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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).

Aptamers are small oligonucleotides that are selected to bind tightly and specifically to a target molecule. We sought to determine whether aptamers have potential for in vivo delivery of radioisotopes or cytotoxic agents.TTA1, an aptamer to the extracellular matrix protein tenascin-C, was prepared in fluorescent and radiolabeled forms. After in vivo administration, uptake and tumor distribution of Rhodamine Red-X-labeled aptamer was studied by fluorescence microscopy. In glioblastoma (U251) and breast cancer (MDA-MB-435) tumor xenografts, biodistribution and imaging studies were performed using TTA1 radiolabeled with (99m)Tc. Tenascin-C levels and tumor uptake were studied in a variety of additional human tumor xenografts. To assess the effect of radiometal chelate on biodistribution, mercapto-acetyl diglycine (MAG(2)) was compared with diethylenetriaminepentaacetic acid and with MAG(2)-3,400-molecular-weight PEG (PEG(3,400)).Intravenous injection of fluorescent aptamer TTA1 produced bright perivascular fluorescence in a xenografted human tumor within 10 min. In the ensuing 3 h, fluorescence diffused throughout the tumor. Labeled with (99m)Tc, TTA1 displayed rapid blood clearance, a half-life of less than 2 min, and rapid tumor penetration: 6% injected dose (%ID)/g at 10 min. Tumor retention was durable, with 2.7 %ID/g at 60 min and a long-lived phase that stabilized at 1 %ID/g. Rapid tumor uptake and blood clearance yielded a tumor-to-blood ratio of 50 within 3 h. Both renal and hepatic clearance pathways were observed. Using the (99m)Tc-labeled aptamer, images of glioblastoma and breast tumors were obtained by planar scintigraphy. Aptamer uptake, seen in several different human tumors, required the presence of the target protein, human tenascin-C. Modification of the MAG(2) radiometal chelator dramatically altered the uptake and clearance patterns.TTA1 is taken up by a variety of solid tumors including breast, glioblastoma, lung, and colon. Rapid uptake by tumors and rapid clearance from the blood and other nontarget tissues enables clear tumor imaging. As synthetic molecules, aptamers are readily modified in a site-specific manner. A variety of aptamer conjugates accumulate in tumors, suggesting imaging and potentially therapeutic applications.

Tenascin-C (TN-C) is an extracellular matrix protein that is overexpressed during tissue remodeling processes, including tumor growth. To identify an aptamer for testing as a tumor-selective ligand, SELEX (systematic evolution of ligands by exponential enrichment) procedures were performed using both TN-C and TN-C-expressing U251 glioblastoma cells. The different selection techniques yielded TN-C aptamers that are related in sequence. In addition, a crossover procedure that switched from tumor cell to purified protein selections was effective in isolating two high-affinity TN-C aptamers. When targeting tumor cells in vitro, the observed propensity of naive oligonucleotide pools to evolve TN-C aptamers may be due to the abundance of this protein. In vivo, TN-C abundance may also be well suited for aptamer accumulation in the tumor milieu. A size-minimized and nuclease-stabilized aptamer, TTA1, binds to the fibrinogen-like domain of TN-C with an equilibrium dissociation constant (K(d)) of 5 x 10(-9) m. At 13 kDa, this aptamer is intermediate in size between peptides and single chain antibody fragments, both of which are superior to antibodies for tumor targeting because of their smaller size. TTA1 defines a new class of ligands that are intended for targeted delivery of radioisotopes or chemical agents to diseased tissues.

