Genetically Encoded, Functional Single‐Strand RNA Origami: Anticoagulant

Abstract

Nucleic acid aptamers selected for thrombin binding have previously been shown to possess anticoagulant activity, however problems with rapid renal clearance and short circulation half-life prevented translation to clinical usefulness. Here, we describe a family of self-folding, functional RNA origami molecules bearing multiple thrombin-binding RNA aptamers and showing significantly improved anticoagulant activity. These constructs may overcome earlier problems preventing clinical use of nucleic acid anticoagulants. RNA origami structures were designed in silico and produced by in vitro transcription from DNA templates. Incorporation of 2'-fluoro-modified C-and U-nucleotides was shown to increase nuclease resistance and stability during long-term storage. We demonstrate 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. The RNA origami constructs show anticoagulant activity seven-fold greater than free aptamer and higher than previous DNA weave tiles decorated with DNA aptamers. Anticoagulation activity was maintained after at least three 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.

A Mini-Review: Clinical Development and Potential of Aptamers for Thrombotic Events Treatment and Monitoring

“…The author also reported the high biocompatibility of the assembly in in vitro cytotoxicity and hemolysis assays [44]. Krissanaprasit et al reported using an in silico design to construct RNA origami containing multiple thrombin-binding RNA aptamers [45]. After introducing the 2’-fluoro modification in C and U nucleotides, the RNA origami structure showed increased resistance to nuclease degradation and also greater anticoagulant activity than free aptamers, which can also be reversed by using ssDNA antidotes [45].…”

“…Krissanaprasit et al reported using an in silico design to construct RNA origami containing multiple thrombin-binding RNA aptamers [45]. After introducing the 2’-fluoro modification in C and U nucleotides, the RNA origami structure showed increased resistance to nuclease degradation and also greater anticoagulant activity than free aptamers, which can also be reversed by using ssDNA antidotes [45]. Amato et al investigated the effect of the different linkers in constructing a dimeric HD1 aptamer [46].…”

Intelligent antithrombotic nanomedicines: Progress, opportunities, and challenges

“…[ 22 ] In addition, highly specific, corresponding reversal agents can be developed to cease undesirable antithrombotic effects. [ 23 ] Thus, such a multifaceted strategy can notably lower bleeding risk, while preserving antithrombotic effects. In the current review, we highlight the recent progress of antithrombotic nanomedicines in three categories, namely, fibrinolytic therapy, anticoagulant therapy, and antiplatelet therapy (Figure 1).…”

A DNA origami-based aptamer nanoarray for potent and reversible anticoagulation in hemodialysis

“…In contrast to traditional nanoparticles, DNA/ RNA nanotechnology offers a variety of nanostructures with rationally designed geometric features, precise spatial addressability and remarkable biocompatibility [19][20][21][22][23][24] . Several aptamertagged DNA/RNA nanostructures have been utilized for immobilizing proteins with defined nanometer spacing and precision [23][24][25][26] . Though the reported nanoarchitectures have achieved substantial anticoagulant effects in plasma in test tubes 24,26 , in vivo study and clinically relevant validation have yet to be reported.…”

“…Several aptamertagged DNA/RNA nanostructures have been utilized for immobilizing proteins with defined nanometer spacing and precision [23][24][25][26] . Though the reported nanoarchitectures have achieved substantial anticoagulant effects in plasma in test tubes 24,26 , in vivo study and clinically relevant validation have yet to be reported.…”