Walk on the origami side: isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon

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. We try to address this large gap between DNA origami and RNA origami development, by introducing biologically inert (bio-orthogonal) RNA origami that is able to fold at constant temperature.

In our previous work, square DNA origami and triangle RNA-DNA hybrid origami were synthesized using uniquely addressable bio-orthogonal De Brujin sequences as scaffold. In this work, we use RNA staples to promote folding of the scaffold into a nanoribbon at physiological temperature (37 °C). The assembly was verified by gel assay, atomic force microscopy, and with a new split Broccoli aptamer system integrated into the staples, that binds a specific fluorophore to mark completed folds. Using in-gel imaging and fluorescence measurements, we found the split aptamer sequences in close proximity once the RNA origami assembly occurs, forming the aptamer and turning on the fluorescence.

In this talk I will describe how we investigated and combined these different aspects: (1) bio-orthogonality, (2) physiologically compatible folding, and (3) monitoring of the assembly through a new split Broccoli RNA aptamer system.