Orientation discrimination of single-stranded DNA inside the !-hemolysin membrane channel

We characterize the voltage-driven motion and the free motion of single-stranded DNA (ssDNA) molecules captured inside the !1.5-nm !-hemolysin pore, and show that the DNA–channel interactions depend strongly on the orientation of the ssDNA molecules with respect to the pore. Remarkably, the voltage-free diffusion of the 3!-threaded DNA (in the trans to cis direction) is two times slower than the corresponding 5!-threaded DNA having the same poly(dA) sequence. Moreover, the ion currents flowing through the blocked pore with either a 3!-threaded DNA or 5! DNA differ by !30%. All-atom molecular dynamics simulations of our system reveal a microscopic mechanism for the asymmetric behavior. In a confining pore, the ssDNA straightens and its bases tilt toward the 5! end, assuming an asymmetric conformation. As a result, the bases of a 5!-threaded DNA experience larger effective friction and forced reorientation that favors co-passing of ions. Our results imply that the translocation process through a narrow pore is more complicated than previously believed and involves base tilting and stretching of ssDNA molecules inside the confining pore.