A tenascin-C aptamer identified by tumor cell SELEX: Systematic evolution of ligands by exponential enrichment

Abstract

The targeting of molecular repertoires to complex systems rather than biochemically pure entities is an accessible approach that can identify proteins of biological interest. We have probed antigens presented by a monolayer of tumor cells for their ability to interact with a pool of aptamers. A glioblastoma-derived cell line, U251, was used as the target for systematic evolution of ligands by exponential enrichment by using a single-stranded DNA library. We isolated specifically interacting oligonucleotides, and biochemical strategies were used to identify the protein target for one of the aptamers. Here we characterize the interaction of the DNA aptamer, GBI-10, with tenascin-C, an extracellular protein found in the tumor matrix. Tenascin-C is believed to be involved in both embryogenesis and oncogenesis pathways. Systematic evolution of ligands by exponential enrichment appears to be a successful strategy for the a priori identification of targets of biological interest within complex systems. T he use of molecular repertoires is becoming increasingly important in the fields of drug discovery and biological research (1-3). These strategies involve the selection of combinatorially derived species. The most accessible techniques are based on the phage display of peptide or antibody libraries (4-8) and the use of libraries of oligonucleotides (9, 10). Systematic evolution of ligands by exponential enrichment (SELEX) is an iterative selection procedure used to identify oligonucleotides with desired properties, most often binding to a molecular target. The starting libraries (11,12) are as large as 10 15 unique sequences, some of which will be able to adopt secondary and tertiary structures (13). High-affinity oligonucleotide ligands to a plethora of high-and low-molecular-weight targets have been identified (3). However, the vast majority of these experiments have targeted biochemically pure entities.The targeting of complex systems with SELEX lends itself to the concept of a priori identification of targets of biological interest and possibly to in vivo efficacy of such bioactive molecules. Here we demonstrate oligonucleotide targeting of the glioblastoma cell line U251. Glioblastomas are the most common of the human brain malignancies (14-16). Their aggressive nature is believed to be due to a combination of hypervascularity, focal necrosis, and rapid cellular proliferation. The glioblastoma remains refractory to therapy because of tumor heterogeneity, local invasion, and nonuniform vascular permeability to drugs. Our goal was to generate oligonucleotide ligands that recognize tumor-associated proteins on͞within living cells, simultaneously identifying target proteins and DNA aptamers. Experimental ProceduresCell SELEX. Synthetic DNA template (10 pmol; Operon Technologies, Alameda, CA) containing 34 random nucleotides flanked by fixed regions 5Ј-GCCTGTTGTGAGCCTCCT-N34-CGCT-TATTCTTGTCTCCC-3Ј complementary to the primers 5Ј-BBB-GCCTGTTGTGAGCCTCCT-3Ј and 5Ј-GGGAGACAA-GA ATA AGCG-3, where BBB denotes three biotin ph...

Tenascin-C: Exploitation and collateral damage in cancer management

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Gene therapy progress and prospects: RNA aptamers

“…In particular, the concept of 'complex-target' selection, where targets are not pure proteins but may include whole organisms, intact cells or human plasma, may lead to the identification of aptamers against proteins that can only be targeted in their physiological milieu. [65][66][67][68] In some biological systems, agonists may be of more therapeutic importance than antagonists and we envision that agonistic aptamers will soon be realized. Furthermore, the increasing focus on the use of aptamers as tools for diagnostics, target validation and drug discovery will maximize the potential of aptamers in therapeutics.…”

Activatable aptamer probe for contrast-enhanced in vivo cancer imaging based on cell membrane protein-triggered conformation alteration

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