Inhibition of receptor binding by high-affinity RNA ligands to vascular endothelial growth factor

Abstract

The proliferation of new blood vessels (angiogenesis) is a process that accompanies many pathological conditions including rheumatoid arthritis and solid tumor growth. Among angiogenic cytokines that have been identified to date, vascular endothelial growth factor (VEGF) is one of the most potent. We used SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505-510] to identify RNA ligands that bind to VEGF in a specific manner with affinities in the low nanomolar range. Ligands were selected from a starting pool of about 10(14) RNA molecules containing 30 randomized positions. Isolates from the affinity-enriched pool were grouped into six distinct families on the basis of primary and secondary structure similarities. Minimal sequence information required for high-affinity binding to VEGF is contained in 29-36-nucleotide motifs. Binding of truncated (minimal) high-affinity ligands to VEGF is competitive with that of other truncated ligands and heparin. Furthermore, truncated ligands from the six ligand families inhibit binding of [125I]VEGF to its cell-surface receptors. Oligonucleotide ligands described here represent an initial set of lead compounds in our ongoing effort toward the development of potent and specific VEGF antagonists.

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF 165

“…The Ca 2ϩ requirement for the formation of the VEGF 165 -aptamer complex is specific to this aptamer family and is not observed in other high-affinity aptamers to VEGF 165 (8,(23)(24)(25). The results here show that Ca 2ϩ is also required for the HBD-aptamer complex formation.…”

Exon repression by polypyrimidine tract binding protein

“…To determine the extent of RNA bound in each complex, we performed boundary analyses to identify the length of RNA needed to assemble into each complex (Jellinek et al 1994;Bevilacqua et al 1998;Darnell et al 2001). A longer RNA encompassing the nucleotides of Probe A was labeled at either its 5Ј or 3Ј end, subjected to partial alkaline hydrolysis, and bound to PTB.…”

In vitro selection of RNAs that bind to the human immunodeficiency virus type-1 gag polyprotein

“…The binding affinity of RNA ligands for various proteins was measured by a nitrocellulose filtration method essentially as described (24,26,28). Radiolabeled RNA (∼2 fmol, ∼10 000 c.p.m.)…”

“…The boundaries of RNA ligands were determined essentially as described by Jellinek et al (28). Ligands were labeled at the 5′ end by dephosphorylating the RNA with bacterial alkaline phosphatase and then labeling the RNA by using [γ-32 P]ATP and T4 polynucleotide kinase.…”