Proximity extension of circular DNA aptamers with real-time protein detection

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

Proximity extension assay performance with different tail–loop hybridization lengths

<p><b>Copyright information:</b></p><p>Taken from "Proximity extension of circular DNA aptamers with real-time protein detection"</p><p>Nucleic Acids Research 2005;33(6):e64-e64.</p><p>Published online 7 Apr 2005</p><p>PMCID:PMC1074748.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Reactions were performed with 40 nM aptamer concentrations in the absence (open circles) or presence (black squares) of 2 nM thrombin

Practical synthesis of sugar monophosphonucleotides

A reliable procedure for the preparation of sugar nucleoside monophosphates is presented, which involves condensation of an activated glycosyl-1-H-phosphonate with an appropriately protected nucleoside and simple end-product isolation via lithium perchlorate-induced precipitation. The utility of these methods is demonstrated by the preparation of a number of purine- and pyrimidine-based sugar nucleoside monophosphate derivatives

Nucleoside triphosphate mimicry: a sugar triazolyl nucleoside as an ATP-competitive inhibitor of B. anthracis pantothenate kinase

The synthesis of a library of nucleoside triphoshate mimetics is described where the Mg2+ chelated triphosphate sidechain is replaced by an uncharged methylene-triazole linked monosaccharide sidechain. The compounds have been evaluated as inhibitors of Bacillus anthracis pantothenate kinase and a competitive inhibitor has been identified with a Ki that is 3-fold lower than the Km value of ATP