Biomolecular　self-assembly　is　a　powerful　approach　for　fabricating　supramolecular　architectures.　Over　the　past　decade,　a　myriad　of　biomolecular　assemblies,　such　as　self-assembly　proteins,　lipids,　and　DNA　nanostructures,　have　been　used　in　a　wide　range　of　applications,　from　nano-optics　to　nanoelectronics　and　drug　delivery.　The　method　of　controlling　when　and　where　the　self-assembly　starts　is　essential　for　assembly　dynamics　and　functionalization.　Here,　train-shaped　DNA　nanostructures　are　actively　self-assembled　using　DNA　tiles　as　artificial　＇carriages,＇　hairpin　structures　as　＇couplers,＇　and　initiators　of　catalytic　hairpin　assembly　(CHA)　reactions　as　＇wrenches.＇　The　initiator　wrench　can　selectively　open　the　hairpin　couplers　to　couple　the　DNA　tile　carriages　with　high　product　yield.　As　such,　DNA　nanotrains　are　actively　prepared　with　two,　three,　four,　or　more　carriages.　Furthermore,　by　flexibly　modifying　the　carriages　with　＇biotin　seats＇　(biotin-modified　DNA　tiles),　streptavidin　＇passengers＇　are　precisely　arranged　in　corresponding　seats.　The　applications　of　the　CHA-triggered　self-assembly　mechanism　are　also　extended　for　assembling　the　large　DNA　origami　dimer.　With　the　creation　of　1D　architectures　established,　it　is　thought　that　this　CHA-triggered　self-assembly　mechanism　may　provide　a　new　element　of　control　for　complex　autonomous　assemblies　from　a　variety　of　starting　materials　with　specific　sites　and　times.　©　2019　WILEY-VCH　Verlag　GmbH　&　Co.　KGaA,　Weinheim