Natl Acad. and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids. INTRODUCTION Aptamers are folded single-stranded nucleic acids that can target given molecules. The concept is based on the ability of short (20C80 mer) sequences to fold, in the presence of a target, into unique 3D structures, which allow the aptamer to bind target molecules with high affinity and specificity (1C3). Therefore, aptamers can be thought of as nucleic-acid analogs to antibodies. RNA aptamers are selected from a combinatorial library of randomized sequences (4C8) by selection, known as SELEX (systematic evolution of ligands by exponential enrichment), and the target molecules can be nucleic acids, proteins, or small organic compounds. Aptamers have therefore many potential uses in medicine and technology. The first aptamer-based therapeutic, Pegaptanib (Macugen), which targets vascular endothelial growth factor, was approved by the FDA in 2004 for the treatment of age related macular degeneration (AMD) (9,10). Considering the basic principles of aptamer selection, the high potential of RNA to create a vast set of tertiary structures, which we referred to as RNA plasticity (11), is conceivable from both the RNA world hypothesis (12) and Alectinib Hydrochloride the concept of molecular mimicry between RNA and protein (13). Whereas RNA aptamers have been examined for their 3D structures by X-ray crystallography or NMR spectroscopy (14), only three high resolution structures of RNA aptamers in complex with target proteins have been reported: RNA aptamers in complex with NF-B solved at 2.45?? (15), with bacteriophage MS2 capsid at 2.8?? (16) and with thrombin at 1.9?? resolutions (17). NF-B and bacteriophage MS2 capsid naturally bind to nucleic acids. The crystal structures of RNA aptamers in complex with NF-B and bacteriophage MS2 capsid indicate that the aptamers bind to the respective nucleic-acid-binding sites and mimic naturally occurring electrostatic interactions (16,18). Thrombin does not naturally bind to nucleic acid, but bears an extensive positively charged surface responsible for heparin binding. The crystal structure of an RNA aptamer in complex with Rabbit Polyclonal to ZADH2 thrombin demonstrated that the RNA aptamer binds to the positively charged surface required for heparin binding (17). Thus, the crystal structures determined to date have suggested that RNA aptamers bind target proteins predominantly through electrostatic forces. Using SELEX, we have previously identified an RNA aptamer containing 2-fluoro pyrimidine nucleotides that binds to the Fc portion of human IgG1 (hFc1) in the presence of Ca2+ and Mg2+ (19). A unique characteristic of the Fc fragments, including hFc1, is the absence of an extensive positively charged molecular surface (20); it is thus tempting to speculate that the RNA aptamer may interact with the hFc1 via non-electrostatic forces. The aptamers high specificity Alectinib Hydrochloride to human IgGs and its requirement of divalent cations for binding activity are additional distinctive characteristics (19). This specificity provides us with an alternative reagent for the mass purification of therapeutic antibodies (19), as described earlier. Protein A affinity chromatography is currently the most common procedure used for purification of humanized or chimeric antibodies (21,22). This process requires an acidic elution step, which may sometime cause unexpected aggregation or denaturing of antibodies (22C24). Aptamer Alectinib Hydrochloride bound IgGs are instead easily released from the aptamer resin under neutral pH conditions using simple elution buffers, such as an EDTA solution (19). This is a potential advantage of aptamers for affinity purification. In this study, to understand the structural basis of the Alectinib Hydrochloride hyperspecificity and high affinity of the anti-hFc1 aptamer as well as prompt release of bound IgG by EDTA, we solved the crystal structure of the aptamerChFc1 complex at the 2 2.15-? resolution. MATERIALS AND METHODS Crystallization and data collection Human Alectinib Hydrochloride IgG1 was purchased from Calbiochem (USA). To produce Fc fragments, human IgG1 was digested with papain (Wako Pure Chemical Industries Ltd, Japan) at 37C for 1?h in 100?mM sodium phosphate (pH 7.2), 10?mM l-cysteine and 2?mM EDTA (molar ratio of IgG1:papain is 17:1).The reaction was stopped by the addition of protease inhibitor. The Fc fragment was purified with Protein A column (GE Healthcare) (25). The chemically synthesized RNA aptamer containing 2-fluoropyrimidines (Figure 1a) was purchased from GeneDesign Inc. (Japan) and purified as described previously (25). The.
