Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System

Abstract

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

Molecular Application of Aptamers in the Diagnosis and Treatment of Cancer and Communicable Diseases

“…Since then, many more useful studies have been piloted in the search for solutions for the treatment of HIV, TB, ZIKA, Ebola, cancer, hepatitis and measles. The use of nanostructures in targeting biomolecules for diagnosis and therapy is increasing; in vivo RNA nanotechnology leads the way in this regard [76,130,131]. This is due to the fact that RNA and proteins are enzymatically synthesized by transcription and translation of analogous gene sequences and the occurrence of RNA nanostructures seen to be interacting with proteins has been found in nature involving ribosomes, spliceosome, RNase P [130].…”

“…Schwarz-Schilling et al [130], has shown that the interaction of RNA with proteins can be mediated by aptamer motifs which have been used in identifying RNA molecules with fluorescent proteins or enzymes. Developing RNA-protein nanostructures involves co-expression of RNA and functional constituents (aptamer motifs, proteins or enzymes) in one reaction [130]. However, this co-expression of multiple aptamers specific for various biomolecules can be a major challenge, since the binding affinities and expression levels of the proteins may vary.…”

“…However, this co-expression of multiple aptamers specific for various biomolecules can be a major challenge, since the binding affinities and expression levels of the proteins may vary. Schwarz-Schilling et al [130], used a 40 nt pRNA 3WJ as the core motif of an RNA-protein nanostructure, which incorporated four aptamers (TAR, PP7, streptavidin and malachite green); two of the aptamers bind specifically to fluorescent proteins (Tat and PCP), one to streptavidin and the other to malachite green (MG). With the aid of fluorescence from the MG aptamer and Förster resonance energy transfer (FRET) between the fluorescing protein molecules, the authors were able to study the binding parameters and the stability of the complex nanostructure.…”

“…These results are evidence to the advancing RNA-based technology, which not only ensure high efficacy of expression, but a better yield under controlled conditions in vitro. This is a great improvement, which is important in the use of RNA aptamer for both diagnostics and therapy [130].…”

Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology

“…In turn, the split GFP system, in which two halves of the GFP are brought within binding proximity for restored fluorescence, has allowed for a conditionally activated fluorescence signal [18] and this approach can potentially be used to validate the formation of NANPs in cells [29,30]. The combination of several fluorescent proteins forming FRET pairs is another possible way for NANP visualizations [31]. However, all these systems require the presence of bulky tags and may be limited to the intracellular compartmentalization of NANPs.…”