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

Abstract

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.

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 HOR 4 (OR 5 ) m R 7 , 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; R 1 , R 2 , R 7 , and R 8  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; R 3  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; R 4  and R 5  are independently selected from the group consisting of methylene, ethylene, propylene, butylene, pentylene, and hexylene; R 6  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 R 7  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.