Selection, binding and design of phosphorothioate duplex aptamers for the transcription factors NF-IL6 and NF-kappaB

Methods have been established for the combinatorial selection of phosphorothioate oligonucleotide aptamers. The protein systems to which this technology has been applied are the transcription factors NF-IL6 and NF-kappaB. Firstly, PCR methods were established for the construction and purification of phosphorothioate combinatorial libraries with monothiophosphate modification 5' to each deoxyadenosine residue. These methods can be extended to obtain libraries containing thiophosphate modification 5' to any and all of the usual nucleotides found in DNA.; Using the purified recombinant proteins, nitrocellulose filter binding methods were developed to separate from the libraries sequences which bind to and compete for these transcription factors. These sequences or "aptamers" have both motifs which resemble their natural binding sites and those which are novel. Interestingly, it appears that while NF-IL6 can select aptamers bearing no resemblance to its natural binding sites, NF-kappaB must retain at least one natural half site. The presence of sulfur appears to enhance the affinity of the aptamers to the NF-kappaB proteins and some results suggest that the selection process involves random sampling of the library for sulfur in critical positions, thus limiting the number of monothiophosphates necessary for enhanced and specific binding.; Binding energetics of p65 and p50 homodimers were studied using a quantitative electrophoretic mobility shift assay or EMSA. As a reference system for competitive aptamer binding, the 22mer phosphoryl binding site known as IgkappaB binds to p50 and p65 with roughly the same affinity (Kd = 4.77 +/- 0.21 nM for p65 and Kd = 0.76 +/- .21 nM for p50).; A global analysis tool for competitive NF-kappaB/IgkappaB binding has been developed and utilized to measure the affinity of a p65 selected aptamer. (Kd = 78.85 +/- 1.87 nM for the thiosubstituted DNA and Kd = 249.12 +/- 1.76 nM for normal, phosphate ester backbone). Qualitative binding experiments demonstrate that the nature of specificity and enhanced affinity can be attributed to the presence of sulfur. Collectively these results demonstrate the feasibility of the aptamer selection technology as a method for producing specific, high affinity ligands to proteins.