ALT In DNA nanotechnology, the assembly and stability of DNA nanostructures typically requires high concentrations of magnesium ions, limiting their applications. While chemically diverse conditions have been explored for DNA nanostructure assembly, only a limited set of ions have been used so far. We investigated the assembly of DNA nanostructures in a wide variety of ions using nanostructures of different sizes and show successful assembly of a majority of these structures in Ca2+, Ba2+, Na+, K+ and Li+. We further show that structures assembled in monovalent ions exhibit up to a 10-fold higher nuclease resistance compared to those assembled in divalent ions. Our work presents new assembly conditions for a wide range of DNA nanostructures that provide enhanced biostability.