NUCLEASE-RESISTANT NUCLEIC-ACID LIGANDS TO VASCULAR-PERMEABILITY FACTOR VASCULAR ENDOTHELIAL GROWTH-FACTOR

Background: Vascular permeability factor/vascular endothelial growth f actor (VPF/VEGF) is a potent inducer of new blood vessel growth (angio genesis) that contributes to the pathology of many angiogenesis-associ ated disease states such as psoriasis, rheumatoid arthritis and cancer . Few molecular entities capable of binding to VPF/VEGF with high affi nity and specificity have been described to date. Result: Nuclease-res istant 2'-amino-2'deoxypyrimidine nucleotide RNA (2'-aminopyrimidine R NA) ligands that bind to VPF/VEGF with high affinity have been identif ied by iterative rounds of affinity-selection/amplification from two i ndependent random libraries. The sequence information that confers hig h affinity binding to VPF/VEGF is contained in a contiguous stretch of 24 nucleotides, 5'-CCCUGAUGGUAGAC-GCCGGGGUG-3' (2'-aminopyrimidine nu cleotides are designated with italic letters). Of the 14 ribopurines i n this minimal ligand, 10 can be substituted with the corresponding 2' -O-methylpurine nucleotides without a reduction in binding affinity to VPF/VEGE In fact, the 2'O-methyl substitution at permissive positions leads to a similar to 17-fold improvement in the binding affinity to VPF/VEGE. The higher affinity results from the reduction in the dissoc iation rate constant of the 2'-O-methyl-substituted RNA ligand from th e protein compared to the unsubstituted ligand. The 2'-O-methyl-substi tuted minimal ligand, which folds into a bulged hairpin motif, is also more thermally stable than the unsubstituted ligand. Nuclease resista nce of the Ligand is further improved by the 2'-O-methyl substitutions and the addition of short phosphorothioate caps to the 3'- and 5'-end s. Conclusions: We have used the SELEX (systematic evolution of ligand s by exponential enrichment) process in conjunction with post-SELEX mo difications to define a highly nuclease-resistant oligonucleotide that binds to VPF/VEGF with high affinity and specificity.