%0 Journal Article
%T Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins
%A Sreerupa Challa
%A Saul Tzipori
%A Abhineet Sheoran
%J Journal of Nucleic Acids
%D 2014
%I Hindawi Publishing Corporation
%R 10.1155/2014/214929
%X Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells. 1. Introduction Infection with Shiga toxin- (Stx-) producing Escherichia coli (STEC) is the most significant cause of hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children [1¨C4]. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS as STEC strains producing this toxin are more frequently associated with HUS than strains that produce both Stx1 and Stx2, while Stx1 alone has rarely been associated with HUS [5¨C7]. Stx1 and Stx2 are similar in basic structure [8], binding specificity [8], and mode of action. Both toxins consist of an A-subunit monomer and a B-subunit pentamer [8¨C10]. The pentameric B subunit binds to its cell surface receptor CD77, also called globotriaosylceramide (Gb3; Gal¦Á1-4Gal¦Â1-4glucosyl ceramide) [11, 12]. This triggers endocytosis of the holotoxin, mainly through clathrin-coated pits [13]. The internalization of catalytically active A-subunit, delivered to cytosol via retrograde transport, causes shut down of protein synthesis and leads to cell death [14, 15]. Nucleic acid aptamers are being developed as therapeutic and diagnostic reagents against biotoxins [16]. Aptamers are short, synthetic, single stranded nucleic acids with unique three-dimensional conformations that bind strongly and selectively to a target molecule via shape specific recognition [17¨C20]. Aptamers are similar in many respects to antibodies except that they are cheaper to produce, have extremely long shelf lives, and thus, are far
%U http://www.hindawi.com/journals/jna/2014/214929/