Folding molecular origami from ribosomal RNA

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Biology

Background:

• Ribosomal RNA (rRNA) constitutes approximately 80% of cellular RNA, presenting an abundant and cost-effective source of long, single-stranded nucleic acids.
• This abundance makes rRNA a promising candidate for fabricating nanoscale structures through molecular origami techniques.

Purpose of the Study:

• To demonstrate the efficient and robust construction of 2D and 3D origami nanostructures using cellular rRNA as a scaffold.
• To develop and present calibrated protocols for folding rRNA into desired shapes using crude total RNA extracts.
• To analyze and enhance the stability of the resulting RNA:DNA origami nanostructures for potential biomedical applications.

Main Methods:

• Utilized cellular rRNA as a scaffold and DNA oligonucleotide staples for nanostructure fabrication.
• Developed and optimized protocols for folding rRNA subunits into contiguous 2D and 3D shapes.
• Investigated the stability of RNA:DNA origami nanostructures under various conditions, including temperature, Mg2+ concentration, human serum, and enzymatic degradation (DNase I, RNase H).
• Applied polylysine-polyethylene glycol coating to enhance nanostructure stability.

Main Results:

• Successfully constructed 2D and 3D origami nanostructures using cellular rRNA and DNA staples.
• Demonstrated efficient folding protocols using crude total RNA extracts, yielding stable structures.
• Showcased the inherent stability of RNA within the folded nanostructures.
• Confirmed enhanced stability of RNA:DNA origami nanostructures when coated with polylysine-polyethylene glycol, particularly under challenging conditions like varying temperatures, low Mg2+ concentrations, human serum, and nuclease presence.

Conclusions:

• Cellular rRNA is a viable and efficient raw material for constructing stable 2D and 3D origami nanostructures.
• The developed protocols enable robust folding of rRNA into desired shapes, suitable for biomedical applications.
• RNA:DNA origami nanostructures exhibit enhanced stability, especially when surface-modified, laying the groundwork for their use in diverse biological environments and therapeutic strategies.