Truncated Twinkle Enzyme Catalyzes Toehold Strand Displacement

The patent introduces an enzyme (helicase or polymerase) as an intermediary catalyst to mediate the toehold binding step. The enzyme binds to the toehold region and facilitates strand displacement, converting the slow spontaneous endothermic process into a fast enzyme-catalyzed reaction. This resolves the contradiction by providing a chemical mediator that accelerates the rate-determining step without changing the thermodynamics of the overall reaction.

The patent changes the physical-chemical parameters of the system by introducing enzymatic catalysis. The enzyme alters the activation energy barrier of the toehold binding step, transforming it from a slow spontaneous process to a fast catalyzed process. This parameter change (from spontaneous to enzyme-catalyzed) directly addresses the speed limitation while maintaining the spontaneous nature of the overall TMSD reaction.

The patent divides the full-length twinkle enzyme into two separate functional domains: the N-terminal domain (responsible for helicase activity and TMSD catalysis) and the C-terminal domain (responsible for DNA binding and stability). By expressing only the N-terminal domain or a truncated version, the patent achieves TMSD catalysis while improving solubility and stability by eliminating the problematic C-terminal region that causes aggregation or instability in certain conditions.

The patent extracts and removes the C-terminal domain from the full-length twinkle enzyme to create a truncated version that retains TMSD catalytic activity but has improved solubility and stability characteristics. This extraction of the problematic domain resolves the contradiction between catalytic function and physical stability.