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

Abstract

The scaffolded origami technique has provided an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in high yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time. Our work suggests that the scaffolded origami technique is a valid, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/864678v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@ff817aorg.highwire.dtl.DTLVardef@8da463org.highwire.dtl.DTLVardef@12381b7org.highwire.dtl.DTLVardef@194733_HPS_FORMAT_FIGEXP M_FIG C_FIG