Figure 3.

Helicase-mediated strand displacement can drive a downstream reaction. (a) Helicase-fueled dissipative strand displacement cycle coupled to a downstream reaction. There is a 10-nucleotide toehold on β in the α:β complex. Rep-X-mediated unwinding of the ground-state hybridized strands (α:β) results in an excited state where α, β, and γ are single-stranded. From the excited state, either α and β can hybridize to form an α/β complex to enter the ground state or α and γ can hybridize to enter the metastable state; the complex α/γ is transient. We tracked the states of this reaction using fluorescence measurements: α has an Iowa black quencher on its 3′ end, β has a 5-FAM fluorophore on its 5′ end, and γ has a 5-Cy3 fluorophore on its 5′ end. (b) Free-energy diagram of the α, β, and γ three-strand system. α:β and γ comprise the ground state (−37 kcal/mol); all single strands in solution comprise the excited state (0 kcal/mol); and α:γ and β comprise the metastable state (−27 kcal/mol). The values refer to the free energies of the hybridization process, starting in the “excited state” where all DNA molecules are single-stranded. The free energies were calculated with NUPACK using a reference concentration of 55.6 M (the concentration of water molecules in water), which is appropriate for an aqueous solution at low solute concentrations. When in the excited state, the system can either move back to the ground state or into the metastable state. The metastable state relaxes back to the equilibrium state over time. (c) Concentrations of β strands unwound by Rep-X with either ATP alone or ATP with regeneration machinery. With 100 nM Rep-X and 100 nM α, β, and γ, either 1 mM ATP (neon green) or 1 mM ATP with ATP regeneration machinery (dark green) was added to the solution. We observed a decline in Rep-X activity immediately after the maximum fraction of β was unwound in the sample containing only ATP. A steady fraction of unwound α/β was maintained for around 5 h in the sample containing ATP regeneration machinery. Both samples returned to or approached their baseline fluorescence after 8 h. (d) Concentrations of γ strands after Rep-X unwinds α:β using either ATP or ATP with regeneration machinery. With 100 nM Rep-X and 100 nM each of α, β, and γ, either 1 mM ATP (orange) or 1 mM ATP and regeneration machinery (dark red) was added to the solution. ∼65 and ∼75% of available γ strands hybridized with free α in the samples containing ATP and ATP with regeneration machinery, respectively. ∼75% of γ strands remained hybridized for about 5 h with ATP with regeneration machinery, whereas with ATP alone, the amount of free γ increased after reaching its minimum and returned to baseline after about 8 h.