Abstract
RNA presents intringuing roles in many cellular processes and its versatility underpins many different applications in synthetic biology. Nonetheless, RNA origami as a method for nanofabrication is not yet fully explored and the majority of RNA nanostructures are based on natural pre-folded RNA. Here we describe a biologically inert and uniquely addressable RNA origami scaffold that self-assembles into a nanoribbon by seven staple strands. An algorithm is applied to generate a synthetic De Bruijn scaffold sequence that is characterized by the lack of biologically active sites and repetitions larger than a predetermined design parameter. This RNA scaffold and the complementary staples fold in a physiologically compatible isothermal condition. In order to monitor the folding, we designed a new split Broccoli aptamer system. The aptamer is divided into two nonfunctional sequences each of which is integrated into the 5′ or 3′ end of two staple strands complementary to the RNA scaffold. Using fluorescence measurements and in-gel imaging, we demonstrate that once RNA origami assembly occurs, the split aptamer sequences are brought into close proximity forming the aptamer and turning on the fluorescence. This light-up ‘bio-orthogonal’ RNA origami provides a prototype that can have potential for in vivo origami applications.
Highlights
A plethora of self-assembled DNA origami and hybrid RNA-DNA origami nanostructures have been synthesized using the basic principle of Watson-Crick base pairing[1,2,3,4,5,6,7,8,9]
While significant advances have been made in DNA origami synthesis, the design and realization of RNA origami has been reported only recently[13]
RNA nanostructures were obtained with different tools and techniques imported from DNA nanotechnology, such as the use of the double cross over (DX) motifs and the DNA origami design
Summary
A plethora of self-assembled DNA origami and hybrid RNA-DNA origami nanostructures have been synthesized using the basic principle of Watson-Crick base pairing[1,2,3,4,5,6,7,8,9]. In contrast to the above mentioned strategies used for the construction of RNA nanoparticles, Yingling and Shapiro[24] introduced a shape-based computational approach to design RNA hexagonal nanoring from two building blocks using stable RNAIi/RNAIIi loop-loop noncovalent intermolecular interactions. In this regard, a computational-based approach promoted the in silico design of a three dimensional cubic RNA-based scaffold that was self-assembled in a one-pot process. Taking into account the powerful origami technique, Geary et al.[30] showed the cotrascriptional folding of an artificially designed single stranded RNA (ssRNA) into a RNA tile: at the end of the RNA tile synthesis, the tiles were released and assembled into hexagonal lattices through kissing loop interactions. A 7-helix bundled RNA tile and a 6-helix bundled RNA origami tube were designed and prepared from single stranded RNA scaffold and multiple staple RNA strands by employing for the first time a direct extension of the DNA origami strategy[13]
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