Figure 1.

Event structure. (a) Crystal structure of M2-NNN MspA. Charged vestibule residues are indicated in blue (negative) or red (positive). (b) A schematic depicting a standard experiment. Roman numerals correspond to positions in the current trace in c. (c) The measured blockage current (I_b) as a fraction of the open pore current (I_o) is shown for a sample event. (i) A single MspA pore (purple) in a lipid bilayer (gray). The template strand (black) contains the sequence to be read. A primer strand (blue) is hybridized to the template’s 3′ end. A blocking oligomer (red) with a 3′ end of several abasic sites is adjacent to the primer. The phi29 DNAP (green) binds to the DNA to form a complex that is driven into MspA. A positive voltage is applied to the trans side. The single stranded 5′ end of the DNA-motor complex threads through MspA and the ionic current drops. (ii) The electric force on the captured strand draws the DNA through the phi29 DNAP, unzipping the blocking oligomer. Arrows show the direction of motion of the DNA template strand. The ionic current exhibits distinct steps while nucleotides pass through the pore. (iii) The blocking oligomer is removed and DNA reverses direction (marked by blue dashed line). (iv) The phi29 DNAP incorporates nucleotides into the primer strand, pulling the template toward the cis side. The current repeats previously observed levels in reverse time-order. Two abasic sites produce a high current peak (~0.6 I_o) indicated by red Xs. This marker is first seen during unzipping and then again during synthesis. When synthesis is complete, the DNA and DNAP escape to the cis volume, marked by the return to I_o.