Bead bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries

Abstract

The present invention includes composition and methods for making and using a combinatorial library having two or more beads, wherein attached to each bead is a unique nucleic acid aptamer that have disposed thereon a unique sequence. The library aptamers may be attached covalently to the one or more beads, which may be polystyrene beads. The aptamers may include phosphorothioate, phosphorodithioate and/or methylphosphonate linkages and may be single or double stranded DNA, RNA or even PNAs.

1 - 31 . (canceled) 
     
     
         32 . A combinatorial oligonucleotide library on supports made by the process comprising the steps of:
 establishing a reaction column for each subunit oligonucleotide base species that will be used to synthesize a random collection of oligonucleotide base sequences and distributing a set of activated or prederivitized supports in each reaction column;   attaching a single base species to the supports in each of the columns;   mixing the supports from the columns together;   splitting the mixed supports back into each of the columns;   adding a new single base species to the mixed supports in each of the columns; and   repeating the steps of mixing the supports together, splitting the supports and adding a new base to each of the oligonucleotides on each support in each of the columns until the oligonucleotide library is complete, wherein each support comprises many copies of a single unique oligonucleotide sequence.   
     
     
         33 . The library of  claim 32 , wherein the supports are beads. 
     
     
         34 . The library of  claim 32 , wherein the supports are nanoparticles. 
     
     
         35 . The library of  claim 32 , further comprising the step of attaching a predetermined subunit oligonucleotide having a known sequence to each oligonucleotide on each support prior to mixing and splitting. 
     
     
         36 . The library of  claim 32 , wherein the oligonucleotides are attached to the supports from the 3′ end. 
     
     
         37 . The library of  claim 32 , wherein the oligonucleotides are attached to the supports from the 5′ end. 
     
     
         38 . The library of  claim 32 , wherein the oligonucleotides are attached to the supports using a chemical linker that is not cleaved upon oligonucleotide deprotection. 
     
     
         39 . The library of  claim 32 , wherein the oligonucleotides are attached to the supports using a chemical entity that allows cleavage of the oligonucleotides from the supports. 
     
     
         40 . The library of  claim 32 , wherein the oligonucleotides are a mixture of standard DNA and chemically-modified DNA. 
     
     
         41 . The library of  claim 32 , wherein the oligonucleotides are a mixture of standard RNA and chemically-modified RNA. 
     
     
         42 . The library of  claim 32 , wherein the oligonucleotides are a mixture of DNA and RNA, either standard or chemically-modified. 
     
     
         43 . The library of  claim 32 , wherein the oligonucleotides cannot be amplified using polymerase chain reaction. 
     
     
         44 . The library of  claim 32 , further comprising the step of converting the oligonucleotide on the supports to a double-stranded oligonucleotide with a DNA polymerase I Klenow fragment. 
     
     
         45 . The library of  claim 32 , wherein the oligonucleotides further comprise a fluorophor. 
     
     
         46 . The library of  claim 32 , wherein the oligonucleotides further comprise one or more primers at a 3′, a 5′ or both the 3′ and 5′ ends. 
     
     
         47 . The library of  claim 32 , wherein the oligonucleotides further comprise one or more fluorophors attached to a 5′ end, a 3′ end or internally within the oligonucleotide. 
     
     
         48 . The library of  claim 32 , wherein the base further comprises a monothiophosphate or a dithiophosphate backbone substitution. 
     
     
         49 . The library of  claim 32 , wherein the library is double-stranded. 
     
     
         50 . The library of  claim 32 , wherein the library of random sequence oligonucleotides on supports is exposed to a tagged target and the individual supports that bind the target are separated from the library for further analysis. 
     
     
         51 . The library of  claim 32 , wherein a mixture of subunit base species are added to one or more columns for random attachment to the oligonucleotide sequences on the supports to create a sublibrary of oligonucleotide sequences on each support member of the combinatorial oligonucleotide library.