We have identified a group of DNA molecules that bind to platelet-derived growth factor (PDGF)-AB with subnanomolar affinity from a randomized DNA library using in vitro selection. Individual ligands cloned from the affinity-enriched pool bind to PDGF-AB and PDGF-BB with comparably high affinity (Kd approximately 10(-10) M) and to PDGF-AA with lower affinity (> 10(-8) M), indicating specific recognition of the PDGF B-chain in the context of the hetero- or homodimer. The consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a three-nucleotide loop at the branch point. Photo-cross-linking experiments with 5-iodo-2'-deoxyuridine-substituted ligands establish a point contact between a thymidine nucleotide in the helix junction loop region and phenylalanine 84 of the PDGF-B chain. Representative minimal DNA ligands inhibit the binding of 125I-PDGF-BB but not of 125I-PDGF-AA to PDGF alpha- or beta-receptors expressed in porcine aortic endothelial (PAE) cells in a concentration-dependent manner with half-maximal effects of approximately 1 nM. The same ligands also exhibit a similar inhibitory effect on PDGF-BB-dependent [3H]thymidine incorporation in PAE cells expressing the PDGF beta-receptors. These DNA ligands represent a novel class of specific and potent antagonists of PDGF-BB and, by inference, PDGF-AB.

We have used the SELEX (systematic evolution of ligands by exponential enrichment) process in conjunction with post-SELEX modifications to define a highly nuclease-resistant oligonucleotide that binds to VPF/VEGF with high affinity and specificity.

Stanniocalcin (STC) is a glycoprotein hormone that is secreted by the corpuscle of Stannius, an endocrine gland of bony fish, and is involved in calcium and phosphate homeostasis. The related mammalian proteins, STC1 and STC2, are expressed in a wide variety of tissues. The ovaries have the highest level of STC1, and this increases during pregnancy and lactation. STC1 is present in breast ductal epithelium, and its expression is induced by BRCA1, a tumor suppressor gene that has an important role in breast and ovarian cancer. The expression of STC2 is induced by estrogen, and there is a positive correlation between the level of expression of estrogen receptor and expression of both STC1 and STC2 in breast cancer. This article reviews the data currently available regarding the mammalian STCs, and discusses the roles they may play in normal physiology and in breast and other cancers.

The proliferation of new blood vessels (angiogenesis) is a process that accompanies many pathological conditions including rheumatoid arthritis and solid tumor growth. Among angiogenic cytokines that have been identified to date, vascular endothelial growth factor (VEGF) is one of the most potent. We used SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505-510] to identify RNA ligands that bind to VEGF in a specific manner with affinities in the low nanomolar range. Ligands were selected from a starting pool of about 10(14) RNA molecules containing 30 randomized positions. Isolates from the affinity-enriched pool were grouped into six distinct families on the basis of primary and secondary structure similarities. Minimal sequence information required for high-affinity binding to VEGF is contained in 29-36-nucleotide motifs. Binding of truncated (minimal) high-affinity ligands to VEGF is competitive with that of other truncated ligands and heparin. Furthermore, truncated ligands from the six ligand families inhibit binding of [125I]VEGF to its cell-surface receptors. Oligonucleotide ligands described here represent an initial set of lead compounds in our ongoing effort toward the development of potent and specific VEGF antagonists.

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)

Reiterative in vitro selection-amplification from random oligonucleotide libraries allows the identification of molecules with specific functions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resistance. Libraries of RNA molecules containing 2'amino-(2'NH2)- or 2'fluoro-(2'F)-2'-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is because the intramolecular helices containing 2'NH2 have lower melting temperatures (Tm) compared with helices containing 2'F, giving them thermodynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human keratinocyte growth factor from libraries containing modified RNA molecules with either 2'NH2 or 2'F pyrimidines. We demonstrated that 2'F RNA ligands have affinities (Kd approximately 0.3-3 pM) and bioactivities (Ki approximately 34 pM) superior to 2'NH2 ligands (Kd approximately 400 pM and Ki approximately 10 nM). In addition, 2'F ligands have extreme thermo-stabilities (Tm approximately 78 degrees C in low salt, and specificities).

We In cell culture, bFGF binds to low-affinity and high-affinity sites. The low-affinity sites are composed of cell-associated heparan sulfate proteoglycans to which bFGF binds with approximately nanomolar affinity (6). All biological effects of bFGF appear to be mediated through its interaction with the tyrosine kinase FGF receptors, which represent the highaffinity binding sites (Kd = 10-100 pM) (2). There is now substantial evidence that the low-affinity and the high-affinity receptor sites act cooperatively in determining the overall affinity of bFGF (7,8 ment (SELEX) is a prototypic method for isolating rare nucleic acid molecules that have high affinity for a target molecule from a large randomized sequence set by iterative rounds of affmity selection and amplification (13). This procedure has so far yielded tight-binding oligonucleotide ligands for a number of target molecules, including nucleic acid binding proteins (13-17), non-nucleic-acid binding proteins (12,18), and organic dyes (19,20). In this paper we describe and characterize a set of high-affinity RNA ligands for bFGF that were selected from a pool of 1014 molecules and show that these ligands inhibit binding ofthe growth factor to its cell-surface receptors.EXPERIMENTAL PROCEDURES Materials. Human recombinant bFGF (Mr 18,000, 154 amino acids) was obtained from Bachem California. Other reagents and chemicals were at least reagent grade and were purchased from commercial sources.SELEX. Essential features of the SELEX protocol have been described in detail (13,21). Initial selections were done with 200 pmol (1014 molecules) of RNA that contained a region of 30 randomized positions (Fig. 1). All selections were done at 37°C in phosphate-buffered saline (PBS = 10.1 mM Na2HPO4/1.8 mM KH2PO4/137 mM NaCl/2.7 mM KCl, pH 7.4).Equilibrium Dissociation Constants. Affinity of RNA ligands for bFGF was determined by nitrocellulose filter binding (13). To eliminate dimerization and other higher order structure formation, all RNA solutions were diluted to about 1 nM in PBS, heated to 90°C for 2 min, and then cooled on ice prior to incubation with the protein. After [1] where q is the fraction of bound RNA at equilibrium, Pt and Rt are total bFGF (protein) and total RNA concentrations, andfreflects the efficiency of retention of the protein-RNA complexes on nitrocellulose filters. The average value offin our assays was 0.82. A number of RNA ligands clearly exhibited biphasic binding to bFGF. For those ligands, binding of RNA to bFGF is described by a model where total RNA is assumed to be partitioned between two components (R1 and R2) that do not interconvert on the time scale of the experiment and that bind to bFGF with different affinities.Abbreviations: bFGF, basic fibroblast growth factor; SELEX, systematic evolution of ligands by exponential enrichment.

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with (32)P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the in vivo kinase. STC2 was phosphorylated in vitro by casein kinase II (CK2), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization-time-of-flight MS to be a single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.