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(alphaS)'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.
King
D J
DJ
Sealy Center for Structural Biology, Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston 77555-1157, USA. david@nmr.utmb.edu
Ventura
D A
DA
Brasier
A R
AR
Gorenstein
D G
DG
D001483:Base Sequence; D001667:Binding, Competitive / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D016415:Sequence Alignment; D013873:Thionucleotides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides