Screening of random oligonucleotide libraries with SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L.(1990) Science 249, 505—510] has emerged as a powerful method for identifying high-affinity nucleic acid ligands for a wide range of molecular targets. Nuclease sensitivity of unmodified RNA and DNA, however, imposes considerable restrictions on their use as therapeutics or diagnostics. Modified RNA in which pyrimidine 2'-hydroxy groups have been substituted with 2'-amino groups (2'-aminopyrimidine RNA) is known to be substantially more resistant to serum nucleases. We report here on the use of SELEX to identify high-affinity 2'-aminopyrimidine RNA ligands 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 1014 molecules containing 30 or 50 randomized positions. Compared to unmodified RNA with the same sequence, 2'-aminopyrimidine ligands are at least 1000-fold more stable in 90% human serum. The sequence information required for high-affinity binding to bFGF is contained within 24-26 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 deoxycytidine) bindsto bFGF with an apparent dissociation constant (Kf) of (3.5±0.3) x 10-10 M at 37 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\a=

5.9 min). The calculated value for the association rate constant (kon= k0ff/Kd) was 5.6 x 106 M_1 s_1. Highly specific binding of m21A to bFGF was observed: binding to denatured bFGF, five proteins from the FGF family (acidic FGF, FGF-4, FGF-5, FGF-6, and FGF-7), and four other heparin binding proteins is substantially weaker under the same conditiqns with XdbFGF/Xdprotem values rangingfrom (4.1±1.4) x 10-2 to> 10-6. Heparin but not chondroitin sulfate competed for binding of m21A to bFGF. In cell culture, m21A inhibited [125I] bFGF binding to both low-affinity sites (ED50~ 1 nM) and high-affinity sites (ED50% 3 nM) on CHO cells expressing transfected FGF receptor-1. Basic FGF-dependent migration of bovine aortic endothelial cells as well as bFGF-induced proliferation of human umbilical vein endothelial cells was also inhibited by m21A in a concentration-dependent manner with ED50 values of 50—100 nM. The 2'-aminopyrimidine RNA ligand m21A thereforerepresents a useful lead compound in our efforts to develop potent oligonucleotide-based angiogenesis antagonists.