Mechanical Modulation of Enzyme Activity by Rationally Designed DNA Tweezers: From the Ensemble to the Single-Molecule Level

Abstract

Switchable nanomachines provide a platform to control dynamic functional states by altering distances at the nanoscale on demand. Recently, a tweezers-like DNA device was used to control the activity of an enzyme/cofactor pair juxtaposed on the two arms of the tweezers. Initial studies focused on bulk properties of the tweezers-mediated reactions, and hence lacked insight into the mechanism of enzymatic activation. Here, we used site-specific fluorophore labeling of the tweezers to monitor the arm-to-arm distance through single-molecule fluorescence resonance energy transfer (smFRET). Consistent with AFM measurements, smFRET showed that the tweezers only partially close in the proposed “closed” state and exhibit conformational sub-states. MD simulations showed bending and twisting of the tweezers arms, rationalizing the sub-states. Additionally, smFRET experiments on the isolated Holliday junction hinge suggested that the isomer resulting in the tightest closing of the tweezers (isomer-I) is relatively disfavored, further explaining the only partial closing. We rationally improved the closing by increasing the stem length of the DNA hairpin bridging and actuating the tweezers from 3 to 4 base pairs, and by redesigning Holliday junction(s) of the tweezers to favor the optimal isomer-I. The performance of the new tweezers was quantitatively assessed by juxtaposing glucose-6-phosphate dehydrogenase (G6pDH) with its cofactor NAD+ on the tweezers arms and measuring the G6pDH activity through a coupled enzymatic cascade. Using our optimized tweezers, we were able to enhance the bulk activity of G6pDH upon tweezers closure to up to ∼12-fold. Currently, we are exploring the tweezers-manipulated enzymatic reaction at the single-molecule level. Our results suggest that G6pDH stochastically fluctuates between active—inactive states, favoring the active state upon closure of the tweezers. Our discovery may represent a general approach for refining nanodevices for advanced applications.