially comprises a small plurality of units derived, actually or conceptually, from molecules of lower relative molecular mass, it may be described as oligomeric, or by oligomer used adjectivally. Oligomers are typically comprised of monomers. Moreover, a “mer” number is the number of monomer units in an oligomer.

The term “co-oligomer” means an oligomer derived from more than one species of monomer. The term oligomer includes co-oligomers. As examples of oligomers, a single stranded DNA molecule consisting of deoxyadenylate (A), deoxyguanylate (G), deoxycytidylate (C), and deoxythymidylate (T) units in the following sequence, AGCTGCTAT is a co-oligomer, and a single stranded DNA molecule consisting of 10-T units is an oligomer; however, both are referred to as oligomers.

The term “monomer” means a molecule that can undergo polymerization thereby contributing constitutional units to the essential structure of a macromolecule such as an oligomer, co-oligomer, polymer, or co-polymer. Examples of monomers for oligonucleotides include A, C, G, T, adenylate, guanylate, cytidylate, and uridylate. Monomers for other oligomers, including polypeptides, include amino acids, vinyl chloride, and other vinyls.

The term “polymer” means a substance composed of macromolecules, which is a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass. In many cases, especially for synthetic polymers, a molecule can be regarded as having a high relative molecular mass if the addition or removal of one or a few of the units has a negligible effect on the molecular properties. This statement fails in the case of certain macromolecules for which the properties may be critically dependent on fine details of the molecular structure. If a part or the whole of the molecule has a high relative molecular mass and essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass, it may be described as either macromolecular or polymeric, or by polymer used adjectivally.

The term “copolymer” means a polymer derived from more than one species of monomer. Copolymers that are obtained by copolymerization of two monomer species are sometimes termed bipolymers, those obtained from three monomers terpolymers, those obtained from four monomers quaterpolymers, etc. The term polymer includes co-polymers.

The term “polyethylene glycol” (PEG) means an organic chemical having a chain consisting of the common repeating ethylene glycol unit [—CH2—CH2—O—]n. PEG's are typically long chain organic polymers that are flexible, hydrophilic, enzymatically stable, and biologically inert, but they do not have an ionic charge in water. In general, PEG can be divided into two categories. First, there is polymeric PEG having a molecular weight ranging from 1000 to greater than 20,000. Second, there are PEG-like chains having a molecular weight that is less than 1000. Polymeric PEG has been used in bioconjugates, and numerous reviews have described the attachment of this linker moiety to various molecules. PEG has been used as a linker, where the short PEG-like linkers can be classified into two types, the homo-[X—(CH2—CH2—O)n]—X and heterobifunctional [X—(CH2—CH2—O)n]—Y spacers.

The term “PEG derivative” means an ethylene glycol derivative having the common repeating unit of PEG. Examples of PEG derivatives include, but are not limited to, diethylene glycol (DEG), tetraethylene glycol (TEG), polyethylene glycol having primary amino groups, di(ethylene glycol) mono allyl ether, di(ethylene glycol) mono tosylate, tri(ethylene glycol) mono allyl ether, tri(ethylene glycol) mono tosylate, tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol) mono trityl ether, tri(ethylene glycol) mono chloro mono methyl ether, tri(ethylene glycol) mono tosyl mono allyl ether, tri(ethylene glycol) mono allyl mono methyl ether, tetra(ethlyne glycol) mono allyl ether, tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) mono tosyl mono allyl ether, tetra(ethylene glycol) mono tosylate, tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) mono trityl ether, tetra(ethylene glycol) mono 1-hexenyl ether, tetra(ethylene glycol) mono 1-heptenyl ether, tetra(ethylene glycol) mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether, tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol) mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether, penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethylene glycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allyl ether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol) mono benzyl ether, hexa(ethylene glycol) mono trityl ether, hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono 1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether, hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono 1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono 1-undecenyl ether, hepta(ethylene glycol) mono allyl ether, hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) mono tosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methyl ether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol) mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether, undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) mono allyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methyl ether, undeca(ethylene glycol) mono allyl ether, octadeca(ethylene glycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol), dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethylene glycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol) allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether, benzophenone-4-hexa(ethylene glycol) octenyl ether, benzophenone-4-hexa(ethylene glycol) decenyl ether, benzophenone-4-hexa(ethylene glycol) undecenyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and 4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether.

The term “polyethylene glycol having primary amino groups” refers to polyethylene glycol having substituted primary amino groups in place of the hydroxyl groups. Substitution can be up to 98% in commercial products ranging in molecular weight from 5,000 to 20,000 Da.

The term “alkyl” means a straight or branched chain alkyl group containing up to approximately 20 but preferably up to 8 carbon atoms. Examples of alkyl groups include but are not limited to the following: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, isohexyl, n-hexyl, n-heptyl, and n-octyl. A substituted alkyl has one or more hydrogen atoms substituted by other groups or a carbon replaced by a divalent, trivalent, or tetravalent group or atom. Although alkyls by definition have a single radical, as used herein, alkyl includes groups that have more than one radical to meet valence requirements for substitution.

The term “alkenyl” means a straight or branched chain alkyl group having at least one carbon-carbon double bond, and containing up to approximately 20 but preferably up to 8 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-butenyl, 1,3-butadienyl, 2-pentenyl, 2,4-hexadienyl, 4-(ethyl)-1,3-hexadienyl, and 2-(methyl)-3-(propyl)-1,3-butadienyl. A substituted alkenyl has one or more hydrogen atoms substituted by other groups or a carbon replaced by a divalent, trivalent, or tetravalent group or atom. Although alkenyls by definition have a single radical, as used herein, alkenyl includes groups that have more than one radical to meet valence requirements for substitution.

The term “alkynyl” means a straight or branched chain alkyl group having a single radical, having at least one carbon-carbon triple bond, and containing up to approximately 20 but preferably up to 8 carbon atoms. Examples of alkynyl groups include, but are not limited to, the ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 4-pentynyl, 5-hexynyl, 6-heptynyl, 7-octynyl, 1-methyl-2-butynyl, 2-methyl-3-pentynyl, 4-ethyl-2-pentynyl, and 5,5-methyl-1,3-hexynyl. A substituted alkynyl has one or more hydrogen atoms substituted by other groups or a carbon replaced by a divalent, trivalent, or tetravalent group or atom. Although alkynyls by definition have a single radical, as used herein, alkynyl includes groups that have more than one radical to meet valence requirements for substitution.

The term “cycloalkyl” means an alkyl group forming at least one ring, wherein the ring has approximately 3 to 14 carbon atoms. Examples of cycloalkyl groups include but are not limited to the following: cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A substituted cycloalkyl has one or more hydrogen atoms substituted by other groups or a carbon replaced by a divalent, trivalent, or tetravalent group or atom. Although cycloalkyls by definition have a single radical, as used herein, cycloalkyl includes groups that have more than one radical to meet valence requirements for substitution.

The term “cycloalkenyl” means an alkenyl group forming at least one ring and having at least one carbon-carbon double bond within the ring, wherein the ring has approximately 3 to 14 carbon atoms. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, 1,3-cyclopentadienyl, and cyclohexenyl. A substituted cycloalkenyl has one or more hydrogens substituted by other groups or a carbon replaced by a divalent, trivalent, or tetravalent group or atom. Although cycloalkenyls by definition have a single radical, as used herein, cycloalkenyl includes groups that have more than one radical to meet valence requirements for substitution.

The term “cycloalkynyl” means an alkynyl group forming at least one ring and having at least one carbon-carbon triple bond, wherein the ring contains up to approximately 14 carbon atoms. A group forming a ring having at least one triple bond and having at least one double bond is a cycloalkynyl group. An example of a cycloalkynyl group includes, but is not limited to, cyclooctyne. A substituted cycloalkynyl has one or more hydrogen atoms substituted by other groups. Although cycloalkynyls by definition have a single radical, as used herein, cycloalkynyl includes groups that have more than one radical to meet valence requirements for substitution.

