First, we simply assembled a DNA origami mix (M13 scaffold + set of staples coding for sharp triangles), without any thermal pretreatment, and let it incubate at 25°C for several hours (Fig. ). When this was done in the conventionally used buffer (Trizma-base 40 mM, acetic acid 20 mM, MgCl2 12.5 mM), with or without EDTA, we did not observe any properly shaped objects, regardless of the incubation time (Fig. , left and middle), in agreement with a previous report. We attribute this effect to the formation of kinetically trapped structures, for which the magnesium-stabilized base pairing would require a much higher thermal energy to allow structure reconfigurability. We thus opted for an alternative buffer, referred to as TANa buffer, which was solely composed of tris-acetate buffer (Trizma-base 40 mM, acetic acid 20 mM), without EDTA nor magnesium to promote staple exchange and reconfiguration. It was supplemented instead with a monovalent salt (NaCl) to ensure enough electrostatic screening between the repulsive anionic DNA strands. Remarkably, with [NaCl] = 100 mM (Debye length λD = 0.8 nm) , we observed the progressive formation of properly folded triangles in a few hours at 25 °C (Fig. ). After 24 hours, the yield was comparable to that with thermal annealing operating in the same buffer (Fig. ) and the resulting structures were stable over several days (Fig. ). We repeated the isothermal procedure with the same origami mix in TANa buffer but for various fixed temperatures and NaCl concentrations. For each condition, we systematically established the ratio of mis-/unfolded (no triangular shape), partially folded (defined as incomplete triangular shapes with at least one well-formed corner) and fully folded (defined as triangles with three well-formed corners) origamis obtained after 24 h of selfassembly (Fig. ). Without or with a high amount (500 mM) of NaCl, we could not detect any properly folded origamis for all the tested temperatures. In contrast, for intermediate concentrations ranging from [NaCl] = 50 mM to 250 mM, a significant fraction of partially or fully folded origamis was obtained for different temperatures between 15 °C and 60 °C (Fig. , Fig. , Table ). Sufficient electrostatic screening was thus necessary to allow base pairing between anionic DNA strands but should not be too high to maintain some electrostatic repulsion at short distance and hamper the formation of mismatched assemblies. In this NaCl concentration range, origamis properly assembled up to a temperature increasing from 35 °C to 60 °C when NaCl increased from 50 mM to 250 mM, which was attributed to the electrostatic stabilization by salts against DNA melting. This led to a broad range of temperatures and NaCl concentrations for which isothermal self-assembly led to properly folded origamis after 24 h of incubation (Fig. ). It was found to be particularly efficient for [NaCl] = 100 -150 mM, for which a majority of fully folded origamis was obtained when the assembly temperature was set in the range 25 -40 °C with [NaCl] = 100 mM and 15 -55 °C with [NaCl] = 150 mM (Figures ). We found that this was not specific to the triangle origami shape, as simply changing the initial staple composition allowed us to successfully produce origamis of other target shapes, such as rectangles without staple edges to avoid inter-origami stacking or smileys, by isothermal self-assembly at 25 °C (Figures S7). Close-up AFM analysis revealed that, although the resulting origamis were not perfectly flawless, their overall shape and internal organization showed excellent similarity with the target structure (Figure ) and were comparable to what can be obtained with conventional thermal annealing (Figure ). All these results demonstrate that isothermal self-assembly in TANa buffer allows one to generate well-defined and stable origamis of target morphology in several hours, in a broad window of working temperatures including room and body temperature. We then explored the applicability of our method to generate complex assemblies of various functionality, nature, shapes and sizes, by isothermal self-assembly at 25 °C in the TANa buffer. First, we achieved spatial control over the assembly progress by successfully programming the stepwise assembly of origamis by successive addition of different fractions of the staple set in the self-assembling mixture. This allowed us, for instance, to construct triangles either from a corner to the opposite edge or from an edge to the opposite corner (Fig. ). Then, we targeted the preparation of protein-functionalized origamis. To this end, we directly mixed the M13 scaffold, staples coding for triangles and streptavidin as a model protein, and let the system self-assemble at 25 °C for 24 h. We replaced the staples in the initial mix with up to 6 biotinylated ones to direct the streptavidin binding during the isothermal self-assembly and analyzed the localization of tethered streptavidin in the obtained origamis (Figs. ). In the case of single-site functionalization, we found that 93% of available biotin sites were occupied by a streptavidin (Fig. ) and all the bound streptavidin proteins were at the prescribed position (Fig. , left). For multiple site functionalization, this occupation rate was slightly lower with 65, 70 and 71% of available sites occupied when we prepared origamis with 2, 3 and 6 binding sites, respectively (Fig. ), but the large majority of detected proteins were bound to the origamis at the prescribed positions (Fig. , middle and right). Isothermal selfassembly was thus shown to be perfectly compatible with in situ site-specific protein functionalization, offering new solutions for the self-assembly of functional hybrid DNAprotein structures involving in particular temperature-sensitive entities. With origamis, the large excess of staples with respect to the DNA scaffold favored the folding into the desired shape at a high