Abstract

To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-alpha aptamer named T1-T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-alpha. At first, 54 mer T1-T4 aptamers with 5 '-NH2 groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-alpha aptamer and its dissociation constant (K-d) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-alpha activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1-T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1-T4 aptamer significantly improved its anti-TNF-alpha activity compared to non-modified aptamers (P < 0.0001) from 40 to 60. The determination of K-d showed that trimerization could effectively enhance K-d of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 +/- 1.2 mm to 1.6 +/- 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-alpha aptamer with a novel approach could improve the anti-TNF-alpha activity and therapeutic efficacy. According to our findings, a new anti-TNF-alpha aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases.