DNA nanostructures are typically assembled by thermal annealing in buffers containing magnesium. We demonstrate the assembly of DNA nanostructures at constant temperatures ranging from 4° to 50°C in solutions containing different counterions. The choice of counterions and the assembly temperature influence the isothermal assembly of several DNA motifs and designed three-dimensional DNA crystals. Molecular dynamics simulations show more fluctuations of the DNA structure in select monovalent ions (Na and K) compared to divalent ions (Mg and Ca). A key highlight is the successful assembly of DNA motifs in nickel-containing buffer at temperatures below 40°C, otherwise unachievable at higher temperatures or using an annealing protocol. DNA nanostructures isothermally assembled in different ions do not affect the viability of fibroblasts, myoblasts, and myotubes or the immune response in myoblasts. The use of ions other than the typically used magnesium holds key potential in biological and materials science applications that require minimal amounts of magnesium.
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