Multivalent Aptamer‐Functionalized Single‐Strand RNA Origami as Effective, Target‐Specific Anticoagulants with Corresponding Reversal Agents

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.

Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds

“…Owing to their rigidity arising from their double helix structure, DNA nanostructures with specific shapes have emerged as scaffolds for multivalent interactions of biosensors. Various recognition systems have been designed by introducing aptamers [15,99,[108][109][110] or ligands [111,112] into specific positions of 2D or 3D DNA nanostructures such as DNA origami, tiles, and tetrahedral nanostructures capable of a trivalent binding [7,18,82,[113][114][115][116][117][118].…”

Multivalent Aptamer Approach: Designs, Strategies, and Applications

“…Spatial arrangements can be achieved by either altering the distance between two adjacent aptamers [ 92 , 93 ] or positioning aptamers in a desired geometry [ 5 , 94 ]. Distance arrangements can be achieved by positioning aptamers onto rigid scaffolds (i.e., dsDNA, dsRNA [ 95 ], or DNA origami [ 96 ]). By conjugating two different anti-thrombin aptamers onto multi-helix DNA tiles and DNA origami scaffolds at various distances, Rinker et al demonstrated that placing two different thrombin binding aptamers at an optimal distance on a rigid DNA tile nanostructure could lead to significant improvements in binding affinity, with results suggesting an estimated 50-fold enhancement in binding strength compared to aptamer monomer approach [ 97 ].…”

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN): The Present and Future of the Burgeoning Field

“…This research has also led to enhancing the stability of DNA origami for biomedical applications, , which can additionally be conjugated with fluorophores and antibodies . Thomas LaBean at North Carolina State University and co-workers at iNano in Aarhus, Denmark, have explored using RNA origami as an anticoagulant . Thomas Hermann’s lab at the University of California, San Diego, has developed a library of RNA–DNA hybrid nanoshapes that self-assemble from modules , and can be programmed to do so only in the presence of a target ligand …”

“…57 Thomas LaBean at North Carolina State University and co-workers at iNano in Aarhus, Denmark, have explored using RNA origami as an anticoagulant. 58 Thomas Hermann's lab at the University of California, San Diego, has developed a library of RNA− DNA hybrid nanoshapes that self-assemble from modules 59,60 and can be programmed to do so only in the presence of a target ligand. 61 As a functional output, RNA is advantageous not only as a therapeutic moiety, but also as a biosensing component.…”