The term “aryl” means an aromatic carbon ring group having a single radical and having approximately 4 to 20 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthryl. A substituted aryl has one or more hydrogen atoms substituted by other groups. Although aryls by definition have a single radical, as used herein, aryl includes groups that have more than one radical to meet valence requirements for substitution. An aryl group can be a part of a fused ring structure such as N-hydroxysuccinimide bonded to phenyl (benzene) to form N-hydroxyphthalimide.

The term “hetero” when used in the context of chemical groups, or “heteroatom” means an atom other than carbon or hydrogen. Preferred examples of heteroatoms include oxygen, nitrogen, phosphorous, sulfur, boron, silicon, and selenium.

The term “heterocyclic ring” means a ring structure having at least one ring moiety having at least one heteroatom forming a part of the ring, wherein the heterocyclic ring has approximately 4 to 20 atoms connected to form the ring structure. An example of a heterocyclic ring having 6 atoms is pyridine with a single hereroatom. Additional examples of heterocyclic ring structures having a single radical include, but are not limited to, acridine, carbazole, chromene, imidazole, furan, indole, quinoline, and phosphinoline. Examples of heterocyclic ring structures include, but are not limited to, aziridine, 1,3-dithiolane, 1,3-diazetidine, and 1,4,2-oxazaphospholidine. Examples of heterocyclic ring structures having a single radical include, but are not limited to, fused aromatic and non-aromatic structures: 2H-furo[3,2-b]pyran, 5H-pyrido[2,3-d]-o-oxazine, 1H-pyrazolo[4,3-d]oxazole, 4H-imidazo[4,5-d]thiazole, selenazolo[5,4-f]benzothiazole, and cyclopenta[b]pyran. Heterocyclic rings can have one or more radicals to meet valence requirements for substitution.

The term “polycyclic” or “polycyclic group” means a carbon ring structure having more than one ring, wherein the polycyclic group has approximately 4 to 20 carbons forming the ring structure and has a single radical. Examples of polycyclic groups include, but are not limited to, bicyclo[1.1.0]butane, bicyclo[5.2.0]nonane, and tricycle[5.3.1.1]dodecane. Polycyclic groups can have one or more radicals to meet valence requirements for substitution.

The term “halo” or “halogen” means fluorine, chlorine, bromine, or iodine.

The term “heteroatom group” means one heteroatom or more than one heteroatoms bound together and having two free valences for forming a covalent bridge between two atoms. For example, the oxy radical, —O— can form a bridge between two methyls to form CH3—O—CH3 (dimethyl ether) or can form a bridge between two carbons to form an epoxy such as cis or trans 2,3-epoxybutane,

As used herein and in contrast to the normal usage, the term heteroatom group will be used to mean the replacement of groups in an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl and not the formation of cyclic bridges, such as an epoxy, unless the term cyclic bridge is used with the term heteroatom group to denote the normal usage.

Examples of heteroatom groups, using the nomenclature for hetero bridges (such as an epoxy bridge), include but are not limited to the following: azimino (—N═N—HN—), azo (—N═N—), biimino (—NH—NH—), epidioxy (—O—O—), epidithio (—S—S—), epithio (—S—) epithioximino (—S—O—NH—), epoxy (—O—), epoxyimino (—O—NH—), epoxynitrilo (—O—N═), epoxythio (—O—S—), epoxythioxy (—O—S—O—), furano (—C1H2O—), imino (—NH—), and nitrilo (—N═). Examples of heteroatom groups using the nomenclature for forming acyclic bridges include but are not limited to the following: epoxy (—O—), epithio (—S—), episeleno (—Se—), epidioxy (—O—O—), epidithio (—S—S—), lambda4-sulfano (—SH2—), epoxythio (—O—S—), epoxythioxy (—O—S—O—), epoxyimino (—O—NH—), epimino (—NH—), diazano (—NH—NH—), diazeno (—N═N—), triaz[1]eno (—N═N—NH—), phosphano (—PH—), stannano (—SnH2—), epoxymethano (—O—CH2—), epoxyethano (—O—CH2—CH2—), epoxyprop[1]eno

The term “bridge” means a connection between one part of a ring structure to another part of the ring structure by a hydrocarbon bridge. Examples of bridges include but are not limited to the following: methano, ethano, etheno, propano, butano, 2-buteno, and benzeno.

The term “hetero bridge” means a connection between one part of a ring structure to another part of the ring structure by one or more heteroatom groups, or a ring formed by a heterobridge connecting one part of a linear structure to another part of the linear structure, thus forming a ring.

The term “oxy” means the divalent radical —O—.

The term “oxo” means the divalent radical ═O.

The term “carbonyl” means the group

wherein the carbon has two radicals for bonding.

The term “amide” or “acylamino” means the group

wherein the nitrogen has one single radical for bonding and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “alkoxy” means the group —O—R, wherein the oxygen has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. Examples of alkoxy groups where the R is an alkyl include but are not limited to the following: methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy, 1,1-dimethylethoxy, 1,1-dimethylpropoxy, 1,1-dimethylbutoxy, 1,1-dimethylpentoxy, 1-ethyl-1-methylbutoxy, 2,2-dimethylpropoxy, 2,2-dimethylbutoxy, 1-methyl-1-ethylpropoxy, 1,1-diethylpropoxy, 1,1,2-trimethylpropoxy, 1,1,2-trimethylbutoxy, 1,1,2,2-tetramethylpropoxy. Examples of alkoxy groups where the R is an alkenyl group include but are not limited to the following: ethenyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy, 3-butenyloxy, 1-methyl-prop-2-enyloxy, 1,1-dimethyl-prop-2-enyloxy, 1,1,2-trimethyl-prop-2-enyloxy, and 1,1-dimethyl-but-2-enyloxy, 2-ethyl-1,3-dimethyl-but-1-enyloxy. Examples of alkyloxy groups where the R is an alkynyl include but are not limited to the following: ethynyloxy, 1-propynyloxy, 2-propynyloxy, 1-butynyloxy, 2-butynyloxy, 3-butynyloxy, 1-methyl-prop-2-ynyloxy, 1,1-dimethyl-prop-2-ynyloxy, and 1,1-dimethyl-but-2-ynyloxy, 3-ethyl-3-methyl-but-1-ynyloxy. Examples of alkoxy groups where the R is an aryl group include but are not limited to the following: phenoxy, 2-naphthyloxy, and 1-anthyloxy.

The term “acyl” means the group

wherein the carbon has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. Examples of acyl groups include but are not limited to the following: acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, acryloyl, propioloyl, mathacryloyl, crotonoyl, isocrotonoyl, benzoyl, and naphthoyl.

The term “acyloxy” means the group

wherein the oxygen has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. Examples of acyloxy groups include but are not limited to the following: acetoxy, ethylcarbonyloxy, 2-propenylcarbonyloxy, pentylcarbonyloxy, 1-hexynylcarbonyloxy, benzoyloxy, cyclohexylcarbonyloxy, 2-naphthoyloxy, 3-cyclodecenylcarbonyloxy.

The term “oxycarbonyl” means the group

wherein the carbon has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. Examples of oxycarbonyl groups include but are not limited to the following: methoxycarbonyl, ethoxycarbonyl, isopropyloxycarbonyl, phenoxycarbonyl, and cyclohexyloxycarbonyl.

The term “acyloxycarbonyl” means the group

wherein the carbon has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “alkoxycarbonyloxy” means the group

wherein the oxygen has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “carboxy” means the group —C(O)OH, wherein the carbon has a single radical.

The term “imino” or “nitrene” means the group ═N—R, wherein the nitrogen has two radicals and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “amino” means the group —NH2, where the nitrogen has a single radical.

