We chose DNA nanotubes assembling from double-crossover (DX) tiles requiring the presence of only five short, distinct DNA strands designed to interact as depicted in Figure left. These strands form two DNA double helices held together as three of the strands (yellow, blue, and gray in Figure ) cross over from one helix to the other, forming two junctions that confer rigidity to the tile. The 5'-end of the strand in the center of the tile (blue in Figure ) was modified with the Cy3 dye to enable fluorescence microscopy observation. Tiles interact via complementary single stranded domains known as sticky ends (at the 5' and 3' end of the yellow and gray strands). The inter-tile crossover distance is chosen so that individual tiles bind to each other at an angle, and thus form micrometer-scale tubular structures rather than flat lattices . Because these DNA nanotubes can be engineered to work as scaffolds with the capacity to respond to biochemical and physical stimuli, they are excellent components to build an "artificial cytoskeleton" for synthetic cells and responsive biomaterials . A notable limitation toward this has been the requirement to thermally anneal the DX tiles , which is at odds with the goal of building life-like systems operating at constant temperature. With the objective to build nanostructures that can grow under life-compatible conditions, we placed the DNA strands in a buffer exclusively composed of monovalent cations, dubbed "TANa" buffer (Trizma base 40 mM, acetic acid 20 mM, 100 mM NaCl), which was recently shown to enable successful assembly of complex DNA nanostructures at constant room temperature. Doing so, we questioned not only whether DNA nanotubes could assemble isothermally at room temperature but also how they would self-organize over time (Figure middle, right). To evaluate the performance and understand the advantage of using monovalent salts, we compared the results to those obtained with the magnesium-containing buffers conventionally used for DNA self-assembly, either TAMg (Trizma base 40 mM, acetic acid 20 mM, 12.5 mM MgCl 2 ) or TAEMg (TAMg + 1 mM EDTA).