yield. We thus challenged the possibility to prepare another kind of user-defined but scaffold-free DNA nanostructure. We chose the single-stranded tile (SST) technology, in which stoichiometric arrangement of the DNA strands leads to much lower yield but allows one to reach a large variety of shapes by simply removing strands from the same initial mix. We thus simply prepared the SST mix of the so-called R4 rectangle in the TANa buffer and let it selfassemble at 25 °C. We followed the evolution of the system by AFM. After a few hours, we observed the formation of the first fully assembled R4 rectangles coexisting with a large number of ill-defined objects. The fraction of fully folded R4 rectangles increased slowly but substantially with time (Fig. , middle), as confirmed by a characteristic band of increasing intensity observed by gel electrophoresis (Fig. , bottom left). The purification of this band led to well-defined R4 rectangles (Fig. , bottom right), demonstrating successful isothermal self-assembly at 25 °C of this reference SST system. Finally, to prepare objects of extended dimensions, we mixed 9 DNA strands that could organize in self-repeating units of an extended nanogrid, and let the system self-assemble at 25 °C in the TANa buffer. After 24 h of assembly, nanogrids of large dimensions were successfully obtained in the presence of NaCl, with a particularly sharp square motif at [NaCl] = 150 mM (Figs. ). By allowing programmable DNA self-assembly at room temperature in systems of drastically different nature and composition (protein-modified origamis, well-folded SST structures, nanogrids), the isothermal self-assembly principle thus appears as a tool of remarkable robustness and versatility. To characterize the mechanism of the isothermal self-assembly, we devised a method to follow in situ and in real time the folding pathway of DNA origamis. To keep most of the dynamic features possible upon surface adsorption for AFM imaging, we used a mica substrate coated by a supported lipid bilayer. It was shown in previous works that origamis could adsorb on such substrates through electrostatic interactions mediated by magnesium ions and keep their mobility once adsorbed. To avoid the presence of magnesium ions, we chose another adsorption method by using a few cholesterol-modified staples to anchor parts of the origamis to the bilayer-coated surface. We prepared Λ-shaped origamis by thermal annealing in the TANa buffer by using an origami mix for a triangular shape with cholesterol modifications on two of the sides but depleted from the staples of the third side (Fig. ), and absorbed them on the lipid bilayer. Each adsorbed object was therefore composed of two anchored folded sides and an unfolded, unmodified single-stranded M13 fragment with no prescribed folding constrain and minimal interactions with the surface. This system was then maintained at 25 °C in the TANa buffer containing unadsorbed Λ structures and their excess of staples. We added to this solution the staples of the triangle missing side and followed in situ and in real time the folding of the M13 fragment (Movie S1). At t = 0 (addition of staples), all adsorbed objects had the characteristic Λ shape. Strikingly, with increasing time, we observed that some of the adsorbed structures evolved to form well-defined triangular origamis, indicating complete folding of the M13 fragment into a perfectly arranged triangle side (Fig. , white circles). Contrary to experiments performed so far allowing to make snapshots at a given time of a population of origamis, here we provide the whole temporal evolution of individual structures from an unfolded M13 fragment to the folded state at equilibrium. Notably, several fully folded triangles adsorbed in the field of view during imaging, indicating that isothermal folding also occurred in bulk and at a higher speed than on the surface (Fig. , blue circle). Incompletely folded entities also adsorbed and tended to continue their folding process once adsorbed (Fig. , yellow circle). A detailed look at the initially adsorbed origamis evolving to fully folded structures revealed that individual origamis followed markedly different pathways (Figs ). One observed pathway consisted in a folding process nucleating at one triangle corner, i.e., an extremity of the free M13 fragment, and progressing toward the opposite extremity (Fig. , movie S2), a mechanism reminiscent to the DNA assembly nucleation-growth process in SST revealed in the past by computer simulation. Folding parallel to the triangle edge was also observed in two different directions, either from the inside to the outside (Fig. ) or from the outside to the inside (Fig. , movie S4). These observations reveal that attaining the equilibrium structure for an individual origami is not constrained to one specific folding pathway but to multiple ones (Fig. ). The apparent diversity of these evolutions emphasizes that, contrary to experiments in a magnesium-containing buffer (Fig. ), the system in TANa buffer is not kinetically trapped. We thus conclude that isothermal origami formation in our conditions is a thermodynamically controlled process where the equilibrium state constituted by properly folded origamis can be spontaneously reached by self-assembly. ) with a partial or fully folded rectangular shape (red) or triangular shape with 1 (green), 2 (light blue) or 3 (dark blue) well-formed corners, after an increasing incubation time, with 0 (top), 20 (middle) or 48 (bottom) shortened staples in the rectangle. The remaining fraction of each histogram, which corresponds to mis-shaped or ill-defined objects, is not plotted in this graph. The bars for triangular shapes are shown in a stacked manner; the red and blue lines indicate the cumulative fraction of rectangular and triangular shapes, respectively. [M13] = 0.25 nM; each staple concentration is 10 nM (rectangle) or 100 nM (triangle).