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Titolo:
POTENT 2'-AMINO-2'-DEOXYPYRIMIDINE RNA INHIBITORS OF BASIC FIBROBLASTGROWTH-FACTOR
Autore:
JELLINEK D; GREEN LS; BELL C; LYNOTT CK; GILL N; VARGEESE C; KIRSCHENHEUTER G; MCGEE DPC; ABESINGHE P; PIEKEN WA; SHAPIRO R; RIFKIN DB; MOSCATELLI D; JANJIC N;
Indirizzi:
NEXSTAR PHARMACEUT INC,2860 WILDERNESS PL BOULDER CO 80301 NEXSTAR PHARMACEUT INC BOULDER CO 80301 NYU,MED CTR,RAYMOND & BEVERLY SACKLER FDN LAB,KAPLAN CTR NEW YORK NY 10016 NYU,MED CTR,RAYMOND & BEVERLY SACKLER FDN LAB,DEPT CELL BIOL NEW YORKNY 10016
Titolo Testata:
Biochemistry
fascicolo: 36, volume: 34, anno: 1995,
pagine: 11363 - 11372
SICI:
0006-2960(1995)34:36<11363:P2RIOB>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
PLASMINOGEN-ACTIVATOR PRODUCTION; ENDOTHELIAL-CELL-GROWTH; NUCLEOTIDE-SEQUENCE; FACTOR RECEPTOR; BINDING-SITES; DNA-SYNTHESIS; AFFINITY; ANGIOGENESIS; LIGANDS; POLYMERASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
69
Recensione:
Indirizzi per estratti:
Citazione:
D. Jellinek et al., "POTENT 2'-AMINO-2'-DEOXYPYRIMIDINE RNA INHIBITORS OF BASIC FIBROBLASTGROWTH-FACTOR", Biochemistry, 34(36), 1995, pp. 11363-11372

Abstract

Screening of random oligonucleotide libraries with SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 595-510] 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 havebeen 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 atleast 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 (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 witha 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 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 K-d(bFGF)/K-d(protein) 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 [I-125]bFGF binding to both low-affinity sites (ED(50) approximate to 1nM) and high-affinity sites (ED(50) approximate to 3 nM) on CHO cellsexpressing 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 ED(50) values of 50-100 nM. The 2'-aminopyrimidine RNA ligand m21A therefore represents a useful lead compound in our efforts to develop potent oligonucleotide-based angiogenesis antagonists.

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Documento generato il 04/12/20 alle ore 16:42:00