Simian virus 40 capsid protein (VP1) is a unique system for studying substrate-dependent assembly of a nanoparticle. Here, we investigate a simplest case of this system where 12 VP1 pentamers and a single polyanion, e.g., RNA, form a T = 1 particle. To test the roles of polyanion substrate length and structure during assembly, we characterized the assembly products with size exclusion chromatography, transmission electron microscopy, and single-particle resistive-pulse sensing. We found that 500 and 600 nt RNAs had the optimal length and structure for assembly of uniform T = 1 particles. Longer 800 nt RNA, shorter 300 nt RNA, and a linear 600 unit poly(styrene sulfonate) (PSS) polyelectrolyte produced heterogeneous populations of products. This result was surprising as the 600mer PSS and 500-600 nt RNA have similar mass and charge. Like ssRNA, PSS also has a short 4 nm persistence length, but unlike RNA, PSS lacks a compact tertiary structure. These data indicate that even for flexible substrates, shape as well as size affect assembly and are consistent with the hypothesis that work, derived from protein-protein and protein-substrate interactions, is used to compact the substrate.