The term “secondary amino” means the group —NH—R, wherein the nitrogen has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “tertiary amino” means the group

wherein the nitrogen has a single radical and R1 and R2 are independently selected from the group consisting of unsubstituted and substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group.

The term “hydrazi” means the group —NH—NH—, wherein the nitrogens have single radicals bound to the same atom. The term “hydrazo” means the group —NH—NH—, wherein the nitrogens have single radicals bound to the different atoms.

The term “hydrazino” means the group NH2—NH—, wherein the nitrogen has a single radical.

The term “hydrazono” means the group NH2—N═, wherein the nitrogen has two radicals.

The term “hydroxyimino” means the group HO—N═, wherein the nitrogen has two radicals.

The term “alkoxyimino” means the group R—O—N═, wherein the nitrogen has two radicals and R is an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “azido” means the group N3—, wherein the nitrogen has one radical.

The term “azoxy” means the group —N(O)═N—, wherein the nitrogens have one radical.

The term “alkazoxy” means the group R—N(O)═N—, wherein the nitrogen has one radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. Azoxybenzene is an example compound.

The term “cyano” means the group —CN. The term “isocyano” means the group —NC. The term “cyanato” means the group —OCN. The term “isocyanato” means the group —NCO. The term “fulminato” means the group —ONC. The term “thiocyanato” means the group —SCN. The term “isothiocyanato” means the group —NCS. The term “selenocyanato” means the group —SeCN. The term “isoselenocyanato” means the group —NCSe.

The term “carboxyamido” or “acylamino” means the group

wherein the nitrogen has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “acylimino” means the group

wherein the nitrogen has two radicals and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “nitroso” means the group O═N—, wherein the nitrogen has a single radical.

The term “aminooxy” means the group —O—NH2, wherein the oxygen has a single radical.

The term “carxoimidioy” means the group

wherein the carbon has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “hydrazonoyl” means the group

wherein the carbon has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “hydroximoyl” or “oxime” means the group

wherein the carbon has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “hydrazino” means the group

wherein the nitrogen has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “amidino” means the group

wherein the carbon has a single radical.

The term “sulfide” means the group —S—R, wherein the sulfur has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “thiol” means the group —S—, wherein the sulfur has two radicals. Hydrothiol means —SH.

The term “thioacyl” means the group —C(S)—R, wherein the carbon has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group.

The term “sulfoxide” means the group

wherein the sulfur has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. The term “thiosulfoxide” means the substitution of sulfur for oxygen in sulfoxide; the term includes substitution for an oxygen bound between the sulfur and the R group when the first carbon of the R group has been substituted by an oxy group and when the sulfoxide is bound to a sulfur atom on another group.

The term “sulfone” means the group

wherein the sulfur has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. The term “thiosulfone” means substitution of sulfur for oxygen in one or two locations in sulfone; the term includes substitution for an oxygen bound between the sulfur and the R group when the first carbon of the R group has been substituted by an oxy group and when the sulfone is bound to a sulfur atom on another group.

The term “sulfate” means the group

wherein the oxygen has a single radical and R is hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, or polycyclic group. The term “thiosulfate” means substitution of sulfur for oxygen in one, two, three, or four locations in sulfate.

The term “phosphoric acid ester” means the group R1R2PO4—, wherein the oxygen has a single radical and R1 is selected from the group consisting of hydrogen and unsubstituted and substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group, and R2 is selected from the group consisting of unsubstituted and substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group.

The term “substituted” or “substitution,” in the context of chemical species, means independently selected from the group consisting of (1) the replacement of a hydrogen on at least one carbon by a monovalent radical, (2) the replacement of two hydrogens on at least one carbon by a divalent radical, (3) the replacement of three hydrogens on at least one terminal carbon (methyl group) by a trivalent radical, (4) the replacement of at least one carbon and the associated hydrogens (e.g., methylene group) by a divalent, trivalent, or tetravalent radical, and (5) combinations thereof. Meeting valence requirements restricts substitution. Substitution occurs on alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic groups, providing substituted alkyl, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, substituted cycloalkynyl, substituted aryl group, substituted heterocyclic ring, and substituted polycyclic groups.

The groups that are substituted on an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic groups are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, polycyclic group, halo, heteroatom group, oxy, oxo, carbonyl, amide, alkoxy, acyl, acyloxy, oxycarbonyl, acyloxycarbonyl, alkoxycarbonyloxy, carboxy, imino, amino, secondary amino, tertiary amino, hydrazi, hydrazino, hydrazono, hydroxyimino, azido, azoxy, alkazoxy, cyano, isocyano, cyanato, isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato, selenocyanato, carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide, thiol, sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate, hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl, nitrilo, nitro, aci-nitro, nitroso, semicarbazono, oxamoyl, pentazolyl, seleno, thiooxi, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfinyl, sulfo, sulfoamino, sulfonato, sulfonyl, sulfonyldioxy, hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono, thiocarbonohydrazido, thiocarbonyl, thiocarboxy, thiocyanato, thioformyl, thioacyl, thiosemicarbazido, thiosulfino, thiosulfo, thioureido, thioxo, triazano, triazeno, triazinyl, trithio, trithiosulfo, sulfinimidic acid, sulfonimidic acid, sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid, sulfonohydroximic acid, and phosphoric acid ester, and combinations thereof.

As an example of a substitution, replacement of one hydrogen atom on ethane by a hydroxyl provides ethanol, and replacement of two hydrogens by an oxo on the middle carbon of propane provides acetone (dimethyl ketone.) As a further example, replacement the middle carbon (the methenyl group) of propane by the oxy radical (—O—) provides dimethyl ether (CH3—O−CH3.) As a further example, replacement of one hydrogen atom on benzene by a phenyl group provides biphenyl.

As provided above, heteroatom groups can be substituted inside an alkyl, alkenyl, or alkylnyl group for a methylene group (:CH2) thus forming a linear or branched substituted structure rather than a ring or can be substituted for a methylene inside of a cycloalkyl, cycloalkenyl, or cycloalkynyl ring thus forming a heterocyclic ring. As a further example, nitrilo (—N═) can be substituted on benzene for one of the carbons and associated hydrogen to provide pyridine, or and oxy radical can be substituted to provide pyran.

The term “unsubstituted” means that no hydrogen or carbon has been replaced on an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, or aryl group.

The term “linker” means a molecule having one end attached or capable of attaching to a solid surface and the other end having a reactive group that is attached or capable of attaching to a chemical species of interest such as a small molecule, an oligomer, or a polymer. A linker may already be bound to a solid surface and/or may already have a chemical species of interest bound to its reactive group. A linker may have a protective group attached to its reactive group, where the protective group is chemically or electrochemically removable. A linker may comprise more than one molecule, where the molecules are covalently joined in situ to form the linker having the desired reactive group projecting away from a solid surface.

The term “spacer” or “linker moiety” means a molecule having one end attached or capable of attaching to the reactive group of a linker or porous reaction layer and the other end having a reactive group that is attached or capable of attaching to a chemical species of interest such as a small molecule, an oligomer, or a polymer. A spacer may already be bound to a linker or a porous reaction layer and/or may already have a chemical species of interest bound to its reactive group. A spacer may have a protective group attached to its reactive group, where the protective group is chemically or electrochemically removable. A spacer may be formed in situ on a linker or porous reaction layer. A spacer may be formed and then attached to a linker already attached to a solid surface or attached to a porous reaction layer on the solid suface. A spacer may be externally synthesized on a chemical species of interest followed by attachment to a linker already attached to a solid surface or attached to a porous reaction layer on the solid suface. A chemical species of interest may be attached to a spacer that is attached to a linker where the entire structure is then attached to a solid surface at a reactive sight on the solid surface. The purpose of a spacer is to extend the distance between a molecule of interest and a solid surface.

