The microtubule-based motor cytoplasmic dynein powers the transport of a diverse array of cargo, allowing cells to organize their contents, move, divide and respond to signals. Dynein is a ∼1 MDa homodimer that can take many consecutive (processive) steps along its microtubule track by converting ATP hydrolysis into mechanical motion. It remains largely unknown how a dynein dimer coordinates the respective mechano-chemical cycles of its two motor domains to produce processive motion and how ensembles of motors work together to move cargo in concert inside cells. We have developed novel tools using DNA nanotechnology for dissecting the coordination of dynein both at the single molecule and ensemble levels.To determine how dynein coordinates its two motor domains to achieve processive motion, we have developed a novel method to create stable dynein heterodimers by utilizing the hybridization of complementary DNA oligonucleotides. Two-color, high-precision, single molecule experiments reveal that dynein, unlike kinesin and myosin, uses both a stochastic and a coordinated stepping mechanism dependent upon the distance between the motor domains within the dimer.To determine the mechanism of cargo motility by ensembles of motors, we have used DNA origami to build a synthetic cargo scaffold to which precise numbers and arrangements of motors can be attached. Our preliminary results in an unloaded system suggest that increasing the number of dynein motors enhances cargo processivity but does not affect velocity. Studies of opposite polarity motors are underway to determine whether cargos driven by both dynein and kinesin exhibit stochastic bidirectional movements.