Therapeutic and diagnostic applications have been envisioned for aptamers, a class of oligonucleotide ligands that bind their target molecules with high affinity and specificity (Gold, J. Biol. Chem. 270, 13581-13584, 1995). To identify parameters that are important for the in vivo activity of aptamers acting on intravascular targets, we have studied binding characteristics in vitro, pharmacokinetic parameters in Sprague-Dawley rats, and inhibitory activity in a SCID mouse/human lymphocyte model of lymphocyte trafficking for both 2'F pyrimidine 2'OH purine RNA and ssDNA anti-human L-selectin aptamers. The data indicate that aptamers with low nanomolar affinity are suitable candidates for use as in vivo reagents and that nonspecific binding to vascular cells is not an issue for efficacy. As is often observed for other reagents, plasma clearance is biphasic. Both the distribution phase and the clearance rate strongly affect in vivo activity. Pharmacokinetic parameters and in vivo activity are significantly improved by conjugating aptamers to a carrier molecule, such as polyethylene glycol (PEG). Most active in vivo is 1d40, a 2'F pyrimidine 2'OH purine aptamer conjugated to 40 kDa PEG. At a dose of 5.4 nmol/kg body weight, its duration of effect (time to 50% inhibition) is 11.2 hours, and at 1 mg or 90 nmol/kg, its plasma clearance rate (CL) is 0.4 ml/min/kg. Its ED50 is estimated to be 80 pmol/kg in preinjection dose-response experiments, compared with 4 pmol/kg for the dimeric anti-L-selectin antibody DREG56. Further improvement of in vivo activity is expected from nucleotide modifications that increase resistance to nuclease digestion for aptamers where mass is not rate limiting for clearance. Because the relationship of clearance to conjugate molecular weight (MW) is not the same for all aptamers, it is advisable to determine the relationship at the outset of in vivo studies. In summary, the data suggest that properly formulated aptamers have the capacity to be effective therapeutic agents against intravascular targets.

Oligonucleotide ligands that bind a protein or a small molecule of interest are readily isolated by in vitro selection and amplification of rare sequences from combinatorial libraries of sequence-randomized oligonucleotides (Gold et al., 1995). Classic systematic evolution of ligands by exponential enrichment (SELEX) protocols are affinity based (Tuerk and Gold, 1990), but because many problems and applications require antagonists, protocols for selecting inhibitors are both desirable and valuable. A widely applicable approach for isolating inhibitors is competitive elution with a molecule that binds the targeted molecule's active or binding site. We have used this approach to isolate antagonists of wheat germ agglutinin (WGA) from a library of 2'NH2-pyrimidine, 2'OH-purine oligonucleotides by elution with N N' N"-triacetylchitotriose, (GlcNAc)3. The highest affinity aptamers have equilibrium dissociation constants of 1 nM-20 nM for WGA, a 10(3)-10(4)-fold improvement relative to (GlcNAc)3, and unlike the carbohydrate, are highly specific. In addition to competing for binding with (GlcNAc)3, aptamers inhibit WGA-mediated agglutination of sheep erythrocytes, demonstrating that they are able to compete with natural ligands presented on the surfaces of cells. These results illustrate the feasibility of isolating high-affinity, high-specificity antagonists by competitive elution with low molecular weight, relatively low-affinity, and low-specificity small molecules.

A family of sequence-related 2'-aminopyrimidine, 2'-hydroxylpurine aptamers, developed by oligonucleotide-based combinatorial chemistry, SELEX (systematic evolution of ligand by exponential enrichment) technology, binds human nonpancreatic secretory phospholipase A2 (hnps-PLA2) with nanomolar affinities and inhibits enzymatic activity. Aptamer 15, derived from the family, binds hnps-PLA2 with a Kd equal to 1.7 +/- 0.2 nM and, in a standard chromogenic assay of enzymatic activity, inhibits hnps-PLA2 with an IC50 of 4 nM, at a mole fraction of substrate concentration of 4 x 10(-6) and a calculated Ki of 0.14 nM. Aptamer 15 is selective for hnps-PLA2, having a 25- and 2500-fold lower affinity, respectively, for the unrelated proteins human neutrophil elastase and human IgG. Contractions of guinea pig lung pleural strips induced by hnps-PLA2 are abolished by 0.3 microM aptamer 15, whereas contractions induced by arachidonic acid are not altered. The structure that is essential for binding and inhibition appears to be a 40-base hairpin/loop motif with an asymmetrical internal loop. The affinity and activity of the aptamers demonstrate the ability of the SELEX process to isolate antagonists of nonnucleic-acid-binding proteins from vast oligonucleotide combinatorial libraries.

The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.

Milk proteins are crucial for the development of all newborn mammals. Caseins that constitute the bulk of the protein in mammalian milk have been shown to be members of a multigene family in at least two species. They are among the most rapidly diverging groups of proteins, and their numbers vary widely among species. beta- and kappa-Caseins are the only caseins present in human milk. Using polymerase chain reaction on genomic DNA from somatic cell hybrids, we have localized the human beta-casein gene (CSN2) to 4pter----q21.