Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

Abstract

Rational design of nucleic acid molecules yields self-assembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. Here we demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 μm in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowly annealed, and a one-pot transcription and anneal procedure. We identify the tile nick position as a structural requirement for lattice formation. Our results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components.

Figure 1.

Design of DX RNA tiles (A) Schematic of DX RNA tile (29) and tile abstraction (gray). We tested tiles where the nick in strand Sb can be centered (SbC) or shifted two bases to the right (SbR) or to the left (SbL) of the midpoint between crossovers. (B) Three-dimensional rendering of the DX RNA tile motif. (C) Expected lattice geometry, where tiles assemble in an alternate facing up (O-marked tile) and facing down (X-marked tile) pattern. (D) Example, AFM image of lattices obtained with tiles presenting a right-shifted nick (design D1R described in the text). Scalebar is 50 nm.

Figure 2.

Assembly methods and results (A) Gel extraction and anneal protocol, showing templates with T7 promoter (black lines with bent arrows), RNA transcripts (red lines), and tile assembly. (B) Example, AFM image of assemblies obtained from tile D1R. (C) Representative image of small lattices obtained from tile D2R (additional images are in Supplementary Figure S20). (D) One-pot transcription and anneal protocol. (E) Example, AFM image of tubular structure produced from one-pot assembly of tiles D1R. (F) Lattices formed by one-pot assembly of tiles D2R. Scale bar: 250 nm.

Figure 3.

Example, AFM images of chiral tubular structures formed by D1R tiles. The chirality feature is presumably promoted by a combination of sequence content and tile geometry. A detailed discussion of handedness is provided in (SI section 13). Scale bar is 50 nm. Right: rendering of a left-handed chiral sheet as a guide to the eye.

Figure 4.

Gel analysis of assembling structures. Strands were gel extracted and annealed prior to loading them into the gel. (A1, A2 and A3) Non-denaturing PAGE gels comparing complexes that form as part of tile variants D1R, D1L and D1C. Each lane was loaded with annealed strands as annotated on top of the gel. Ratio 1:1 indicates that both strands were annealed at a 1 μm concentration; ratio 2:1 indicates concentrations 1 μm : 500 nm; ratio 2:1:2 indicates concentrations 1 μm : 500nm : 1 μm. Bands forming in lanes 1 and 2 provide information on the formation of the core of the tile; Sa and Sb in stoichiometric amounts are expected to form smaller complexes relative to the case where Sa and Sb are in the 2:1 ratio required for tile formation. In variant D1C, the two cases are indistinguishable, which suggests improper formation of the tile core during annealing. (B) Agarose gel of annealed tile variants D1R, D1L and D1C, compared to non-multimerized annealed tile variant D1R*. A significant fraction of annealed tile D1C runs roughly as the D1R* control complex, indicating that D1C assemblies are not as robust as in the other variants.