POTENT 2'-AMINO-2'-DEOXYPYRIMIDINE RNA INHIBITORS OF BASIC FIBROBLASTGROWTH-FACTOR

Screening of random oligonucleotide libraries with SELEX [systematic e volution of ligands by exponential enrichment; Tuerk, C., & Gold, L. ( 1990) Science 249, 595-510] has emerged as a powerful method for ident ifying high-affinity nucleic acid ligands for a wide range of molecula r targets, Nuclease sensitivity of unmodified RNA and DNA, however, im poses considerable restrictions on their use as therapeutics or diagno stics. Modified RNA in which pyrimidine 2'-hydroxy groups have been su bstituted with 2'-amino groups (2'-aminopyrimidine RNA) is known to be substantially more resistant to serum nucleases, We report here on th e use of SELEX to identify high-affinity 2'-aminopyrimidine RNA ligand s to a potent angiogenic factor, basic fibroblast growth factor(bFGF). High-affinity ligands with the same consensus primary structure have been isolated from two independent libraries of approximately 6 x 10(1 4) molecules containing 30 or 50 randomized positions. Compared to unm odified RNA with the same sequence, 2'-aminopyrimidine ligands are at least 1000-fold more stable in 90% human serum. The sequence informati on required for high-affinity binding to bFGF is contained within 24-2 6 nucleotides. The minimal ligand m21A (5'-GGUGUGUGGAAGACAGCGGGUGGUUC- 3'; G = guanosine, A = adenosine, C = 2'-amino-2'-deoxycytidine, U = 2 '-amino-2'-deoxyuridine, and C = 2'-amino-2'-deoxycytidine or deoxycyt idine) binds to bFGF with an apparent dissociation constant (K-d) of ( 3.5 +/- 0.3) x 10(-10) M at 37 degrees C in phosphate-buffered saline (pH 7.4). Dissociation of m21A from bFGF is adequately described with a first-order rate constant of (1.96 +/- 0.08) x 10(-3) s(-1) (t(1/2) = 5.9 min). The calculated value for the association rate constant (k( on) = k(off)/K-d) was 5.6 x 10(6) M(-1) s(-1). Highly specific binding of m21A to bFGF was observed: binding to denatured bFGF, five protein s from the FGF family (acidic FGF, FGF-4, FGF-5, FGF-6, and FGF-7), an d four other heparin binding proteins is substantially weaker under th e same conditions with K-d(bFGF)/K-d(protein) values ranging from (4.1 +/- 1.4) x 10(-2) to > 10(-6). Heparin but not chondroitin sulfate co mpeted for binding of m21A to bFGF. In cell culture, m21A inhibited [I -125]bFGF binding to both low-affinity sites (ED(50) approximate to 1 nM) and high-affinity sites (ED(50) approximate to 3 nM) on CHO cells expressing transfected FGF receptor-1. Basic FGF-dependent migration o f bovine aortic endothelial cells as well as bFGF-induced proliferatio n of human umbilical vein endothelial cells was also inhibited by m21A in a concentration-dependent manner with ED(50) values of 50-100 nM. The 2'-aminopyrimidine RNA ligand m21A therefore represents a useful l ead compound in our efforts to develop potent oligonucleotide-based an giogenesis antagonists.