Tenascin-C aptamers are generated using tumor cells and purified protein.

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.

A high throughput platform for systematic evolution of ligands by exponential enrichment (SELEX).

The systematic evolution of ligands by exponential enrichment (SELEX) process is a combinatorial chemistry method for the isolation of nucleic acid ligands (aptamers) that bind to a desired target molecule with high affinity. In order to increase throughput via automation, we have adapted the SELEX process for protein targets to a robotics-compatible microtiter plate format. A remarkable feature of the platform is that targets are immobilized by hydrophobic adsorption onto the plate surface. Hydrophobic immobilization procedures are simple and require no specialized modification of the protein target. This format was tested by manually performing four independent SELEX experiments. All were concluded within 8 rounds of selection and yielded aptamers that bind in solution to their respective protein target, calf intestinal alkaline phosphatase, human alpha-thrombin or human platelet derived growth factor, with equilibrium dissociation constants below 3 x 10-10 M.

Post-SELEX combinatorial optimization of aptamers.

In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine.

The Oxytricha telomere protein specifically recognizes single-stranded telomeric DNA, forming an extremely salt resistant and kinetically stable nucleoprotein complex. The absence of information on how this heterodimeric protein binds to DNA prompted this photo-cross-linking study. Multiple protein-DNA photo-cross-links are formed upon UV irradiation of Oxytricha telomeres reconstituted with a synthetic oligonucleotide terminating in 5'-T16T15T14T13G12G11G10G9T8T7T6T5G4G3G2G1-3'. Site-specific substitution of certain nucleotides with 5-bromodeoxyuridine (BrdU) greatly increased the photo-cross-linking yield, each substitution favoring a specific protein-DNA cross-link. For example, substitution of BrdU for T7 resulted in 25% cross-linking of the bound DNA, a 10-fold increase over the unsubstituted DNA. Both subunits of the telomere protein cross-link to, and are therefore near, the DNA. Three point contacts within this nucleoprotein complex, involving the alpha subunit, were established using BrdU substitution: Tyr239, Tyr142, and His292 cross-link to G3, T15, and T7, respectively. One photo-cross-link, Tyr239-G3, occurs amid a short acidic stretch of the alpha subunit, counter to expectations for amino acids that approach the polyanionic DNA. The two remaining cross-links are to amino acids in hydrophobic regions of the primary polypeptide sequence, consistent with the hypothesis that hydrophobic interactions account for the salt resistance (> 2 M NaCl) of this protein-DNA complex. These two photo-cross-links suggest that the telomere protein may bind telomeric single-stranded DNA by intercalation of aromatic residues into a nucleotide lattice.

Photocrosslinking of 5-iodouracil-substituted RNA and DNA to proteins.

5-Iodouracil-substituted RNA and DNA were crosslinked regiospecifically to associated proteins in yields of 70 to 94% of bound nucleic acid. Irradiation of the iodouracil chromophore with monochromatic, long-wavelength ultraviolet radiation (325 nanometers) eliminates excitation of other nucleic acid and protein chromophores. The combination of high crosslinking yields, excellent specificity, and elimination of photodamage to other chromophores represents an important advance toward the precise identification of contacts in nucleoprotein complexes.

Two versions of the gene encoding the 41-kilodalton subunit of the telomere binding protein of Oxytricha nova.

Macronuclear chromosomes of the ciliated protozoan Oxytricha nova terminate with a single-stranded (T4G4)2 overhang. The (T4G4)2 telomeric overhang is tenaciously bound by a protein heterodimer. We have cloned and sequenced the gene encoding the 41-kDa subunit of this telomere binding protein. The predicted amino acid sequence comprises two distinct regions, a carboxyl-terminal two-thirds that is 23% lysine and bears similarity to histone H1 and an amino-terminal one-third containing a hydrophobic stretch of about 15 amino acids. Two macronuclear versions of the gene differ in nucleotide sequence at several positions, but the derived polypeptides differ only at a single position, Ser-110 or Ala-110. Both versions harbor a small intron. The existence of this intron demonstrates that, despite the elimination of 95% of the micronuclear genome from the developing macronucleus, at least some noncoding DNA is retained during macronuclear development of hypotrichous ciliates.

Stereoselective arginine binding is a phylogenetically conserved property of group I self-splicing RNAs.

We have examined the reaction of GTP with RNA polymerase transcripts containing the self-splicing RNA precursors from the Neurospora crassa Cob1 intron, and from introns in the sunY, nrdB and td genes of bacteriophage T4. In each case, we find a low Km for GTP (between 0.8 and 11 microM), accompanied by competitive inhibition of the GTP reaction by L-arginine, as was found for the previously examined Tetrahymena nuclear pre-rRNA intron. Trials with the 20 standard amino acids show that inhibition in all cases is specific to the arginine side-chain. L-arginine binds with similar affinity to all introns studied, the Ki's ranging from 4.3 to 21 mM. Strikingly, the relative binding preference of the RNAs for L- versus D-arginine is highly conserved: the ratio of L-arg Ki/D-arg Ki, the stereoselectivity, is always close to 2. Because of the conservation of GTP and arginine binding constants and particularly because of the conserved stereoselectivity, we conclude that the evolution of an effective group I RNA transesterification catalyst necessarily produces a specific and stereoselective RNA binding site for a single amino acid. This suggests that selection for an ancient group I RNA could have fortuitously initiated the specific association of RNA sequences with amino acids, a first step toward the genetic code.

Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA.

The telomere binding protein from macronuclei of Oxytricha nova binds macronuclear DNA in vitro, protecting the 3'-terminal single-stranded (T4G4)2 tail from chemical and enzymatic probes. We have used synthetic oligodeoxynucleotides to study the binding properties of the telomere protein. It binds at the 3' end of single-stranded oligonucleotides that have the sequence (T4G4)n, where n greater than or equal to 2, reconstituting the methylation protection seen with macronuclear DNA. Three oligonucleotide.protein complexes are resolved in nondenaturing gels, all specific for this sequence. Single-stranded oligonucleotides that have one or more repeats of the sequence C4A4 are also recognized, forming a single complex. The dissociation constant for (T4G4)4 is about 19 nM, and for macronuclear DNA is at least 20-fold lower. The basis for this difference is not fully understood, but it is not simply due to the absence of a (C4A4)2.5.(G4T4)2.5 region on the oligonucleotide. Transversions of T's to A's or of G's to C's in the 3' tail portion prevent binding. Changing T's to dU's does not prevent binding, indicating that the hydrophobic 5-methyl group is not required for binding as had been suggested from the salt-stability of the complex. The properties of the DNA-protein complex suggest a revised model for telomere synthesis in Oxytricha.

Catalytic and noncatalytic nucleotide binding sites of the Escherichia coli F1 ATPase. Amino acid sequences of beta-subunit tryptic peptides labeled with 2-azido-ATP.

Under appropriate conditions tight, noncovalent binding of 2-azido-adenine nucleotides to either catalytic or noncatalytic binding sites on the E. coli F1-ATPase occurs. After removal of unbound ligands, UV-irradiation results primarily in the covalent incorporation of nucleotide moieties into the beta-subunit in both catalytic and noncatalytic site labeling experiments. Minor labeling of the alpha-subunit was also observed. After trypsin digestion and purification of the labeled peptides, microsequencing studies identified two adjacent beta-subunit tryptic peptides labeled by 2-azido-ADP or -ATP. These beta-subunit peptides were labeled on tyrosine-331 (catalytic sites) and tyrosine-354 (noncatalytic sites) in homology with the labeling patterns of the mitochondrial and chloroplast enzymes.