Faculty Bibliography

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 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 (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 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 1 nM) and high-affinity sites (ED(50) approximate to 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 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

Two cases are described in which patients with type I Gaucher disease developed extraosseous soft tissue masses consisting of Gaucher cell deposits. In one instance the mass destroyed the posterior cortex of the left distal femur and protruded into the soft tissues. In the second case the lesion involved the proximal tibia and gradually extended into the soft tissues. While the incidence of neoplastic disorder such as lymphoproliferative disease appears to be more common in Gaucher disease patients than in the general population, lesions of benign etiology that mimic these aggressive processes should be considered in the differential diagnosis when cortical destruction with coexisting soft tissue most is found in these patients