This second edition provides new and updated methods that detail new DNA nanotechnology techniques. Chapters focus on DNA origami nanostructures for arranging matter in the nanoscale or on their manipulation with the aid of other technologies, on procedures for making nucleic acids nanostructures of different kinds, and methods to simulate complex nanostructures or to use them in biosensing. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.