The term “combination linker and spacer” means a linker having both the properties of a linker and a spacer. A combination linker and spacer may be synthesized in situ or synthesized externally and attached to a solid surface.

The term “coating” means a thin layer of material that is chemically and/or physically bound to a solid surface. A coating may be attached to a solid surface by mechanical interlocking as well as by van der Waals forces (dispersion forces and dipole forces), electron donor-acceptor interactions, metallic coordination/complexation, covalent bonding, or a combination of the aforementioned. A coating can provide a reactive group for direct attachment of a chemical species of interest, attachment of a linker, or attachment of a combination linker and spacer. A coating can be polymerized and/or cross-linked in situ.

The term “reactive” or “reaction” as used in reactive or reaction coating or reactive or reaction layer means that there is a chemical species or bound group within the layer that is capable of forming a covalent bond for attachment of a linker, spacer, or other chemical species to the layer or coating.

The term “porous” as used in porous reactive layer or coating means that there are non-uniformities within the layer or coating to allow molecular species to diffuse into and through the layer or coating.

The term “adsorption” or “adsorbed” means a chemical attachment by van der Waals forces (dispersion forces and dipole forces), electron donor-acceptor interactions, or metallic coordination/complexation, or a combination of the aforementioned forces. After adsorption, a species may covalently bind to a surface, depending on the surface, the species, and the environmental conditions.

The term “microarray” refers to, in general, planer surface having specific spots that are usually arranged in a column and row format, wherein each spot can be used for some type of chemical or biochemical analysis, synthesis, or method. The spots on a microarray are typically smaller than 100 micrometers. The term “electrode microarray” refers to a microarray of electrodes, wherein the electrodes are the specific spots on the microarray.

The term “synthesis quality” refers to, in general, the average degree of similarity between a desired or designed chemical or biochemical species and the species actually synthesized. The term can refer to other issues in a synthesis such as the effect of a layer or coating on the synthesis quality achieved.

The term “solvation” means a chemical process in which solvent molecules and molecules or ions of a solute combine to form a compound, wherein the compound is generally a loosely bound complex held together by van der Waals forces (dispersion forces and dipole forces), acid-base interactions (electron donor acceptor interactions), ionic interaction, or metal complex interactions but not covalent bonds. In water, the pH of the water can affect solvation of dissociable species such as acids and bases. In addition, the concentration of salts as well as the charge on salts can affect solvation.

The term “agarose” means any commercially available agarose. Agarose is a polysaccharide biopolymer and is usually obtained from seaweed. Agarose has a relatively large number of hydroxyl groups, which provide for high water solubility. Agarose is available commercially in a wide ranger of molecular weights and properties.

The term “controlled pore glass” means any commercially available controlled pore glass material suitable for coating purposes. In general, controlled pore glass (CPG) is an inorganic glass material having a high surface area owing to a large amount of void space.

The term “monosaccharide” means one sugar molecule unlinked to any other sugars. Examples of monosaccharides include allose, altrose, arabinose, deoxyribose, erythrose, fructose (D-Levulose), galactose, glucose, gulose, idose, lyxose, mannose, psicose, ribose, ribulose, sedoheptulose, D-sorbitol, sorbose, sylulose, L-rhamnose (6-Deoxy-L-mannose), tagatose, talose, threose, xylulose, and xylose.

The term “disaccharide” means two sugars linked together to form one molecule. Examples of disaccharides include amylose, cellobiose (4-β-D-glucopyranosyl-D-glucopyranose), lactose, maltose (4-O-α-D-glucopyranosyl-D-glucose), melibiose (6-O-α-D-Galactopyranosyl-D-glucose), palatinose (6-O-α-D-Glucopyranosyl-D-fructose), sucrose, and trehalose (a-D-Glucopyranosyl-α-D-glucopyranoside).

The term “trisaccharide” means three sugars linked together to form one molecule. Examples of a trisaccharides include raffinose (6-O-α-D-Galactopyranosyl-D-glucopyranosyl-β-D-fructofuranoside) and melezitose (O-α-D-glucopyranosyl-(1→3)-β-D-fructofuranosyl-α-D-glucopyranoside).

The term “polysaccharide” means more than three sugars linked together to form one molecule, but more accurately means a sugar-based polymer or oligomer. Examples of polysaccharides include inulin, dextran (polymer composed of glucose subunits), starches, and cellulose.

The present invention provides a microarray having a base-labile cleavable succinate linker. The cleavable link optionally has oligomers attached thereto by in situ synthesis. Preferably, the oligomers are oligonucleotides attached to cleavable linkers. Other moieties may be attached to the cleavable linkers. FIGS. 2A and 2B provide a schematic of the construction of such a microarray. The microarray has a solid surface with known locations that have hydroxyl groups. The hydroxyl groups are shown in FIG. 2A in the first step as not reacted; however, the second step shows the hydroxyl groups reacted. The density of the known locations is greater than approximately 100 locations per square centimeter. Density of the known locations can be approximately 1,000 to 1,000,000 locations per square centimeter. Only one known location with one hydroxyl is shown. Amino moieties are attached to the hydroxyl groups. Preferably, the attachment is through a phosphorous-oxygen bond between the phosphorous of amino amidite moieties and the oxygen of the hydroxyl groups as shown in the second step of FIG. 2A. Generally, the hydroxyl groups are referred to as reacted hydroxyl groups after attachment of the amino moieties. The amino moieties have an amine group and a hydroxyl reactive group. The hydroxyl reactive group bonds to the hydroxyl groups at the known locations.

The succinate moieties are attached to the amino moieities through amide bonds as shown in the last step in FIG. 2A. Prior to attachment of the succinate, the microarray is capped to cap unreacted hydroxyl groups followed by deprotection to remove the protecting group on the amine. The protecting group is preferably monomethoxytrityl (MMT) although, generally, any acid-labile protecting group will work such as those disclosed in Montgomery I, II, or III, including dimethoxytrityl (DMT). The resulting structure forms cleavable linkers attached to the microarray. The cleaving point is shown in FIG. 2B. Oligomers are attached to the cleavable linkers as shown in FIG. 2B. If the oligomers are DNA or RNA and cleaved from the microarray, the resulting oligonucleotide has a 3′ hydroxyl. FIG. 2B provides an example structure on a microarray. The succinate moieties have a succinate group bonded to a sugar group and a base bonded to the sugar group. Preferably, the sugar group is ribose and the base group is selected from the group consisting of adenine, guanine, cytosine, and uracyl, or the sugar group is deoxyribose and the base group is selected from the group consisting of adenine, guanine, cytosine, and thymine.

Preferably, the oligomers are selected from the group consisting of DNA, RNA, and polypeptide, and combinations thereof, whereby the oligomers comprising DNA and RNA have a 3′ hydroxyl after cleaving from the solid surface. Preferably, the cleaving base is selected from the group consisting of ammonium hydroxide, electrochemically generated base, sodium hydroxide, potassium hydroxide, methylamine, and ethylamine and combinations thereof.

Preferably, the solid surface has electrodes such as on an electrode microarray. An example of an electrode microarray is a CombiMatrix CustomArray™ 12K, which has over 12,000 electrodes and an electrode density of approximately 17,778 electrodes per square centimeter.

