Square-shaped RNA particles from different RNA folds.

Area of Science:

• Biotechnology and Nanotechnology
• Synthetic Biology
• RNA Structural Biology

Background:

• Natural RNA motifs can encode structural information for specific three-dimensional (3D) conformations.
• This principle can be applied to artificial RNA sequences for controlling shape and self-assembly in nanotechnology and synthetic biology.
• Three natural RNA motifs inducing ~90-degree helical bends were identified: a five-way transfer RNA (tRNA) junction, a three-way junction, and a two-helix bend.

Purpose of the Study:

• To investigate the use of three natural RNA motifs in rationally designed RNA (tectoRNA) for generating square-shaped tetrameric RNA nanoparticles.
• To compare the ability of each motif to direct supramolecular assembly formation.
• To demonstrate how different 90-degree motifs can be programmed to direct the formation of either 2D or 3D assemblies.

Main Methods:

• Incorporation of three distinct 90-degree RNA bending motifs into tectoRNA scaffolds.
• Characterization of supramolecular assembly formation using native gel electrophoresis.
• Visualization and analysis of nanoparticle structure and assembly using atomic force microscopy (AFM).

Main Results:

• All three motifs were successfully embedded within tectoRNA to create square-shaped tetrameric nanoparticles.
• Native gel assays and AFM revealed differences in the biophysical behavior of supramolecular complexes formed by each motif.
• Structural assembly programming demonstrated that specific 90-degree motifs preferentially directed the formation of either 2D or 3D assemblies.

Conclusions:

• Natural RNA motifs can be effectively utilized to program the self-assembly of artificial RNA nanoparticles with controlled shapes.
• The choice of 90-degree motif influences the thermodynamics and molecular dynamics, leading to distinct supramolecular assembly behaviors.
• This work provides a framework for designing RNA-based nanostructures with predictable 2D or 3D architectures for diverse applications.