Illustration of the variable 1D sequences but conserved 2D and 3D structures of different ncRNA nanostructures for tracking the evolution and origin of organisms.

Viruses and other microorganisms are constantly mutating and emerging in different regions. Here, we apply nanotechnology to examine the primary, secondary, and tertiary structures of novel noncoding RNA nanoparticles and find that primary sequences (1D) vary widely between viruses, while secondary (2D) and tertiary structures (3D) are highly conserved. The uniqueness of the phi29 system makes this reported study possible. RNA evolution studies are relevant to the concept of RNA as the origin of life. The recent discovery that RNA is a motile and recombinant entity supports the hypothesis that RNA is the substance of life. Previously, we reported noncoding RNA nanoparticles packaging RNA (pRNA) of the Bacillus virus phi29 (Nature Nanotechnology, 2011, PMID: 21909084; Nature Nanotechnology, 2010, PMID: 21102465; Science, 1987, PMID: 3107124). Phi29 can infect spore-forming Bacillus subtilis, which has long been hidden in the soil with slow or no evolution due to the protection of spores. They are living fossils. The pRNA of the phi29 DNA packaging motor drives the viral motor for DNA transport. In this study, we used existing datasets to search for more pRNAs. Their primary sequence diversity makes it challenging to identify them from the whole genomes of other species. Using the top-down (1D) approach and the bottom-up assembly (3D) approaches, we found that their 2D and 3D structures are highly conserved. Structural conservation enabled us to apply the two-dimensional structure-based approach to find these ncRNAs from databases and identify 12 new pRNAs. The presence of two additional components in the genome, a motor channel protein and a motor ATPase, further confirmed the authenticity of these pRNAs and supported the conclusion that these pRNAs are motor-driven components. Highly conserved and paired left and right loops for assembling the pRNA hexamer were identified in all 12 pRNAs. Artificial modification of the pairing and determination of the virion production activity of the mutated phi29 pRNA further confirmed the conclusion that the secondary and tertiary structures are highly conserved. Understanding the retention, conservation, and variation of viral non-coding RNA sequences and structure can help us trace the evolutionary history of the virus, find lineage information, and provide important information about the origin of the viruses. It can also provide knowledge for the design of disease prevention and treatment by providing the background for in vivo RNA nanotechnology.