DNA Nanoparticles Programmed from the Top Down with Variable Design Motifs

Abstract

A hallmark of naturally evolved RNA assemblies, such as the ribosome, ribonuclease-P, and tRNAs, is their ability to fold from a single nucleic acid strand into complex, functional 3D structures with diverse biological functions in the cell. A long-standing aim of synthetic structural biology has been to mimic these properties of self-folding and functionality using a single strand of DNA, as a stepping stone to RNA, because of its fewer secondary structure conformations and increased preference to hybridize in solution. Deriving the rules for such self-assembly also allows for the ability to extend the design space to structures beyond the set of naturally evolved molecules. Toward this end, we have developed a top-down sequence design approach based on the principle of scaffolded DNA origami. First, we present a fully autonomous algorithm to produce a single-stranded DNA scaffold and complementary staple strands, which fold into nearly arbitrary target 3D objects, from Platonic solids to non-spherical topologies, with near quantitative synthetic yield, demonstrated for a dozen structures experimentally (Veneziano, Ratanalert, et al., Science, 2016). Second, we present a powerful approach that eliminates staple strands entirely, offering the ability to program a single DNA molecule to fold into these arbitrary 3D shapes on its own, similar to natural RNA assemblies. We examine the folding pathways of each of these design modalities using quantitative PCR and present a thermodynamic model to optimize sequence design, folding temperature, and yield (Ratanalert et al., in prep, 2016). Together, these algorithms solve a long-standing challenge of synthetic structural biology to program nearly arbitrary 3D geometries using synthetic nucleic acids.