Cryo-electron microscopy revealed a highly regular internal structure of the nanotubes with well-defined tiles aligned with the principal axis of the nanotubes (Figure ), confirming that the circumference of a nanotube generally includes 6-7 tiles, an internal structure comparable to that obtained by thermal annealing in the presence of magnesium (Figure ). However, we noted that the diameter obtained by isothermal assembly in TANa (22 ± 4 nm, Figure ) was shifted to higher values compared to the one obtained by thermal annealing in TAMg (12 ± 2 nm, Figure ) probably due to higher flexibility provided by the monovalent salt conditions. Isothermal assembly thus allowed both faithful assembly of DNA strands and sustained growth into soft and well-defined individual DNA nanotubes.