Fig. 1. Experimental setup.

A ALternative Isoinformational ENgineered (ALIEN) DNA (right) consists of four anthropogenic bases (Z, P, S, B) that form orthogonal base pairs (Z:P and S:B) through size and hydrogen bond complementarity. ALIEN DNA is structurally similar to canonical ACGT-DNA (left) and is “isoinformational”, also carrying two bits of information at each site. B A single Mycobacterium smegmatis porin A (MspA) nanopore (yellow) is embedded in a lipid bilayer. A single DNA molecule bound by a Hel308 helicase (green) is drawn into the pore by an electric field. The ion current through the pore is modulated by the bases in the pore constriction and is measured to sequence the strand. C Schematic for the DNA design used throughout this manuscript. Each strand had a 5′ region composed of ACGT DNA designed for ion current calibration and 3′ region designed for motor enzyme loading. The GA tandem repeat on the 3′ end was selected to aid in sequence-to-conductance alignment. We found previously that every 4th step, on the ATP-independent step, Hel308 helicase has increased propensity for backstepping. We used these backsteps in the raw data to help ensure correct registration of the DNA bases to the measured conductance curves. D A representative variable-voltage nanopore trace of a DNA strand section containing 28 consecutive ALIEN bases. Conductance (current/voltage) is displayed instead of ion current for variable-voltage traces due to the variation of the applied voltage. Each curve segment represents one enzyme step of the Hel308 helicase, which feeds DNA through the pore in two steps per nucleotide, roughly equal in length. For ease of visualization, enzyme backsteps have been filtered and recombined in software (Supplementary Information).