Aptamer medicinal chemistry

Abstract

The invention relates generally to the field of nucleic acids and more particularly to aptamers useful as therapeutics, diagnostics and in research such as for target validation. The invention further relates to materials and methods for enhancing aptamers for use in therapeutics, diagnostics and research.

1 ) A method of identifying a substituted single stranded aptamer comprising the steps of: 
 a) substituting a phosphorothioate or phosphorodithioate for a phosphate at an internucleotide linkage position in a single stranded aptamer;    b) assaying the substituted single stranded aptamer for affinity to the target; and    c) identifying the substituted single stranded aptamer having increased affinity for the target relative to that of a starting aptamer which is identical to the modified single stranded aptamer except that it lacks the phosphorothioate substitution.    
     
     
         2 ) The method of  claim 1 , wherein the substituting step further comprises incorporating a phosphorothioate at the internucleotide linkage position by chemical synthesis.  
     
     
         3 ) The method of  claim 2 , wherein the substituting step does not comprise incorporating the phosphorothioate substitution at the internucleotide linkage between the two nucleotides at the 3′ or 5′ end of the single stranded aptamer.  
     
     
         4 ) The method of  claim 2 , wherein the substituting step comprises incorporating a single phosphorothioate substitution into the single stranded aptamer.  
     
     
         5 ) The method of  claim 4 , wherein the substituted single stranded aptamer comprises a binding affinity for the target that is at least two fold higher than that of the starting aptamer.  
     
     
         6 ) A substituted single stranded aptamer identified by the method of  claim 5 .  
     
     
         7 ) The method of  claim 4 , further comprising the step of incorporating an additional substitution into the substituted single stranded aptamer to result in a twice substituted single stranded aptamer.  
     
     
         8 ) The method of  claim 7 , further comprising assaying the twice substituted single stranded aptamer for affinity to the target and identifying the twice substituted single stranded aptamer that has an affinity equal to or higher than that of the starting unsubstituted single stranded aptamer.  
     
     
         9 ) The method of  claim 7 , further comprising assaying the twice substituted single stranded aptamer for affinity to the target and identifying the twice substituted single stranded aptamer that has an affinity equal to or higher than that of the phosphorothioate substituted single stranded aptamer.  
     
     
         10 ) A twice substituted single stranded aptamer identified by the method of  claim 9 .  
     
     
         11 ) The method of  claim 7 , wherein the additional substitution is an additional phosphorothioate substitution at a phosphate position different than that of the first phosphorothioate substitution.  
     
     
         12 ) The method of  claim 7 , wherein the additional substitution is selected from the group consisting of: substituting a nucleotide modified at a base position, substituting a nucleotide modified at a sugar position, and substituting a nucleotide a modified at phosphate position.  
     
     
         13 ) The method of  claim 12 , wherein the additional substitution is selected from the group consisting of: a phosphorodithioate substitution at a phosphate position, an inosine substitution for another nucleotide; a 2′-deoxy dihydro uridine substitution for a uridine; a 2′-deoxy-5-methyl nucleotide substitution for another nucleotide; a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide; a 2′-OMe nucleotide substitution for a 2′-deoxy nucleotide; and a 2-aminopurine substitution for a purine.  
     
     
         14 ) The method of  claim 13 , wherein the additional substitution is selected from the group consisting of: a 2′-deoxy inosine or 2′-OMe inosine substitution for another nucleotide; a 2′-deoxy dihydrouridine substitution for a uridine; a 2′-deoxy-5-methyl cytidine substitution for a cytidine; and a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide.  
     
     
         15 ) A method of identifying a substituted aptamer comprising the steps of: 
 a) incorporating a substitution into a starting aptamer wherein the substitution is selected from the group consisting of: an inosine substitution for another nucleotide, a 2′-deoxy dihydrouridine substitution for a uridine, a 2′-deoxy-5-methyl cytidine for a cytidine, a 2-amino purine substitution for a purine and a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide    b) assaying the substituted aptamer for affinity to the target; and    c) identifying the substituted aptamer having increased affinity for the target relative to that of the starting aptamer which is identical to the substituted aptamer except that it lacks the substituted nucleotide.    
     