Preferably, the known locations are associated with the electrodes by being on the same surface as the electrodes, on an opposing surface to the electrodes, or on an overlayer over the electrodes. Preferably, the electrodes are electronically addressable such as through a computer control system having software to control the electrodes. Optionally, the solid surface is glass such as a glass slide that has been treated with an amino silane coupling agent to allow attachment by in situ synthesis of the structure as shown in FIGS. 2A and 2B. Preferably, the amino silane coupling agent selected from the group consisting of aminopropyltrimethoxysilane, aminopropyltriethoxysilane, aminopropylmethyldiethoxysilane, aminopropylmethyldiethoxysilane hydrozylate, m-aminophenyltrimethoxysilane, phenylaminopropyltrimethoxysilane, 1,1,2,4-tetramethyl-1-sila-2-azacyclopentane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltriethoxysilane, aminoethylaminopropylmethyldimethoxysilane, aminoethylaminopropyltrimethoxysilane hydrolyzate, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane hydrolyzate, trimethoxysilylpropyldiethylenetriamine, vinylbenzylethylenediaminepropyltrimethoxysilane monohydrochloride, vinylbenzylethylenediaminepropyltrimethoxysilane, benzylethylenediaminepropyltrimethoxysilane monohydrochloride, benzylethylenediaminepropyltrimethoxysilane, and allylethylenediaminepropyltrimethoxysilane monohydrochloride and combinations thereof.

Preferably, the amino amidite moieties are made from an amino amidite selected from the group consisting of 3-(trifluoroacetylamino)propyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 12-(4-monomethoxytritylamino)dodecyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and 6-(trifluoroacetylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and combinations thereof. The amine group on the amino amidite moiety is protected by a protecting group. Generally, amino amidite moieties bonded to the surface are referred to as reacted amino amidite moieties. Such protection groups must be removed before a succinate moiety can be reacted to form an amide linkage between the amino amidite and the succinate moiety. Preferably, the protecting groups are removed on an electrode microarray by the generation of acidic protons at the locations associated with an activated electrode. Alternatively, acidic solution may be used. Alternatively, photolabile protecting groups on the amine may be used such as those disclose in Fodor (cited previously).

Preferably, the succinate moieties are selected from a salt of a chemical selected from the group consisting of 5′dimethoxytrityl-N-benzoyl-2′-deoxycytidine-3′-O-succinate, 5′dimethyoxytrityl-N-isobutyryl-2′-deoxyguanosine-3′-O-succinate, 5′-dimethoxytrityl-thymidine-3′-O-succinate, and 5′-dimethoxytrityl-N-benzoyl-2′-deoxyadenosine-3′-O-succinate, and combinations thereof. Generally, succinate moieties reacted to the amino amidite moieties are referred to as reacted succinate moieties. Preferably, the salt is a pyridinium salt as shown in FIG. 3, Compound B. Other salts of the succinate moieties may be used such as triethyl ammonium salt (Pierce Chemical Company), lutidine salt, or imidizole salt and salts having the form HN(R1R2R3)+, wherein R1, R2, and R3 are alkyl groups. HBTU/HOBT activation of the succinate moiety is the preferred embodiment. Other procedures to activate the succinate can be used and include use of a carbodiimide such as N,N′-dicyclohexyl carbodiimide (DCC) or diisopropylcarbodiimide (DIC) both with or without N-hydrooxybenzotriazole (HOBt) or by forming a symmetrical anhydride. Use of other peptide coupling reagents such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), O—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophasphate (BOP), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), or 1,1′-carbonyl-diimidazole (CDI).

Optionally, a spacer having reactive hydroxyl groups is bound to the hydroxyl groups on the solid surface, wherein the amino amidite moieties are bound to the reactive hydroxyl groups of the spacers. The reactive hydroxyl groups are at the opposite end to the spacer end bound to the solid surface. Preferably, the spacer is selected from the group consisting of DNA, RNA, polyethylene glycol, and polypeptides, and combinations thereof. Preferably, the spacer is from approximately 1 to 35 mers.

Preferably, the oligomers are synthesized in situ using electrochemical synthesis. Electrochemical synthesis of DNA uses standard phosphoramidite chemistry coupled with electrochemical deblocking of the protecting groups on the synthesized DNA for the addition of each nucleotide contained in the oligonucleotide. For attachment of the phosphoramidites, the microarray has hydroxyl groups that allow attachment of the first phosphoramidite. Electrochemical deblocking involves turning on an electrode to generate acidic conditions that are sufficient to remove the protecting group only at the active electrode. Buffer in the solution used for deblocking and natural diffusion prevents deblocking at non-activated electrodes. Removal of the protecting groups allows addition of the next phosphoramidite.

Optionally, the oligomers are synthesized in situ by a method selected from the group consisting of (a) printing reagents via ink jet or other printing technology and using regular phosphoramidite chemistry, (b) maskless photo-generated acid controlled synthesis and using regular phosphoramidite chemistry, (c) mask-directed parallel synthesis using photo-cleavage of photolabile protecting groups, and (d) maskless parallel synthesis using photo-cleavage of photolabile protecting groups and digital photolithography.

Preferably, a porous reaction layer attached to the locations provides the hydroxyl groups on the solid surface, wherein the porous reaction layer comprises a chemical species or mixture of chemical specie, wherein the chemical species is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, polyethylene glycol, polyethylene glycol derivative, N-hydroxysuccinimide, formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, and combinations thereof, wherein formula I is

formula II is

formula III is HOR4(OR5)mR7 formula IV is

formula V is

formula VI is

and formula VII is

wherein in each formula III is an integer from 1 to 4; R1, R2, R7, and R8 are independently selected from the group consisting of hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl, acyloxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino, tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato, isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato, selenocyanato, carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide, sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate, hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino, hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono, thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato, thiosemicarbazido, thiosulfino, thiosulfo, thioureido, triazano, triazeno, triazinyl, trithiosulfo, sulfinimidic acid, sulfonimidic acid, sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid, sulfonohydroximic acid, and phosphoric acid ester; R3 is selected from the group consisting of heteroatom group, carbonyl, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; R4 and R5 are independently selected from the group consisting of methylene, ethylene, propylene, butylene, pentylene, and hexylene; R6 forming a ring structure with two carbons of succinimide and is selected from the group consisting of substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; and R7 is selected from the group consisting of amino and hydroxyl.

Preferably, the monosaccharide is selected from the group consisting of allose, altrose, arabinose, deoxyribose, erythrose, fructose, galactose, glucose, gulose, idose, lyxose, mannose, psicose, L-rhamnose, ribose, ribulose, sedoheptulose, D-sorbitol, sorbose, sylulose, tagatose, talose, threose, xylulose, and xylose. Preferably, the disaccharide is selected from the group consisting of amylose, cellobiose, lactose, maltose, melibiose, palatinose, sucrose, and trehalose Preferably, the triaccharide is selected from the group consisting of raffinose and melezitose. Most preferably, the porous reaction layer is sucrose.

Preferably, the polyethylene glycol derivative is selected from the group consisting of diethylene glycol, tetraethylene glycol, polyethylene glycol having primary amino groups, 2-(2-aminoethoxy)ethanol, ethanol amine, di(ethylene glycol) mono allyl ether, di(ethylene glycol) mono tosylate, tri(ethylene glycol) mono allyl ether, tri(ethylene glycol) mono tosylate, tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol) mono trityl ether, tri(ethylene glycol) mono chloro mono methyl ether, tri(ethylene glycol) mono tosyl mono allyl ether, tri(ethylene glycol) mono allyl mono methyl ether, tetra(ethlyne glycol) mono allyl ether, tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) mono tosyl mono allyl ether, tetra(ethylene glycol) mono tosylate, tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) mono trityl ether, tetra(ethylene glycol) mono 1-hexenyl ether, tetra(ethylene glycol) mono 1-heptenyl ether, tetra(ethylene glycol) mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether, tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol) mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether, penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethylene glycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allyl ether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol) mono benzyl ether, hexa(ethylene glycol) mono trityl ether, hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono 1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether, hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono 1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono 1-undecenyl ether, hepta(ethylene glycol) mono allyl ether, hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) mono tosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methyl ether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol) mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether, undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) mono allyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methyl ether, undeca(ethylene glycol) mono allyl ether, octadeca(ethylene glycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol), dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethylene glycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol) allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether, benzophenone-4-hexa(ethylene glycol) octenyl ether, benzophenone-4-hexa(ethylene glycol) decenyl ether, benzophenone-4-hexa(ethylene glycol) undecenyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and 4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether. Preferably, the polyethylene glycol has a molecular weight of approximately 1,000 to 20,000.

