Thio modified aptamer synthetic methods and compositions

Abstract

The present invention provides a method for concurrent achiral nucleotide modification and amplification using PCR. Provided by this method are NF-kB specific thioaptamers of novel sequence. This invention further provides methods of post-selection aptamer modification wherein one or more selected nucleotides of aptamers of known sequence are substituted with modified achiral nucleotides, particularly achiral thiophosphate nucleotides, wherein the substitution results in increased nuclease resistance while retaining binding efficiency and selectivity. Thiosubstitution of post-selection aptamers with specificity for the nuclear factor, NF-κB, produced in accordance with this method have increased binding affinity and specificity in addition to nuclease resistance. Also provided are methods for fractionating oligonucleotides depending on their degree of thiosubstitution by anion exchange chromatography.

1 - 17 . (canceled)  
     
     
         18 . An achiral oligonucleotide that specifically binds a target comprising a sequence of nucleotides one or more nucleotides in sequence are thiophosphate modified.  
     
     
         19 . The achiral oligonucleotide of  claim 18 , wherein the one or more thio-modified nucleotide in the achiral oligonucleotide is selected from the group consisting of dATP(αS), dTTP(αS), dCTP(αS) and dGTP(αS), dATP(S 2 ), dTTP(S 2 ), dCTP(S 2 ) and dGTP(S 2 ).  
     
     
         20 . The achiral oligonucleotide of  claim 18 , wherein not all like nucleotides have a thiomodified phosphate group.  
     
     
         21 . The achiral oligonucleotide of  claim 18 , wherein only one nucleotide has a thiomodified phosphate group.  
     
     
         22 . The achiral oligonucleotide of  claim 18 , wherein two nucleotides have a thiomodified phosphate group.  
     
     
         23 . An achiral oligonucleotide that specifically binds a target comprising a sequence of nucleotides wherein one or more of the nucleotides in sequence are dithiophosphates.  
     
     
         24 . The achiral oligonucleotide of  claim 23 , wherein the one or more thio-modified nucleotide in the achiral oligonucleotide is selected from the group consisting of dATP(αS), dTTP(αS), dCTP(αS), dGTP(αS), dATP(S 2 ), dTTP(S 2 ), dCTP(S 2 ) and dGTP(S 2 ).  
     
     
         25 . The achiral oligonucleotide of  claim 23 , wherein not all like nucleotides have a thiomodified phosphate group.  
     
     
         26 . The achiral oligonucleotide of  claim 23 , wherein only one nucleotide has a thiomodified phosphate group.  
     
     
         27 . The achiral oligonucleotide of  claim 23 , wherein two nucleotides have a thiomodified phosphate group.  
     
     
         28 . The achiral oligonucleotide of  claim 23 , wherein between approximately 10 and 80% of the nucleotides in the sequence are thiomodified.  
     
     
         29 - 39 . (canceled)  
     
     
         40 . The achiral oligonucleotide of  claim 18 , further comprising one or more pharmaceutically acceptable salts.  
     
     
         41 . The achiral oligonucleotide of  claim 18 , further comprising a diluent.  
     
     
         42 . The achiral oligonucleotide of  claim 18 , wherein the achiral oligonucleotide binds specifically to an NF-kB/Rel protein  
     
     
         43 . The achiral oligonucleotide of  claim 18 , wherein the achiral oligonucleotide binds specifically to an NF-kB homodimer.  
     
     
         44 . The achiral oligonucleotide of  claim 18 , wherein the achiral oligonucleotide binds specifically to an NF-kB heterodimer.  
     
     
         45 . The achiral oligonucleotide of  claim 18 , wherein the oligonucleotide further comprises a detectable label.  
     
     
         46 . The achiral oligonucleotide of  claim 18 , wherein the achiral oligonucleotide is single stranded.  
     
     
         47 . The achiral oligonucleotide of  claim 23 , further comprising one or more pharmaceutically acceptable salts.  
     
     
         48 . The achiral oligonucleotide of  claim 23 , further comprising a diluent.  
     
     
         49 . The achiral oligonucleotide of  claim 23 , wherein the achiral oligonucleotide binds specifically to an NF-kB/Rel protein  
     
     
         50 . The achiral oligonucleotide of  claim 23 , wherein the achiral oligonucleotide binds specifically to an NF-kB homodimer.  
     
     
         51 . The achiral oligonucleotide of  claim 23 , F-kB heterodimer.  
     
     
         52 . The achiral oligonucleotide of  claim 23 , wherein the aptamer comprises one or more thiomodifications as set forth in SEQ ID NOS: 32, 33 and 38.  
     
     
         53 . The achiral oligonucleotide of  claim 23 , wherein the achiral oligonucleotide further comprises a detectable label.  
     
     
         54 . The achiral oligonucleotide of  claim 23 , further comprising one or more pharmaceutically acceptable salts.  
     
     
         55 . The achiral oligonucleotide of  claim 23 , further comprising a diluent.  
     
     
         56 . The achiral oligonucleotide of  claim 23 , wherein the achiral oligonucleotide is single stranded.  
     
     
         57 . A partially thio-modified achiral aptamer that binds to a human NF-κB protein complex.