Synthetic nanopores made from DNA replicate the key biological processes of transporting molecular cargo across lipid bilayers. Understanding transport across the confined lumen of the nanopores is of fundamental interest and of relevance to their rational design for biotechnological applications. Here we reveal the transport principles of organic molecules through DNA nanopores by synergistically combining experiments and computer simulations. Using a highly parallel nanostructured platform, we synchronously measure the kinetic flux across hundreds of individual pores to obtain rate constants. The single-channel transport kinetics are close to the theoretical maximum, while selectivity is determined by the interplay of cargo charge and size, the pores’ sterics and electrostatics, and the composition of the surrounding lipid bilayer. The narrow distribution of transport rates implies a high structural homogeneity of DNA nanopores. The molecular passageway through the nanopore is elucidated via coarse-grained constant-velocity steered molecular dynamics simulations. The ensemble simulations pinpoint with high resolution and statistical validity the selectivity filter within the channel lumen and determine the energetic factors governing transport. Our findings on these synthetic pores’ structure–function relationship will serve to guide their rational engineering to tailor transport selectivity for cell biological research, sensing, and drug delivery.     «

Synthetic nanopores made from DNA replicate the key biological processes of transporting molecular cargo across lipid bilayers. Understanding transport across the confined lumen of the nanopores is of fundamental interest and of relevance to their rational design for biotechnological applications. Here we reveal the transport principles of organic molecules through DNA nanopores by synergistically combining experiments and computer simulations. Using a highly parallel nanostructured platform, we...     »