The siRNA oligonucleotides disclosed herein are complementary to their respective viral mRNA sequences. Without being bound by theory, the inventive complementary siRNA sequences can hybridize, under physiological conditions, to cause inhibition of viral replication within an infected host cell. Since the inventive composition and pharmaceutical comprise a pool of different siRNA compounds that bind to a plurality of different viral mRNAs, the present invention achieves its effect combating viral resistance to treatment. Thus, the present invention includes methods of treating virally infected individuals by administering a pool of siRNA sequences that can cause inhibition of viral replication in infected host cells.

Small inhibitory RNA oligonucleotides of the invention can be supplied to a target cell either exogenously as RNA, or endogenously, by supplying a DNA sequence from which the desired small inhibitory RNA oligonucleotide may be expressed by the target cell. In the latter case, the DNA to be expressed may be supplied to the target cell, as a recombinant nucleic acid (for example, a DNA molecule) containing a sequence complementary to the viral RNA (siRNA), which in turn is substantially complementary to viral proteins and the mRNA sequences encoding them. Therefore, expression of the different plurality of siRNA's is capable of inhibiting viral replication in a cell host. The invention also provides a composition of matter consisting essentially of at least one small inhibitory RNA oligonucleotide substantially complementary to a viral mRNA sequence.

The methods of the present invention can be utilized to prevent viral infection as well as to combat viral infections. These may be administered to prevent a virus infection or to combat the virus once it has entered the host. The siRNAs are contemplated to be used in an admixture or in chemical combination with one or more other materials, including other “antisense” oligonucleotides and other small inhibitory RNA to viral RNA, materials that increase the biological stability of the oligonucleotides, or materials that increase their ability to selectively penetrate their cultured cell line target cells and reach and hybridize with their target RNA. Furthermore, the term “oligonucleotide” includes derivatives thereof, such as backbone modifications, e.g., phosphorothioate derivatives, employed to stabilize the oligonucleotides. All such modifications are contemplated equivalents of the small inhibitory RNA oligonucleotides of the invention. For example, the small inhibitory RNA oligonucleotides may be provided in stabilized form, e.g., with phosphotriester linkages, or by blocking groups to prevent exonuclease attack (Anticancer Research 10:1169-1182, 1990). For small inhibitory RNA oligonucleotides supplied exogenously, increased selectivity for cultured cell lines may be achieved by linking small inhibitory RNA oligonucleotides of the invention to natural ligands or to synthetic ligands that will bind to the cell surface receptor.

In general, a high efficiency cell specific delivery system for in vivo therapeutic use may utilize a number of approaches, including the following: (i) specific delivery through a cultured cell line-specific receptor, (ii) delivery of small inhibitory RNA oligodeoxynucleotides in liposomes with or without specific targeting with monoclonal antibodies directed against specific cell surface receptors; (iii) retroviral-mediated transfer of DNA expressing the small inhibitory RNA construct of interest; (iv) direct targeting to cells of oligonucleotides via conjugation to monoclonal antibodies that are specific for cell surface receptors that function in a receptor-mediated endocytotic process; and (v) specific delivery to cultured cell lines via a replication-defective viral vector.

The pools or a plurality of different small inhibitory RNA compositions of the invention may be administered as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. The dosage administered will vary depending upon known pharmacokinetic/pharmacodynamic characteristics of the particular agent, and its mode and route of administration, as well as the age, weight, and health (including renal and hepatic function) of the recipient; the nature and extent of disease; kind of concurrent therapy; frequency and duration of treatment; and the effect desired. Usually a daily dose of active ingredient can be about 0.1 to 100 mg per kilogram of body weight. Ordinarily 0.5 to 50 and preferably 1 to 10 mg per kg of body weight per day given in divided doses or in sustained release form (including sustained intravenous infusion) will be effective to achieve the desired effects. Dosage forms suitable for internal administration generally contain about 1 milligram to about 500 milligrams of active ingredient per unit. The active ingredient will ordinarily be present in an amount of about 0.5% to 95% by weight of the total pharmaceutical preparation. The small inhibitory RNA oligonucleotide compositions of the invention may be administered parenterally (e.g., intravenously, preferably by intravenous infusion). For parenteral administration, the compositions will be formulated as a sterile, non-pyrogenic solution, suspension, or emulsion. The preparations may be supplied as a liquid formulation or lyophilized powder to be diluted with a pharmaceutically acceptable sterile, non-pyrogenic parenteral vehicle of suitable tonicity, e.g., water for injection, normal saline, or a suitable sugar-containing vehicle, e.g., D5W, D5/0.45, D5/0.2, or a vehicle containing mannitol, dextrose, or lactose. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field, or the USP/NF.

EXAMPLE 1 Cleavable Linker Using Amino Amidite and T-Succinate

In this example, a CombiMatrix CustomArray™ 12K microarray was used to synthesize oligonucleotides attached to the microarray through a base-cleavable linker. The microarray had approximately 12,000 platinum electrodes on a solid surface having a porous reaction layer, wherein each electrode was electronically addressable via computer control. The oligonucleotides were DNA and were synthesized in situ using electrochemical synthesis at locations associated with the electrodes on the microarray. Electrochemical synthesis used standard phosphoramidite chemistry coupled with electrochemical deblocking of the protecting groups on the synthesized DNA for the addition of each nucleotide contained in the oligonucleotide. For attachment of the phosphoramidites, the microarray had organic reactive hydroxyl groups that allowed attachment of the first phosphoramidite. Electrochemical deblocking involved turning on an electrode to generate acidic conditions at the electrode that were sufficient to remove the protecting group only at the active electrode. Buffer in the solution used for deblocking and natural diffusion prevented deblocking at non-activated electrodes. Removal of the protecting group allowed addition of the next phosphoramidite.

Some electrodes were used as controls while some electrodes were used to synthesize the oligonucleotides. At the non-control locations, a 15-unit deoxythymidylate spacer was synthesized on the reactive hydroxyl groups. At some but not all non-control locations, an amine amidite obtained from Glen Research was attached to the 15-unit spacer. The specific amine amidite was 2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, catalog number 10-1905-xx (5′-Amino-Modifier 5.) The amine amidite had monomethoxytrityl (MMT) protecting groups on the amine. The MMT protecting groups were removed using electrochemical generation of acid by activating selected electrodes.

After removal of the MMT protecting groups, the amine was reacted to a T-succinate to form an amide linkage between the amine groups and the succinate. The specific T-succinate used was 5′-dimethyloxytrityl-thymidine-3′-O-succinate (pyridium salt) obtained from Transgenomic. (Alternatively, A, C, G, succinates could have been used.) The solution to attach the T-succinate to the amine was made by adding 330 milligrams of T-succinate, 150 milligrams of O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), and 60 milligrams of N-hydroxybenztriazole (HOBT) to one milliliter dimethyl formamide (DMF). To this solution, 225 microliters of diisopropylethylamine (DIEPA) was added and the resulting mixture was vortexed to dissolve the material (total mixing time 5-10 min) prior to use.

To attach the T-succinate, the microarray was placed in a manifold, rinsed with anhydrous DMF, and exposed to one-half of the T-succinate coupling mixture for one hour at room temperature. The microarray was washed in the manifold using different solvents successively as follows: 5 milliliters of DMF, 5 milliliters of methylene chloride, and 5 milliliter of DMF. The microarray was exposed to the second half of the coupling mixture for one hour at room temperature. After the completion of the second exposure to the T-succinate reaction mixture, the microarray was washed again using the same washing as above followed by methylene chloride (5 ml) and a stream of ethanol from a squirt bottle. After washing, the microarray was ready for electrochemical synthesis. Synthesis was done on a CombiMatrix bench top synthesizer, wherein oligonucleotides of three different lengths (37, 42, and 47 bp) were synthesized.

