Aptamers are non-naturally occurring structured oligonucleotides that may bind to small molecules, peptides, and proteins. Typically, aptamers are generated by an in vitro selection process referred to as SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind with high affinity and specificity to proteins that reside on the cell surface have potential utility as therapeutic antagonists, agonists, and diagnostic agents. When the target protein requires the presence of the cell membrane (e.g., G-protein-coupled receptors, ion channels) or a co-receptor to fold properly, it is difficult or impossible to program the SELEX experiment with purified, soluble protein target. Recent advances in which the useful range of SELEX has been extended from comparatively simple purified forms of soluble proteins to complex mixtures of proteins in membrane preparations or in situ on the surfaces of living cells offer the potential to discover aptamers against previously intractable targets. Additionally, in cases in which a cell-type specific diagnostic is sought, the most desirable target on the cell surface may not be known. Successful application of aptamer selection techniques to complex protein mixtures can be performed even in the absence of detailed target knowledge and characterization. This Account presents a review of recent work in which membrane preparations or whole cells have been utilized to generate aptamers to cell surface targets. SELEX experiments utilizing a range of target "scaffolds" are described, including cell fragments, parasites and bacteria, viruses, and a variety of human cell types including adult mesenchymal stem cells and tumor lines. Complex target SELEX can enable isolation of potent and selective aptamers directed against a variety of cell-surface proteins, including receptors and markers of cellular differentiation, as well as determinants of disease in pathogenic organisms, and as such should have wide therapeutic and diagnostic utility.

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Chorismate mutase catalyses the [3,3] Claisen rearrangement of chorismate (1) to prephenate (2), the committed step in the biosynthesis of tyrosine and phenylalanine in bacteria, fungi, and higher plants 1 (Scheme 1). Despite more than two decades of studies on this novel biological rearrangement, 2-15 the mechanism of the enzyme-catalyzed reaction remains unclear. Substrate labeling 5,7 and kinetic isotope effect studies 6 demonstrated that both the uncatalyzed and catalyzed reactions proceed through a chairlike transition state in which the C5-O7 bond cleavage precedes C9-C1 bond formation. Mechanistic proposals which have been suggested previously include protonation or deprotonation of the C4 hydroxyl by an active site general acid or base, respectively, nucleophilic catalysis by attack of an active site nucleophile at C5, and simple conformational restriction of the substrate. 9,14 Recently, X-ray crystal structures of monofunctional chorismate mutases from Bacillus subtilis (BsCM) 16,17 Escherichia coli (EcCM), 18 and catalytic antibody 1F7 19 bound to the endo-oxabicyclic transition state analogue 3 11 were solved (Figure 1). Analysis of the active site structures has led to a general mechanistic hypothesis that the enzymes and antibody stabilize the chairlike transition state geometry via a series of electrostatic and hydrogen-bonding interactions, 17-20 which is consistent with the earlier finding that the rearrangement of chorismate and related compounds is more facile in hydrogen-bonding solvents. 8,10 In addition, it has been speculated that enzyme active site residues may stabilize the developing charge on the enol ether oxygen and the cyclohexadiene ring in the polar transition state. 6,10 The availability of the structures combined with the ability to mutate specific residues provides an opportunity to test these mechanistic hypotheses and identify key catalytic residues. We have generated and characterized a series of 16 mutants of the B. subtilis enzyme.The gene encoding BsCM was subcloned from plasmid pBSCM2 21 into the phagemid pAED4 22 under the control of the T7 RNA polymerase promoter. Six histidines were added to the carboxy terminus using PCR 23 to afford plasmid pCM6XH. Mutant genes were constructed using the method of Kunkel, 24 and the resulting proteins were expressed in E. coli strain BL21 which carries the gene for T7 RNA polymerase behind the lacUV5 promoter. 25 Mutant proteins were purified to homogeneity (as determined by SDS-PAGE and Coomassie staining) in a single step using IMAC affinity chromatography on Ni(II)-chelating resin (Novagen). 26,27 The structural integrity of the mutants was assayed by circular dichroism (CD) spectroscopy. The CD spectra of all mutants between 200 and 260 nm were superimposable with that of the wild-type (wt) protein, suggesting that no significant structural changes were caused by the mutations. Activities of the mutants were determined by monitoring the disappearance of chorismate spectrophotometrically at 274 or 304 nm (∆ 274 ) 2340 M -1 cm -1 ...

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