Search for: All records

DNA origami is an excellent tool for building complex artificial nanostructures. Functionalization of these structures provides the possibility of precise organization of matter at the nanoscale. In practice, efforts in this endeavour can be impeded by electrostatic repulsion or other dynamics at the molecular scale, resulting in uncompliant local structures. Using single molecule FRET microscopy combined with coarse-grained Brownian dynamics simulations, we investigated here the local structure around the lid of a DNA origami box, which can be opened by specific DNA keys. We found that FRET signals for the closed box depend on buffer ion concentrations and small changes to the DNA structure design. Simulations provided a view of the global and local structure and showed that the distance between the box wall and lid undergoes fluctuations. These results provide methods to vizualise and improve the local structure of three-dimensional DNA origami assemblies and offer guidance for exercising control over placement of chemical groups and ligands. 

Abstract Anticoagulants are commonly utilized during surgeries and to treat thrombotic diseases like stroke and deep vein thrombosis. However, conventional anticoagulants have serious side‐effects, narrow therapeutic windows, and lack safe reversal agents (antidotes). Here, an alternative RNA origami displaying RNA aptamers as target‐specific anticoagulant is described. Improved design and construction techniques for self‐folding, single‐molecule RNA origami as a platform for displaying pre‐selected RNA aptamers with precise orientational and spatial control are reported. Nuclease resistance is added using 2′‐fluoro‐modified pyrimidines during in vitro transcription. When four aptamers are displayed on the RNA origami platform, the measured thrombin inhibition and anticoagulation activity is higher than observed for free aptamers, ssRNA‐linked RNA aptamers, and RNA origami displaying fewer aptamers. Importantly, thrombin inhibition is immediately switched off by addition of specific reversal agents. Results for single‐stranded DNA (ssDNA) and single‐stranded peptide nucleic acid (PNA) antidotes show restoration of 63% and 95% coagulation activity, respectively. To demonstrate potential for practical, long‐term storage for clinical use, RNA origami is freeze‐dried, and stored at room temperature. Freshly produced and freeze‐dried RNA show identical levels of activity in coagulation assays. Compared to current commercial intravenous anticoagulants, RNA origami‐based molecules show promise as safer alternatives with rapid activity switching for future therapeutic applications. 

Abstract Nucleic acid aptamers selected for thrombin binding have been previously shown to possess anticoagulant activity; however, problems with rapid renal clearance and short circulation half‐life have prevented translation to clinical usefulness. Here, a family of self‐folding, functional RNA origami molecules bearing multiple thrombin‐binding RNA aptamers and showing significantly improved anticoagulant activity is described. These constructs may overcome earlier problems preventing clinical use of nucleic acid anticoagulants. RNA origami structures are designed in silico and produced by in vitro transcription from DNA templates. Incorporation of 2'‐fluoro‐modified C‐ and U‐nucleotides is shown to increase nuclease resistance and stability during long‐term storage. Specific binding to human thrombin as well as high stability in the presence of RNase A and in human plasma, comparatively more stable than DNA is demonstrated. The RNA origami constructs show anticoagulant activity sevenfold greater than free aptamer and higher than previous DNA weave tiles decorated with DNA aptamers. Anticoagulation activity is maintained after at least 3 months of storage in buffer at 4 °C. Additionally, inhibition of thrombin is shown to be reversed by addition of single‐stranded DNA antidotes. This project paves the way for development of RNA origami for potential therapeutic applications especially as a safer surgical anticoagulant.