Isolation and characterization of 2'-amino-modified RNA aptamers for human TNFalpha

Human tumor necrosis factor alpha (hTNFalpha), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFalpha. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 10(15) RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFalpha and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFalpha was confirmed, and their activity to inhibit the cytotoxicity of hTNFalpha on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFalpha with high affinity and blocked the binding of hTNFalpha to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFalpha. Oligonucleotide aptamers described here are potential therapeutics and diagnostics for hTNFalpha-related diseases.