Synthesis of Segmented BSMV Nanorods for Magnetic Separation

• Sebastian Blough, Chemical Engineering, University of Delaware

• Kevin Solomon, Chemical and Biomolecular Engineering, University of Delaware

The ability to precisely organize functional domains along nanoscale scaffolds is critical for developing programmable biomaterials with applications in biosensing, biomolecular separations, and environmental remediation. Barley stripe mosaic virus (BSMV) nanorods provide a genetically programmable platform for constructing high-aspect-ratio protein assemblies. However, methods for controlling nanorod growth at predetermined locations remain limited. Here, we present a DNA-mediated stop-and-go assembly strategy that enables programmable regulation of BSMV nanorod assembly through sequence-specific RNA hybridization. A DNA “stopper” strand was designed to hybridize to engineered regions of the viral RNA template, creating a steric barrier that arrests coat protein assembly at defined positions. RNA/DNA hybrid formation was verified using an RNase H cleavage assay, confirming successful stopper binding to the RNA template. Transmission electron microscopy demonstrated that stopper hybridization significantly reduced nanorod length, producing particles centered near 48 nm compared with approximately 110 nm for untreated controls, demonstrating precise control over BSMV assembly. These findings establish that nucleic acid-directed assembly can reproducibly halt viral nanorod growth at programmable locations. Building on this proof of concept, a complementary release strand will be used to initiate toehold-mediated strand displacement, with the goal of resuming nanorod assembly following programmed arrest. Iterative stop-and-go assembly is expected to generate BSMV nanorods containing spatially patterned functional domains. Inspired by multicomponent magnetic nanorods that demonstrated spatially segregated functional domains for magnetic biomolecular separations, our strategy applies this design principle to genetically encoded BSMV nanorods through selective display of metal-binding peptides on defined nanorod segments. This programmable patterning strategy establishes a foundation for engineering multifunctional BSMV nanorods. By enabling selective placement of metal-binding peptides, this platform could support magnetic separation and recovery of critical metals such as cobalt, nickel, and platinum from complex waste streams while advancing spatially programmed nanobiotechnology.