Multivalent circular aptamers or ‘captamers’ have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the ‘proximity extension’ assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer–thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.

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Here we describe the design and construction of multivalent circular DNA aptamers. Four aptameric binding motifs directed at blood-borne targets are used as a model set from which larger, multidomain aptamers are constructed in a straightforward manner. Intra-or intermolecular ligation of precursor oligonucleotides provides a stabilizing mechanism against degradation by the predominant exonuclease activity of blood products without the need for post-selection chemical modification. In many cases, circular DNA aptamer half-lives are extended beyond 10 h in serum and plasma, making such constructs viable for therapeutic and diagnostic applications. Duplexes and three-way junctions are used as scaffold architectures around which two, three, or four aptameric motifs can be arranged in a structurally defined manner, giving rise to improved binding characteristics through stability and avidity gains. Circular aptamers targeted against thrombin display improved anticoagulant potency with EC 50 values 2-3-fold better than those of the canonical GS-522 thrombin DNA aptamer. Intrinsic duplex regions provide an opportunity to incorporate additional transcription factor binding motifs, whereas ancillary loops can be used to provide further functionality. These anticoagulant aptamers provide a starting point for merging the principles of DNA nanotechnology with aptameric functions.Since the first reports on nucleic acid aptamers more than a decade ago (1-3), this class of molecules has developed steadily toward general therapeutic (4 -6) and diagnostic (5, 7-9) applications. The relative ease of aptamer discovery has allowed for selection against a wide range of targets (10) by processes that have recently been automated (11). Simultaneous advances in signaling and detection strategies have improved the outlook for aptamer arrays in proteomic and biosensor applications. Whereas the high affinity and specificity of aptamers toward their molecular targets make them well suited for these tasks, the susceptibility of natural nucleic acids to nucleolytic degradation is a serious hurdle for uses in biological fluids. Strategies to improve the nuclease stability of aptamers, as for antisense nucleic acids, have included the use of chemical modifications such as phosphorothioate, locked nucleic acid, 2Ј-Omethyl-and 2Ј-fluoro-nucleotides (12), as well as mirror-image Spiegelmers (13). Of most relevance for potential therapeutic applications, some common nucleic acid stabilization strategies have encountered problems associated with toxic degradation products (14). In addition, modification performed post-selection can alter the subtle binding interactions of the selected natural aptamer (15, 16).Circularization of natural aptamers is an attractive alternative to chemical modification for improving aptamer stability. With the majority of nucleic acid degradation activity arising from plasma exonucleases (17), modification of exposed termini often achieves a sufficient improvement in stability for use in vivo (12), and circular constru...

Electroactive nucleoside triphosphates ("electrotides") have been incorporated into primers by DNA polymerase and detected on oligonucleotide surface-assembled monolayers. Four electrotides bearing three different electroactive moieties-ferrocene, vinylferrocene, and anthraquinone-are detected in four alternative formats.

T7 RNA polymerase has been purified to homogeneity from an overproducing clone of Escherichia coli containing pAR1219. Preparations have a zinc content as low as 0.01 mol/mol of enzyme and a high specific activity, 300 000-500 000 units/mg. There are no intrinsic zinc sites. Furthermore, extrinsic Zn2+ does not function as an activator. Supplementation of the assay mix with up to 5 mM ethylenediaminetetraacetic acid has little effect on activity while added Zn2+ is strongly inhibitory at concentrations above 10 microM. This monomeric RNA polymerase is not a zinc metalloenzyme, unlike its multimeric bacterial counterparts. Titration of the urea-denatured protein with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that all 12 Cys residues are present in the free sulfhydryl form, 5 of which are readily accessible to reagent in the native enzyme. More preferential labeling of the sulfhydryls can be achieved with low concentrations of [14C]iodoacetamide, where inactivation of the enzyme proceeds with incorporation of approximately 1.2 mol of [14C]iodoacetamide/mol of polymerase. Amidomethylation primarily occurs at Cys-347, with lesser reaction at Cys-723 and Cys-839. Cys-347 and Cys-723 are in segments of the primary sequence containing numerous basic residues. These same segments have previously been implicated in promoter binding, suggesting that both residues are located within or near the active site region.