In vitro selection of DNA aptamers that bind L-tyrosinamide.
We have applied SELEX (Systematic Evolution of Ligands by EXponential enrichment), a combinatorial method that employs biopolymers for drug discovery, to identify single stranded DNA sequences able to bind L-Tyrosinamide, a simple mimic of Tyrosine, an amino acid essential to the catalytic activity of several enzymes of pharmaceutical interest. After 15 SELEX cycles using L-Tyrosinamide immobilized on an affinity chromatography column, the percentage of aptamers specifically eluted from the affinity column with free L-Tyrosinamide was 55% of the total. Aptamers were subcloned and sequenced, allowing the identification of a highly conserved consensus sequence, and showed a K(d) value for L-Tyrosinamide of 45 microM. The identified aptamer sequence will constitute the basis for further in vitro evolution protocols and structure-based drug design.
Vianini
E
E
Department of Pharmaceutical Sciences, University of Padova, via 5, 35131 Padova, Marzolo, Italy.
Palumbo
M
M
Gatto
B
B
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D020650:Combinatorial Chemistry Techniques; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D015195:Drug Design; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods; D013237:Stereoisomerism; D019914:Taq Polymerase / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D059004:Topoisomerase I Inhibitors; C036387:tyrosinamide; D014443:Tyrosine; D010649:Phenylalanine; C020457:phenylalanine amide; D012313:RNA; D019914:Taq Polymerase; D004264:DNA Topoisomerases, Type I