Bottom-up Assembly of RNA Arrays and Superstructures as Potential Parts in Nanotechnology

Abstract

DNA and protein have been extensively scrutinized for feasibility as parts in nanotechnology, but another natural building block, RNA, has been largely ignored. RNA can be manipulated to form versatile shapes, thus providing an element of adaptability to DNA nanotechnology, which is predominantly based upon a double-helical structure. The DNA-packaging motor of bacterial virus phi29 contains six DNA-packaging RNAs (pRNA), which together form a hexameric ring via loop/loop interaction. Here we report that this pRNA can be redesigned to form a variety of structures and shapes, including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. Three dimensional RNA array formation required a defined nucleotide number for twisting of the interactive helix and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH.

Figure 1

Sequence and structural elucidation of phi29 motor pRNA and related assemblages. (A) Primary and secondary structure of wild-type pRNA I–i′. The binding domain (shaded area) and the DNA translocation domain (the helical region) are marked with bold lines. The four bases in the right and left loops, which are responsible for inter-RNA interactions, are boxed. (B) Three-dimensional structure of wild-type pRNA I–i′ displayed as ribbon. (C) Diagrams depicting the pRNA monomer A–b′ with unpaired right/left loops. (D) pRNA dimers (A–b′)(B–a′). (E) pRNA trimers (A–b′)(B–e′)(E–a′). (F) pRNA monomer with unpaired right/left loops A–b′ and a 6-nucleotide palindromic sequence. (G) pRNA twin A–b′.

Figure 2

Polyacrylamide gel showing monomers, dimers, trimers, twins, tetramers, and arrays. (A) Native and denatured gel. (B) Test of the stability of pRNA dimers under different conditions.

Figure 3

Separation of pRNA monomers, dimers, trimers, twins, and arrays by 5–20% sucrose gradient sedimentation. The [³H]-pRNA monomers, dimers, trimers, and twins were isolated from native polyacrylamide gel (see materials and methods). Arrays were prepared by mixing of equal molar amount of twin (A–b′), twin (B–e′) and twin (E–a′). All particles were loaded onto the top of the gradient and sedimented by ultracentrifugation. Sedimentation is from right to left.

Figure 4

Atomic force microscopy (AFM) showing pRNA monomers (A), dimers (B), trimers (C), and arrays (D) of pRNA. The three insets at the left of each panel contain images with higher magnification, as indicated by the size of the frame. The pRNA monomers folded into a checkmark shape, dimers displayed a rod shape, trimer exhibited triangle shape, and arrays displayed as bundles. Formation of dimers requires Mg²⁺, while the sample on mica was briefly rinsed with water before freezing for cryo-AFM, which resulted in some dissociation of dimers or trimers even when the pRNA was already adsorbed to the activated mica surface. The color within each image reflects the thickness and height of the molecule. The brighter, or whiter, the color, the thicker or taller the molecule; the darker the image, the thinner the molecule.

Figure 5

A mixture of two complementary twins, A–b′ and B–a′, assembled into two distinct supramolecular structures. (A) Two complementary twins were able to form a stable tetramer (double-twins) by assembling into a circular structure. (B) Concatemers of alternating twins formed when a twin interacted with two rather than one complementary twin.