     
         16 ) The method of  claim 15 , wherein the incorporating step further comprises incorporating the substitution into the starting aptamer through chemical synthesis.  
     
     
         17 ) The method of  claim 16 , wherein the identified substituted aptamer is single stranded.  
     
     
         18 ) The method of  claim 15 , wherein the method further comprises the steps of incorporating an additional substitution into the substituted aptamer, wherein the additional substitution is of a different type than the first substitution, to result in a twice substituted aptamer.  
     
     
         19 ) The method of  claim 18 , further comprising assaying the twice substituted aptamer for affinity to the target and identifying the twice substituted aptamer that has an affinity equal to or higher than that of the starting unsubstituted single stranded aptamer.  
     
     
         20 ) The method of  claim 18 , further comprising assaying the twice substituted aptamer for affinity to the target and identifying the twice substituted aptamer that has an affinity equal to or higher than that of the substituted aptamer.  
     
     
         21 ) The method of  claim 20 , wherein the additional substitution is selected from the group consisting of: substituting a nucleotide modified at a base position, substituting a nucleotide modified at a sugar position, and substituting a nucleotide a modified at phosphate position.  
     
     
         22 ) The method of  claim 21 , wherein the additional substitution is selected from the group consisting of: a phosphorodithioate substitution at a phosphate position, an inosine substitution for another nucleotide; a 2′-deoxy dihydro uridine substitution for a uridine, 2′-deoxy-5-methyl nucleotide substitution for a nucleotide, a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide, a 2′-OMe nucleotide substitution for a 2′-deoxy nucleotide, and a 2-aminopurine substitution for a purine and wherein the additional substitution is different from the first substitution.  
     
     
         23 ) The method of  claim 23 , wherein the additional substitution is selected from the group consisting of: a 2-deoxy inosine or 2′-OMe inosine substitution for another nucleotide, a 2′-deoxy dihydrouridine substitution for a uridine, a 2′-deoxy-5-methyl cytidine substitution for a cytidine, and a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide.  
     
     
         24 ) The method of  claim 15 , further comprising incorporating an additional substitution into the substituted aptamer to result in a twice substituted aptamer wherein the additional substitution is of the same type as the first substitution but at a different nucleotide position.  
     
     
         25 ) The method of  claim 24 , wherein the method further comprises assaying the twice substituted aptamer for affinity to the target and identifying the twice substituted aptamer that has an affinity equal to or higher than that of the starting unsubstituted single stranded aptamer and/or the substituted aptamer.  
     
     
         26 ) A twice substituted aptamer identified by the method of  claim 24 .  
     
     
         27 ) The method of  claim 15 , wherein the incorporating step further comprises substituting an inosine for another nucleotide.  
     
     
         28 ) The method of  claim 27 , wherein the inosine substituted aptamer comprise a binding affinity at least two fold higher than that of the starting unsubstituted aptamer.  
     
     
         29 ) The method of  claim 15 , wherein the incorporating step comprises substituting a 2′-deoxy nucleotide for a 2′-OMe nucleotide.  
     
     
         30 ) The method of  claim 29 , wherein the 2′-deoxy substituted aptamer comprise a binding affinity at least two fold higher than that of starting unsubstituted aptamer.  
     
     
         31 ) The method of  claim 20 , wherein the first substitution is 2′-deoxy substitution for 2′-OMe nucleotide and the additional substitution is an inosine substitution.  
     
     
         32 ) The method of  claim 12 , wherein the twice substituted single stranded phosphorothioate aptamer comprises a binding affinity that is at least 2 fold higher than the starting unsubstituted aptamer and/or the substituted aptamer.  
     
     
         33 ) A single stranded aptamer comprising no more than one phosphorothioate backbone substitution wherein the single stranded aptamer binds specifically to a target with a binding affinity for the target that is increased relative to the binding affinity for the target of a second single stranded aptamer identical to the first but lacking the phosphorothioate substitution.  
     
