Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor.
BACKGROUND
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a potent inducer of new blood vessel growth (angiogenesis) that contributes to the pathology of many angiogenesis-associated disease states such as psoriasis, rheumatoid arthritis and cancer. Few molecular entities capable of binding to VPF/VEGF with high affinity and specificity have been described to date.


RESULTS
Nuclease-resistant 2'-amino-2'-deoxypyrimidine nucleotide RNA (2'-aminopyrimidine RNA) ligands that bind to VPF/VEGF with high affinity have been identified by iterative rounds of affinity-selection/amplification from two independent random libraries. The sequence information that confers high affinity binding to VPF/VEGF is contained in a contiguous stretch of 24 nucleotides, 5'-CCCUGAUGGUAGACGCCGGGGUG-3' (2'-aminopyrimidine nucleotides are designated with italic letters). Of the 14 ribopurines in this minimal ligand, 10 can be substituted with the corresponding 2'-O-methylpurine nucleotides without a reduction in binding affinity to VPF/VEGF. In fact, the 2'-O-methyl substitution at permissive positions leads to a approximately 17-fold improvement in the binding affinity to VPF/VEGF. The higher affinity results from the reduction in the dissociation rate constant of the 2'-O-methyl-substituted RNA ligand from the protein compared to the unsubstituted ligand. The 2'-O-methyl-substituted minimal ligand, which folds into a bulged hairpin motif, is also more thermally stable than the unsubstituted ligand. Nuclease resistance of the ligand is further improved by the 2'-O-methyl substitutions and the addition of short phosphorothioate caps to the 3'- and 5'-ends.


CONCLUSIONS
We have used the SELEX (systematic evolution of ligands by exponential enrichment) process in conjunction with post-SELEX modifications to define a highly nuclease-resistant oligonucleotide that binds to VPF/VEGF with high affinity and specificity.
Green
L S
LS
NeXstar Pharmaceuticals, Boulder, Colorado 80301, USA.
Jellinek
D
D
Bell
C
C
Beebe
L A
LA
Feistner
B D
BD
Gill
S C
SC
Jucker
F M
FM
Janjić
N
N
D000818:Animals; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D016228:Endothelial Growth Factors / Q000737:chemistry* / Q000652:urine; D006801:Humans; D008024:Ligands; D008222:Lymphokines / Q000737:chemistry* / Q000652:urine; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009696:Nucleic Acids / Q000737:chemistry* / Q000652:urine; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D019151:Peptide Library; D011687:Purines / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry* / Q000652:urine; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008024:Ligands; D008222:Lymphokines; D009696:Nucleic Acids; D009841:Oligonucleotides; D019151:Peptide Library; D011687:Purines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012260:Ribonucleases