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Throughout the cell, motor proteins work together to drive numerous molecular processes and functions. For example, ensembles of myosin motors collectively transport vesicles and organelles, maintain membrane homeostasis, and drive muscle contraction. Studying these motors in groups has become increasingly important with work demonstrating the emergence of ensemble behavior distinct from individual motor behavior. One powerful technique that has been used in the last decade is DNA nanotechnology, which provides precise control over the spacing and organization of patterned motor proteins. Until recently, however, most studies combining DNA nanostructures and molecular motors have been confined to discrete DNA structures with limited attachment points for motor proteins. In this chapter, we describe a new approach for making synthetic motor filaments using DNA nanotubes. To begin, we present methods for preparing myosin VI-labeled nanotubes and testing these nanotubes using a general in vitro motility setup. We then describe how to modify this system to pattern motor proteins and effectors on the nanotubes. Overall, these nanotubes can easily be used to study large ensembles of molecular motors, such as muscle myosin or ciliary dynein, both proteins that work in large motor ensembles to drive key cellular functions., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a pre-formed prohead shell in ~5 min at room temperature in vitro. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers. Additionally, we describe techniques for conducting rapid solution exchange measurements that can be used to probe nucleotide-dependent motor-DNA interactions, stall and restart the motor to probe DNA conformational relaxation, and to probe the dynamics of release of the DNA, akin to ejection, in conditions where the motor fully releases its grip. Three viral packaging systems are described in detail: bacteriophages phi29 (φ29), lambda (λ), and T4. Two different approaches are described: 1. With φ29 and T4, prohead-motor complexes can be pre-assembled in bulk and packaging can be initiated in the optical tweezers by "feeding" a single DNA molecule to one of the complexes; 2. with φ29 and λ, packaging can be initiated in bulk then stalled, and a single prohead-motor-DNA complex can then be captured with optical tweezers and restarted. In both cases, the prohead is ultimately attached to one trapped microsphere and the end of the DNA being packaged is attached to a second trapped microsphere such that packaging of the DNA pulls the two microspheres together and the rate of packaging and force generated by the motor is directly measured in real time. These protocols allow for the effect of many experimental parameters on packaging dynamics to be studied such as temperature, ATP concentration, ionic conditions, structural changes to the DNA substrate, and mutations in the motor proteins. Procedures for capturing microspheres with the optical traps and different measurement modes are also described., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)