Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation.From the Clinical EditorThrombin inhibition via DNA aptamers has recently become a possibility. In this study, thrombin-binding DNA aptamers were further optimized by nanoscale organization on DNA nanostructures. The authors have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than that of free aptamer. Reversal of thrombin inhibition was also achieved by single-stranded DNA antidotes, enabling significant control over the coagulation pathway.

Graphical AbstractThe anticoagulant activity of thrombin-binding DNA aptamers was studied by placing the aptamers on DNA weave-tiles, thereby controlling the inter-aptamer distance. Exosite I (magenta) and exosite II (yellow) of thrombin (A) bind Aptamer A (B) and Aptamer B (C), respectively. High anticoagulant activity of Aptamer A was combined with high binding affinity of Aptamer B by positioning them on DNA weave-tiles at two of the four possible locations (D). The resultant construct was found to be a very effective anticoagulant for an optimum inter-aptamer distance, relative orientation and choice of the tile.Figure optionsDownload high-quality image (202 K)Download as PowerPoint slide