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Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon

Lookup NU author(s): Dr Emanuela Torelli, Dr Jurek Kozyra, Dr Jingying Gu, Professor Ulrich Stimming, Professor Natalio Krasnogor


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A plethora of self-assembled DNA origami and hybrid RNA-DNA origami have been synthesized using the basic principle of Watson-Crick base pairing. Despite the RNA functional capacity, the synthesis of RNA nanostructures via 'scaffold' and 'staple' strands is underdeveloped and still lacking. While significant advances have been made in the DNA origami synthesis, the design and realization of RNA origami has been reported only recently1. Here we take into account the large gap between DNA origami and RNA origami development and inspired by a bottom-up origami toolkit, we present a light-up biologically inert (i.e. 'bio-orthogonal') RNA origami able to fold at constant temperature2. In our recent work3, square DNA origami and triangle RNA-DNA hybrid origami were synthesized using 'bio-orthogonal' and uniquely addressable De Brujin 'scaffold' sequences (DBS). Unlike biologically derived 'scaffold', these DNA and RNA 'bio-orthogonal' scaffolds do not contain genetic information, restriction enzyme sites or ambiguity in the addressability making them candidates for in vivoapplications. Here RNA 'staple' strands promoted the folding of a short 'bio-orthogonal' RNA 'scaffold' sequence into a nanoribbon: after an initial denaturation step, the self-assembly occurred at the physiological temperature (37 °C). The RNA origami assembly was verified by gel assay, atomic force microscopy (AFM) and using a new split Broccoli aptamer system able to bind the specific fluorophore only after the folding process. The Broccoli aptamer4is 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 in-gel imaging and fluorescence measurements, we demonstrated that once the RNA origami assembly occurs, the split aptamer sequences are in close proximity to form the aptamer and turn on the fluorescence.Herein, we investigate and combine three different aspects: i) 'bio-orthogonality', ii) physiologically compatible folding at 37 °C and iii) assembly monitoring through a new split Broccoli RNA aptamer system. Our resulting RNA origami nanoribbon can open the way to new potential platform for future in vivoapplications when genetically encoded and transcribed RNA are used.References[1]Endo, M. et al. 2014. Preparation of Chemically Modified RNA Origami Nanostructures. Chemistry - A European Journal. 20:15330-15333.[2] Torelli, E. et al. 2018. Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon. Scientific Reports.Accepted.[3] Kozyra, J. et al. 2017. Designing Uniquely Addressable Bio-orthogonal Synthetic Scaffolds for DNA and RNA Origami. ACS Synthetic Biology.6:1140-1149.[4] Filonov, G. S. et al. 2014. Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution. Journal of the American Chemical Society. 136:16299-16308.

Publication metadata

Author(s): Torelli E, Kozyra JW, Gu JY, Stimming U, Piantanida L, Voïtchovsky K, Krasnogor N

Publication type: Conference Proceedings (inc. Abstract)

Publication status: Published

Conference Name: Functional DNA Nanotechnology

Year of Conference: 2018

Acceptance date: 23/03/2018