Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα  

作　　者：XinruiYan XuwenGao ZhiqingZhang 

机构地区：[1]StateKeyLaboratoryforMolecularVirologyandGeneticEngineering,InstituteofViralDiseaseControlandPrevention,ChineseCenterforDiseaseControlandPrevention,Beijing100052,China

出　　处：《Genomics, Proteomics & Bioinformatics》2004年第1期32-42,共11页基因组蛋白质组与生物信息学报（英文版）

摘　　要：Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine withactivities ranging from host defense mechanisms in infection and injury to severe toxicity in septicshock or other related diseases, is a promising target for drug screening. Using the SELEX(systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotideligands (aptamers) with high affinities for hTNFα. Aptamers were selected from a starting pool of40 randomized sequences composed of about 10^(15) RNA molecules. Representative aptamers weretruncated to the minimal length with high affinity for hTNFα and were further modified byreplacement of 2'-OH with 2'-F and 2'-NH_2 at all ribopurine positions. These modified RNA aptamerswere resistant to nuclease. The specificity of these aptamers for hTNFα was confirmed, and theiractivity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Resultsdemonstrated that four 2'-NH_2-modified aptamers bound to hTNFα with high affinity and blocked thebinding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα.Oligonucleotide aptamers described here are potential therapeutics and diagnostics forhTNFα-related diseases.Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine withactivities ranging from host defense mechanisms in infection and injury to severe toxicity in septicshock or other related diseases, is a promising target for drug screening. Using the SELEX(systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotideligands (aptamers) with high affinities for hTNFα. Aptamers were selected from a starting pool of40 randomized sequences composed of about 10^(15) RNA molecules. Representative aptamers weretruncated to the minimal length with high affinity for hTNFα and were further modified byreplacement of 2'-OH with 2'-F and 2'-NH_2 at all ribopurine positions. These modified RNA aptamerswere resistant to nuclease. The specificity of these aptamers for hTNFα was confirmed, and theiractivity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Resultsdemonstrated that four 2'-NH_2-modified aptamers bound to hTNFα with high affinity and blocked thebinding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα.Oligonucleotide aptamers described here are potential therapeutics and diagnostics forhTNFα-related diseases.

关 键 词：SELEX HTNFΑ APTAMER AFFINITY RNA ELONA 

分 类 号：Q51[生物学—生物化学] Q522