After the completion of electrochemical synthesis, the synthetic oligonucleotides on the microarray were deprotected and cleaved by exposure to concentrated ammonium hydroxide in a pressurized cell at 65° C. The concentration of ammonium hydroxide was 28-30%. During this deprotection step, the cleavable succinate linkage was cleaved thus releasing the synthetic oligonucleotides. The oligonucleotides were isolated by evaporating the ammonia solution and were subjected to amplification using polymerase chain reaction (PCR). The oligonucleotides could be amplified with one set of PCR primers due to the presence of primer amplification sites at the ends of the oligonucleotides. The oligonucleotides were dissolved in 75 microliters of Tris buffer at 95° C. for 5 minutes.

To test if the nucleotides were released, a series of PCR reactions were performed to determine if the oligonucleotides were present in solution. PCR reaction products were run on a non-denaturing polyacrylamide gel (20%) for 100 minutes at 200 volts. When the separation of the PCR products was complete, the gel was stained with SYBR green II dye to visualize the PCR product as shown in FIG. 4. Separation of the PCR product by gel electrophoresis revealed that all three products (37, 42, and 47 bp) were present in approximately equal amounts and ran at the calculated molecular weight. The novel linker allowed for the release of oligonucleotides from the microarray surface.

At some locations on the array, the oligonucleotides synthesized were attached without the cleavable linker. Thus, oligonucleotides attached without the cleavable linker would be expected to remain on the microarray after the ammonium hydroxide reaction. To determine whether there were oligonucleotides remaining on the microarray after the ammonium hydroxide reaction, the microarray was exposed to complementary oligonucleotides having a fluorescent label. FIG. 1 shows an image of a portion of the microarray after exposure to the fluorescently labeled oligonucleotide. There are four different areas, A, B, C, and D, shown in the figure. In areas A, B, and C, oligos were synthesized with and without the cleavable linker. As can be seen in the figure, the microarray locations having the cleavable linker between the oligonucleotide and the microarray are completely dark or are mostly dark indicating little or no DNA remains after cleaving. In contrast, those locations that did not have the cleavable linker between the oligonucleotide and the microarray are brighter, which indicates that the oligonucleotide remained on the microarray. In area D, some electrodes had cleavable linker while some did not; however, no oligo was synthesized so that the entire area appears dark.

Claims

1. A process for manufacturing a pool of oligonucleotides using a microarray having base cleavable succinate linkers, the process comprising:

(a) providing a surface having known locations with hydroxyl groups, wherein the amount of the known locations is greater than approximately 100 per square centimeter;
(b) bonding amino moieties to the hydroxyl groups, wherein the amino moieties have an amine group and a hydroxyl bonding group, wherein the hydroxyl bonding group bonds to the hydroxyl groups of the known locations;
(c) bonding succinate moieties to the amine groups through amide bonds to form cleavable linkers attached to the solid surface, wherein the succinate moieties have a succinate group bonded to a sugar group and a base group bonded to the sugar group, wherein the cleavable linkers have a base-labile cleaving site on the succinate group and a reactable hydroxyl group on the sugar group;
(d) synthesizing oligonucleotides onto the reactable hydroxyl groups; and
(e) cleaving at the base-labile cleaving site the oligomers from the solid surface using a cleaving base, whereby the pool of oligonucleotides are recoverable.

2. The process of claim 1, wherein the sugar group is ribose and the base group is selected from the group consisting of adenine, guanine, cytosine, and uracyl, or the sugar group is deoxyribose and the base group is selected from the group consisting of adenine, guanine, cytosine, and thymine.

3. The process of claim 1, wherein the oligonucleotides are selected from the group consisting of single stranded DNA and RNA and combinations thereof.

4. The process of claim 1, wherein the cleaving base is selected from the group consisting of ammonium hydroxide, electrochemically generated base, sodium hydroxide, potassium hydroxide, methylamine, and ethylamine and combinations thereof, whereby the oligonucleotides comprising DNA and RNA have a 3′ hydroxyl after cleaving from the surface.

5. The process of claim 1, wherein the surface has electrodes and each of the known locations are associated with one of the electrodes, wherein the electrodes are electronically addressable.

6. The process of claim 5, wherein the known locations are on the same surface as the electrodes, on an opposing surface to the electrodes, or on an overlayer over the electrodes.

7. The process of claim 5, wherein a porous reaction layer attached to the known locations provides the hydroxyl groups, wherein the porous reaction layer comprises a chemical species or mixture of chemical specie, wherein the chemical species is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, polyethylene glycol, polyethylene glycol derivative, N-hydroxysuccinimide, formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, and combinations thereof, wherein formula I is formula II is formula III is HOR4(OR5)mR7, formula IV is formula V is formula VI is and formula VII is wherein in each formula m is an integer from 1 to 4; R1, R2, R7, and R8 are independently selected from the group consisting of hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl, acyloxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino, tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato, isocyanato, thiocyanato, fulminato, isothiocyanato, isoselenocyanato, selenocyanato, carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide, sulfoxide, thiosulfoxide, sulfone, thiosulfone, sulfate, thiosulfate, hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino, hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono, thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato, thiosemicarbazido, thiosulfino, thiosulfo, thioureido, triazano, triazeno, triazinyl, trithiosulfo, sulfinimidic acid, sulfonimidic acid, sulfinohydrazonic acid, sulfonohydrazonic acid, sulfinohydroximic acid, sulfonohydroximic acid, and phosphoric acid ester; R3 is selected from the group consisting of heteroatom group, carbonyl, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; R4 and R5 are independently selected from the group consisting of methylene, ethylene, propylene, butylene, pentylene, and hexylene; R6 forming a ring structure with two carbons of succinimide and is selected from the group consisting of substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group; and R7 is selected from the group consisting of amino and hydroxyl.

8. The process of claim 7, wherein the monosaccharide is selected from the group consisting of allose, altrose, arabinose, deoxyribose, erythrose, fructose, galactose, glucose, gulose, idose, lyxose, mannose, psicose, L-rhamnose, ribose, ribulose, sedoheptulose, D-sorbitol, sorbose, sylulose, tagatose, talose, threose, xylulose, and xylose.

9. The process of claim 7, wherein the disaccharide is selected from the group consisting of amylose, cellobiose, lactose, maltose, melibiose, palatinose, sucrose, and trehalose