     
         34 ) The single stranded aptamer of  claim 33 , comprising no more than one additional phosphorothioate substitution.  
     
     
         35 ) The singled stranded aptamer of  claim 34  comprising no more than 2 additional phosphorothioate substitutions.  
     
     
         36 ) The single stranded aptamer of  claim 35 , comprising no more than 3 additional phosphorothioate substitutions.  
     
     
         37 ) The single stranded aptamer of  claim 33 , further comprising an additional nucleotide substitution selected from the group consisting of: a substitution with a nucleotide modified at a base position, a substitution with a nucleotide modified at a sugar position and a substitution with a nucleotide modified at phosphate position wherein when the nucleotide substituted comprises a modification at the phosphate position it is not a phosphorothioate substitution.  
     
     
         38 ) The single stranded aptamer of  claim 37 , wherein the additional nucleotide substitution is selected from the group consisting of: a phosphorodithioate substitution at a phosphate position, an inosine substitution for another nucleotide; a 2′-deoxy dihydro uridine substitution for a uridine, a 2′-deoxy-5-methyl nucleotide substitution for another nucleotide, a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide, a 2′-OMe nucleotide substitution for a 2′-deoxy nucleotide, and a 2-aminopurine substitution for a purine.  
     
     
         39 ) A single stranded aptamer comprising a nucleotide substitution selected from the group consisting of: an inosine substitution for another nucleotide, a 2′-deoxy dihydrouridine substitution for a uridine, a 2′-deoxy-5-methyl cytidine for a cytidine, an 2-amino purine substitution for a purine and a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide, wherein the substituted single stranded aptamer binds specifically to a target with a binding affinity for the target that is increased relative to the binding affinity for the target of a second single stranded aptamer identical to the first but lacking the nucleotide substitution.  
     
     
         40 ) The single stranded aptamer of  claim 39 , comprising at least two nucleotide substitutions selected from the group consisting of: an inosine substitution for another nucleotide, a 2′-deoxy dihydrouridine substitution for a uridine, a 2′-deoxy-5-methyl cytidine for a cytidine, an 2-amino purine substitution for a purine and a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide, wherein the twice substituted single stranded aptamer binds specifically to a target with a binding affinity for the target that is increased relative to the binding affinity for the target of a second single stranded aptamer identical to the first but lacking the at least two nucleotide substitutions.  
     
     
         41 ) The single stranded aptamer of  claim 39 , comprising at least three nucleotide substitutions selected from the group consisting of: an inosine substitution for another nucleotide, a 2′-deoxy dihydrouridine substitution for a uridine, a 2′-deoxy-5-methyl cytidine for a cytidine, an 2-amino purine substitution for a purine and a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide, wherein the triple substituted single stranded aptamer binds specifically to a target with a binding affinity for the target that is increased relative to the binding affinity for the target of a second single stranded aptamer identical to the first but lacking the at least three nucleotide substitutions.  
     
     
         42 ) The single stranded aptamer of  claim 40 , wherein twice substituted single stranded aptamer binds specifically to a target with a binding affinity for the target that is increased relative to the binding affinity for the target of a second single stranded aptamer identical to the first but lacking one of the nucleotide substitutions.  
     
     
         43 ) The single stranded aptamer of  claim 41 , wherein the triple substituted single stranded aptamer binds specifically to a target with a binding affinity for the target that is increased relative to the binding affinity for the target of a second single stranded aptamer identical to the first but lacking at least one of the nucleotide substitutions.  
     
     
         44 ) The single stranded aptamer of  claim 39 , wherein the nucleotide substitution is the substitution of 2′-deoxy nucleotide for a 2′-OMe nucleotide.  
     
     
         45 ) The single stranded aptamer of  claim 39 , wherein the nucleotide substitution is the substitution of an inosine for a purine.  
     
