Catalytic DNA Polymerization Can Be Expedited by Active Product Release**

Abstract

The sequence‐specific hybridization of DNA facilitates its use as a building block for designer nanoscale structures and reaction networks that perform computations. However, the strong binding energy of Watson–Crick base pairing that underlies this specificity also causes the DNA dehybridization rate to depend sensitively on sequence length and temperature. This strong dependency imposes stringent constraints on the design of multi‐step DNA reactions. Here we show how an ATP‐dependent helicase, Rep‐X, can drive specific dehybridization reactions at rates independent of sequence length, removing the constraints of equilibrium on DNA hybridization and dehybridization. To illustrate how this new capacity can speed up designed DNA reaction networks, we show that Rep‐X extends the range of conditions where the primer exchange reaction, which catalytically adds a domain provided by a hairpin template to a DNA substrate, proceeds rapidly.

Directing Uphill Strand Displacement with an Engineered Superhelicase

“…Rep-X and CPK were synthesized and purified according to the protocol described in refs and . In short, recombinant Rep and CPK with 6-histidine His-tags were produced in E.…”

“…Therefore, the function and selectivity of Rep-X allows one to design systems in which Rep-X will unwind only specific complexes in each reaction network. 29 In this work, we show how Rep-X's strand-unwinding propensity can be leveraged to trigger transient and energetically uphill DNA strand displacement reactions by releasing the participating strands from a passive hybridized complex. This release, or unwinding of dsDNA, provides the 15 the invading strand binds to the toehold of a partially double-stranded complex and displaces the red output strand, which can participate in downstream reactions.…”

“…Replacing DNA nucleotides on the 3′ end with methylated RNA also drastically reduces unwinding by Rep-X. 29 Second, to develop larger networks, the development of the ODE-based models to predict how the network's performance is affected by imperfect protection from Rep-X unwinding or unintended side reactions will be important. Because the incorporation of methylated RNA affects the reaction thermodynamics and kinetics, such modeling is not trivial and will require input from quantitative experiments.…”

“…Like native Rep, Rep-X can only bind to single-stranded 3′ overhangs to start the unwinding of duplexes (Figure b). Therefore, the function and selectivity of Rep-X allows one to design systems in which Rep-X will unwind only specific complexes in each reaction network …”

A simple method to alter the binding specificity of DNA-coated colloids that crystallize

“…S1, ESI †). 31 The decreased PER rate when the substrate DNA is grafted onto colloidal particles compared to DNA free in solution is likely due to the steric hindrance of the DNA polymerase in the dense DNA coating.…”