Optimization of the Split-Spinach Aptamer for Monitoring Nanoparticle Assembly Involving Multiple Contiguous RNAs

The fact that structural RNA motifs can direct RNAs to fold and self-assemble into predictable pre-defined structures is an attractive quality and driving force for RNA's use in nanotechnology. RNA's recognized diversity concerning cellular and synthetically selected functionalities, however, help explain why it continues to draw attention for new nano-applications. Herein, we report the modification of a bifurcated reporter system based on the previously documented Spinach aptamer/DFHBI fluorophore pair that affords the ability to confirm the assembly of contiguous RNA strands within the context of the previously reported multi-stranded RNA nanoring. Exploration of the sequence space associated with the base pairs flanking the aptamer core demonstrate that fluorescent feedback can be optimized to minimize the fluorescence associated with partially-assembled RNA nanorings. Finally, we demonstrate that the aptamer-integrated nanoring is capable of assembling directly from transcribed DNA in one pot.