10. The process of claim 7, wherein the triaccharide is selected from the group consisting of raffinose and melezitose.

11. The process of claim 7, wherein the polyethylene glycol derivative is selected from the group consisting of diethylene glycol, tetraethylene glycol, polyethylene glycol having primary amino groups, 2-(2-aminoethoxy)ethanol, ethanol amine, di(ethylene glycol) mono allyl ether, di(ethylene glycol) mono tosylate, tri(ethylene glycol) mono allyl ether, tri(ethylene glycol) mono tosylate, tri(ethylene glycol) mono benzyl ether, tri(ethylene glycol) mono trityl ether, tri(ethylene glycol) mono chloro mono methyl ether, tri(ethylene glycol) mono tosyl mono allyl ether, tri(ethylene glycol) mono allyl mono methyl ether, tetra(ethlyne glycol) mono allyl ether, tetra(ethylene glycol) mono methyl ether, tetra(ethylene glycol) mono tosyl mono allyl ether, tetra(ethylene glycol) mono tosylate, tetra(ethylene glycol) mono benzyl ether, tetra(ethylene glycol) mono trityl ether, tetra(ethylene glycol) mono 1-hexenyl ether, tetra(ethylene glycol) mono 1-heptenyl ether, tetra(ethylene glycol) mono 1-octenyl ether, tetra(ethylene glycol) mono 1-decenyl ether, tetra(ethylene glycol) mono 1-undecenyl ether, penta(ethylene glycol) mono methyl ether, penta(ethylene glycol) mono allyl mono methyl ether, penta(ethylene glycol) mono tosyl mono methyl ether, penta(ethylene glycol) mono tosyl mono allyl ether, hexa(ethylene glycol) mono allyl ether, hexa(ethylene glycol) mono methyl ether, hexa(ethylene glycol) mono benzyl ether, hexa(ethylene glycol) mono trityl ether, hexa(ethylene glycol) mono 1-hexenyl ether, hexa(ethylene glycol) mono 1-heptenyl ether, hexa(ethylene glycol) mono 1-octenyl ether, hexa(ethylene glycol) mono 1-decenyl ether, hexa(ethylene glycol) mono 1-undecenyl ether, hexa(ethylene glycol) mono 4-benzophenonyl mono 1-undecenyl ether, hepta(ethylene glycol) mono allyl ether, hepta(ethylene glycol) mono methyl ether, hepta(ethylene glycol) mono tosyl mono methyl ether, hepta(ethylene glycol) monoallyl mono methyl ether, octa(ethylene glycol) mono allyl ether, octa(ethylene glycol) mono tosylate, octa(ethylene glycol) mono tosyl mono allyl ether, undeca(ethylene glycol) mono methyl ether, undeca(ethylene glycol) mono allyl mono methyl ether, undeca(ethylene glycol) mono tosyl mono methyl ether, undeca(ethylene glycol) mono allyl ether, octadeca(ethylene glycol) mono allyl ether, octa(ethylene glycol), deca(ethylene glycol), dodeca(ethylene glycol), tetradeca(ethylene glycol), hexadeca(ethylene glycol), octadeca(ethylene glycol), benzophenone-4-hexa(ethylene glycol) allyl ether, benzophenone-4-hexa(ethylene glycol) hexenyl ether, benzophenone-4-hexa(ethylene glycol) octenyl ether, benzophenone-4-hexa(ethylene glycol) decenyl ether, benzophenone-4-hexa(ethylene glycol) undecenyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-fluorobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) allyl ether, 4-hydroxybenzophenone-4′-tetra(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) allyl ether, 4-morpholinobenzophenone-4′-hexa(ethylene glycol) undecenyl ether, 4-morpholinobenzophenone-4′-tetra(ethylene glycol) allyl ether, and 4-morpholinobenzophenone-4′-tetra(ethylene glycol) undecenyl ether.

12. The process of claim 7, wherein the polyethylene glycol has a molecular weight of approximately 1,000 to 20,000.

13. The process of claim 1, wherein the surface is glass and the amino moieties are an amino silane coupling agent selected from the group consisting of aminopropyltrimethoxysilane, aminopropyltriethoxysilane, aminopropylmethyldiethoxysilane, aminopropylmethyldiethoxysilane hydrozylate, m-aminophenyltrimethoxysilane, phenylaminopropyltrimethoxysilane, 1,1,2,4-tetramethyl-1-sila-2-azacyclopentane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropyltriethoxysilane, aminoethylaminopropylmethyldimethoxysilane, aminoethylaminopropyltrimethoxysilane hydrolyzate, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane, aminoethylaminoisobutylmethyldimethoxysilane hydrolyzate, trimethoxysilylpropyldiethylenetriamine, vinylbenzylethylenediaminepropyltrimethoxysilane monohydrochloride, vinylbenzylethylenediaminepropyltrimethoxysilane, benzylethylenediaminepropyltrimethoxysilane monohydrochloride, benzylethylenediaminepropyltrimethoxysilane, and allylethylenediaminepropyltrimethoxysilane monohydrochloride, and combinations thereof.

14. The process of claim 1, wherein the amino moieties are an amino amidite moiety selected from the group consisting of 3-(trifluoroacetylamino)propyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 12-(4-monomethoxytritylamino)dodecyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and 6-(trifluoroacetylamino)hexyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, and combinations thereof, whereby the hydroxyl bonding group is phosphoramidite and a phosphorous-oxygen bond is formed between phosphorous of the amino moieties and oxygen of the hydroxyl groups.

15. The process of claim 1, wherein the succinate moieties are selected from a salt of a chemical selected from the group consisting of 5′dimethoxytrityl-N-benzoyl-2′-deoxycytidine-3′-O-succinate, 5′dimethyoxytrityl-N-isobutyryl-2′-deoxyguanosine-3′-O-succinate, 5′-dimethoxytrityl-thymidine-3′-O-succinate, and 5′-dimethoxytrityl-N-benzoyl-2′-deoxyadenosine-3′-O-succinate, and combinations thereof.

16. The process of claim 1, wherein spacers having reactive hydroxyl groups are bound to the hydroxyl groups, wherein the amino amidite moities are bound to the reactive hydroxyl groups of the spacers.

17. The process of claim 16, wherein the spacer is selected from the group consisting of DNA, RNA, polyethylene glycol, and polypeptides, and combinations thereof.

18. The process of claim 16, wherein the spacers are from approximately 1 to 35 mers.

19. A pool of oligonucleotides comprising a plurality of double stranded RNA molecules selected from the group consisting of formula I, formula II, and combinations thereof:

(a) wherein formula I is a stem looped single stranded oligonucleotide comprising a first moiety RNA oligonucleotide sequence having 18 to 28 nucleotides, a second moiety oligonucleotide sequence having 2-15 bases and linked to the 5′ end or 3′ end of the first moiety RNA oligonucleotide sequence, and a third moiety RNA nucleotide sequence having 18-28 nucleotides, linked to the second moiety oligonucleotide sequence, and substantially complementary to the first moiety RNA oligonucleotidenucleotide sequence, wherein substantially complementary means up to three base mismatches, wherein the first moiety RNA oligonucleotide sequence matches sequence regions of virally-generated mRNAs, wherein the second moiety oligonucleotide sequences is comprised of single stranded DNA or RNA or combinations thereof;
(b) wherein formula II is a double stranded RNA oligonucleotide comprising a first strand RNA oligonucleotide sequence having 18-28 nucleotides and a second strand RNA oligonucleotide sequence having 18-28 nucleotides, wherein the second strand RNA oligonucleotide sequence is substantially complementary to the first strand RNA oligonucleotide sequence, wherein substantially complementary means up to three base mismatches, wherein the first strand RNA oligonucleotide sequence matches sequence regions of virally-generated mRNAs; and
(c) wherein the pool of oligonucleotides is made by the process of using a microarray having base cleavable succinate linkers, the process comprising: (i) providing a surface having known locations with hydroxyl groups, wherein the amount of the known locations is greater than approximately 100 per square centimeter; (ii) bonding amino moieties to the hydroxyl groups, wherein the amino moieties have an amine group and a hydroxyl bonding group, wherein the hydroxyl bonding group bonds to the hydroxyl groups of the known locations; (iii) bonding succinate moieties to the amine groups through amide bonds to form cleavable linkers attached to the solid surface, wherein the succinate moieties have a succinate group bonded to a sugar group and a base group bonded to the sugar group, wherein the cleavable linkers have a base-labile cleaving site on the succinate group and a reactable hydroxyl group on the sugar group; (iv) synthesizing oligonucleotides onto the reactable hydroxyl groups; and (v) cleaving at the base-labile cleaving site the oligomers from the solid surface using a cleaving base, whereby the pool of oligonucleotides are recoverable.
Patent History
Publication number: 20080125327
Type: Application
Filed: Jul 9, 2007
Publication Date: May 29, 2008
Inventors: Amit Kumar (San Jose, CA), Karl Maurer (Mukilteo, WA)
Application Number: 11/825,979
Classifications
Current U.S. Class: By Measuring The Ability To Specifically Bind A Target Molecule (e.g., Antibody-antigen Binding, Receptor-ligand Binding, Etc.) (506/9)
International Classification: C40B 30/04 (20060101);