Potent 2'-amino-2'-deoxypyrimidine RNA inhibitors of basic fibroblast growth factor.
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 10(14) 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) binds to bFGF with an apparent dissociation constant (Kd) of 3.5 +/- 0.3) x 10(-10) M at 37 degrees C in phosphate-buffered saline (pH 7.4). Disassociation of m21A from bFGF is adequately described with a first-order rate constant of (1.96 +/- 0.08) x 10(-3) s-1 (t1/2 = 5.9 min). The calculated value for the association rate constant (kon = k(off)/Kd) was 5.6 x 10(6) 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 conditions with KdbFGF/Kdprotein values ranging from (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 approximately 1 nM) and high-affinity sites (ED50 approximately 3 nM) on CHO cells expressing transfected FGF receptor-1.(ABSTRACT TRUNCATED AT 400 WORDS)
Jellinek
D
D
NeXstar Pharmaceuticals, Inc., Boulder, Colorado 80301, USA.
Green
L S
LS
Bell
C
C
Lynott
C K
CK
Gill
N
N
Vargeese
C
C
Kirschenheuter
G
G
McGee
D P
DP
Abesinghe
P
P
Pieken
W A
WA
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology*; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D012313:RNA / Q000378:metabolism
D003854:Deoxyribonucleotides; D016222:Fibroblast Growth Factor 2; D012313:RNA; D006493:Heparin; D004247:DNA