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

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.

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

“…The RNA origami ribbon ( Fig. S1 in the ESM) has been designed as previously described [29]: in the present study, RNA staple strands were changed (r1, l1, r2, l2, f and s1) or elongated (s2) at the 5' end of few bases (1 up to 3 bases) in order to start with -GG or -GA (first and second nucleotides of the transcribed region) necessary for an efficient T7 transcription yield.…”

“…The set of RNA staple strands [29] were mixed in 10-fold excess with RNA scaffold in 50 μL of folding buffer (10 mM MgCl 2 , 20 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, [27]). After an initial thermal denaturation step at 75 °C for 1 min and a snap cooling (-1 °C/0.42 sec), the mixture was subjected to a folding step at 37 °C for 20 min.…”

“…To confirm the RNA origami self-assembly by incorporation of Split Broccoli aptamer system, ingel imaging with fluorophore DFHBI-1T [36] was performed with some modifications. Briefly, RNA origami sample, partially folded samples and Broccoli aptamer (prepared as previously described, [29]) were loaded in the polyacrylamide gels. The gels were washed three times for 5 min in RNase free water and then stained for 20-25 min in aptamer buffer containing 1.26 μM DFHBI-1T, 40 mM HEPES pH 7.4, 100 mM KCl, 1 mM MgCl 2 .…”

“…In our previous work, we went a step further to develop a biologically afunctional (i.e. bioorthogonal by design) RNA origami able to fold at constant temperature (37 °C) after an initial denaturation step [29], which lights-up when folding to its target configuration. Seven RNA staple strands promoted the folding of a RNA bio-orthogonal synthetic De Bruijn scaffold sequence (DBS) that does not contain genetic information, restriction enzyme sites or reduced ambiguity in the addressability [30].…”

“…We demonstrated the possibility to combine scaffold bio-orthogonality, physiologically compatible folding and assembly monitoring using a new RNA-based reporter system. The folding was monitored using our new split Broccoli aptamer system [29].…”