Inhibitory DNA ligands to platelet-derived growth factor B-chain.
We have identified a group of DNA molecules that bind to platelet-derived growth factor (PDGF)-AB with subnanomolar affinity from a randomized DNA library using in vitro selection. Individual ligands cloned from the affinity-enriched pool bind to PDGF-AB and PDGF-BB with comparably high affinity (Kd approximately 10(-10) M) and to PDGF-AA with lower affinity (> 10(-8) M), indicating specific recognition of the PDGF B-chain in the context of the hetero- or homodimer. The consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a three-nucleotide loop at the branch point. Photo-cross-linking experiments with 5-iodo-2'-deoxyuridine-substituted ligands establish a point contact between a thymidine nucleotide in the helix junction loop region and phenylalanine 84 of the PDGF-B chain. Representative minimal DNA ligands inhibit the binding of 125I-PDGF-BB but not of 125I-PDGF-AA to PDGF alpha- or beta-receptors expressed in porcine aortic endothelial (PAE) cells in a concentration-dependent manner with half-maximal effects of approximately 1 nM. The same ligands also exhibit a similar inhibitory effect on PDGF-BB-dependent [3H]thymidine incorporation in PAE cells expressing the PDGF beta-receptors. These DNA ligands represent a novel class of specific and potent antagonists of PDGF-BB and, by inference, PDGF-AB.
NeXstar Pharmaceuticals, Boulder, Colorado 80301, USA.
L S
LS
Green
D
D
Jellinek
R
R
Jenison
A
A
Ostman
C H
CH
Heldin
N
N
Janjic
D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008024:Ligands; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; C095477:platelet-derived growth factor AB