Fig. 2. ATP-fueled transient DySS polymerization of dynamic covalent DNA chains with a tunable lifetime.

(A) Time-dependent GE shows transient lifetimes programmed by ATP fuel concentration (0.1 to 1.0 mM ATP, left to right). Lane assignment: 1, 50-bp ladder; 2, 1-kbp ladder; 3, 0 min; 4, 10 min; 5, 1 hour; 6, 9 hours; 7, 24 hours; 8 to 16, 2 to 10 days (daily interval). (B) Gray scale profiles extracted from GE at 0.4 mM ATP (panel ii) quantify the transient, reversible shifts of molecular weight (top, growth; bottom, decline), which is used to calculate the mass-weighted average chain length (${\overline{\mathit{bp}}}_w$) for each kinetic aliquot. (C) The development of ${\overline{\mathit{bp}}}_w$ over time reveals increasing lifetimes of the transient DNA polymerization with a constant steady-state chain length of around 1000 bp under the given enzymatic conditions when increasing the ATP concentration from 0.1 to 1.0 mM. Lines are guides to the eye. (D) The lifetime scales linearly with the amount of supplied ATP. Error bars result from averaging triplicate measurements. Conditions: 0.05 mM M₁, 41.25 WU of T4 DNA ligase, 900 U of BamHI, and varying amounts of ATP at 25°C in the enzyme reaction buffer.