Helicase SPRNTing through the nanopore.

Enzymes that move directionally on single-stranded nucleic acids are at the core of emerging nanopore sequencing technology. Of a particular use are DNA helicases, molecular motors that bind single-stranded DNA (ssDNA) independently of its sequence and use ATP to fuel their directional motion along the DNA (1). In nanopore-based sequencing, a pore formed by a protein channel embedded into a lipid membrane forms an electrical connection between two salt solutions; electrostatic potential applied across the nanopore drives individual ssDNA molecules through the nanopore aperture; the helicase bound to the DNA controls the ssDNA movement by either pulling the DNA out of the nanopore or by feeding it into the nanopore one nucleotide at a time. The ion current through the nanopore reveals the DNA sequence, while changes in this current reflect the ssDNA movement within the helicase (2, 3). Choosing the right enzyme and utilizing its full potential in the nanoporebased sequencing require a detailed understanding of the helicase’s mechanochemical cycle. A report in PNAS (4) delves into a complex mechanism of the ssDNA translocation by HEL308 DNA helicase. The results of the reported investigation offer intriguing insights into the helicase translocation mechanism, as well as information important for improving the nanopore sequencing methodology. Since the discovery that charged molecules, such as ssDNA, could be electrophoresed through an ion channel in a lipid bilayer (5), nanopore technology has developed into a robust method of nucleic acid sequencing (2, 3). Nanopore sequencing utilizes a pore placed in a phospholipid bilayer separating two electrolyte solutions. A voltage applied across the membrane generates an ion current, which drives singlestranded oligonucleotides, ssDNA or ssRNA, through the pore (6). A helicase or polymerase that is bound to the oligonucleotide feeds the nucleic acid strand into or pulls it through the channel in single-nucleotide steps (Fig. 1). Helicases offer an advantage over polymerases as they bind single-stranded nucleic acid molecules at and initiate movement from random positions along the lattice, while polymerases require a partial duplex where the new nucleotides are added to the 3′ end of the primer. Because of the polar nature of single-stranded nucleic acids and because of the directional nature of the helicase movement on their respective substrates (some helicases move with 3′-to-5′ directionality, while others move 5′ to 3′), whether the ssDNA will be pulled through the nanopore with the current or against it depends on which end of the molecule enters the nanopore first. As the oligonucleotide is progressing through the pore, the ion current fluctuates in a manner dependent upon the nucleotides that are within the channel. The fluctuations in current are then analyzed to determine the sequence of the polynucleotide (6). Several challenges come with the optimization of nanopore sequencing. The first challenge presents itself in detection of the sequence. Ion current fluctuations used to determine the sequence depend Fig. 1. Defining the HEL308–ssDNA translocation mechanism by SPRNT. (A) Schematic representation of the SPRNT experiment that utilizes HEL308 DNA helicase as the DNA translocating motor and the reengineered MspA porin as a biological nanopore. (B) Domain organization and motions of HEL308.