Novel combinatorial selection of phosphorothioate oligonucleotide aptamers

A novel combinatorial approach is described for construction and screening of enhanced nuclease-resistant phosphorothioate DNA 'decoys' or 'aptamers.' Aptamers have been selected that bind tightly to the nuclear factor for human IL6 (NF-IL6), a basic leucine zipper transcription factor involved in the induction of acute-phase responsive and cytokine gene promotors in response to inflammation. Using a random combinatorial selection approach and dNTP(αS)'s in the PCR amplification, we can select specific thio-substituted agents which have the highest specificity in binding to target NF-IL6. Using a 22-nucleotide-long duplex random library, nanomolar binding, specific 22-mer thiophosphate backbone substitution sequences (at dA positions only) were selected. These show a different consensus sequence than normal phosphate backbone CCAAT/enhancer binding protein recognition sites for NF-IL6. Unlike the wild-type 10-mer sequences, which bind 1 protein dimer/duplex, these 22-mer thiophosphate aptamers bind with a stoichiometry of 2 dimers/duplex.