     
         46 ) An aptamer that specifically binds to a target, wherein the aptamer comprises a nucleotide sequence having a phosphorothioate modification of the phosphate back bone at a position selected to increase the binding affinity of the aptamer to the target relative to the binding affinity of a second aptamer to the same target, the of: an inosine substitution for a purine, a 2′-deoxy dihydrouridine substitution for a uridine, a 2′-deoxy-5-methyl cytidine for a cytidine, a 2-amino purine substitution for a purine, a second aptamer having the same nucleotide sequence but lacking the phosphorothioate modification.  
     
     
         47 ) A method of stabilizing an aptamer comprising the steps of: 
 a) introducing stablilizing modifications into a starting aptamer to result in a modified aptamer wherein the starting aptamer has a predeterimined binding affinity for a target,    b) assaying modified aptamer for binding affinity to the target and where the binding affinity is less than that of the starting aptamer introducing a nucleotide substitution to result in a substituted aptamer wherein the nucleotide substitution results in the substituted aptamer having a binding affinity for the target higher than that of the modified aptamer.    
     
     
         48 ) The method of  claim 47 , herein the substituted aptamer comprises a binding affinity for the target substantially the same as that of the starting aptamer.  
     
     
         49 ) The method of  claim 47 , wherein the stabilizing modification is a modification to increase aptamer resistance to nuclease resistance.  
     
     
         50 ) The method of  claim 49 , wherein the stabilizing modification comprises substituting a 2′-OMe nucleotide for another nucleotide.  
     
     
         51 ) The method of  claim 50 , wherein the introducing step comprises introducing more than one 2′-OMe substitution for another nucleotide.  
     
     
         52 ) The method of  claim 51 , wherein the substituting step comprises a substitution selected from the group consisting of: a substitution with a nucleotide modified at a base position, with a nucleotide modified at a sugar position and with a nucleotide modified at phosphate position.  
     
     
         53 ) The method of  claim 52 , where in the substitution is selected from the group consisting phosphorothioate substituted nucleotide for an unsubstituted nucleotide, a phosphorodithioate substituted nucleotide for an unsubstituted nucleotide and a 2′-deoxy nucleotide substitution for a 2′-OMe nucleotide.  
     
     
         54 ) The method of  claim 53 , wherein the substitution is a phosphorothioate substituted nucleotide for an unsubstituted nucleotide.  
     
     
         55 ) A method of identifying a substituted aptamer that binds to a target, wherein the substituted aptamer has a higher binding affinity for the target than that of an identical aptamer but for the substitution, comprising the steps of 
 a) substituting a single nucleotide modified at a base, sugar or phosphate position for an unsubstituted nucleotide, and    b) assaying the substituted aptamer for binding affinity to the target.    
     
     
         56 ) The method of  claim 55 , wherein the substituting step further comprises substituting at least two nucleotides modified at the same position for at least two unmodified nucleotides.  
     
     
         57 ) The method of  claim 56 , wherein the substituting step further comprises substituting at least three nucleotides substituted at the same position for at least three unmodified nucleotides.  
     
     
         58 ) The method of  claim 55 , where the substituting step further comprises substituting at least two nucleotides modified at different positions for at least two unmodified nucleotides.  
     
     
         59 ) The method of  claim 58 , where the substituting step further comprises substituting at least three nucleotides wherein at least two of the nucleotides to be substituted are modified at different positions for at least three unmodified nucleotides.  
     
     
         60 ) An aptamer that specifically binds to a target, wherein the aptamer comprises a nucleotide sequence having a substituted nucleotide selected to increase the binding affinity of the aptamer to the target relative to the binding affinity of a second aptamer to the same target, the second aptamer having the same nucleotide sequence but lacking the substituted nucleotide, wherein the substituted nucleotide comprises a modification at a phosphate, sugar or base position.  
     
     
         61 ) A single stranded aptamer comprising no more than one nucleotide substitution wherein the substituted nucleotide comprises a chemical modification at a base, a sugar or phosphate position and wherein the single stranded aptamer binds specifically to a target with a binding affinity for the target that is increased relative to the binding affinity for the target of a second single stranded aptamer identical to the first but lacking the substitution.