Computer simulation and design of DNA-nanoprobe for fluorescence imaging DNA repair enzyme in living cells

In situ imaging of DNA repair enzymes in living cells gives important insights to diagnosis and explore the formation of various diseases. Fluorescent probes have become a powerful and widely used technique for their high sensitivity and real-time capabilities, but empirical design and optimization of the corresponding probes can be blind and time-consuming. Herein, we report a strategy combining experimental studies with molecular simulation techniques for the rapid and rational design of sensitive fluorescent DNA probes for a representative DNA repair enzyme human apurinic/apyrimidinic endonuclease 1 (APE1). Extended-system Adaptive Biasing Force (eABF) was applied to study the interaction mechanism between DNA probes with respect to the enzyme, based on which a novel sensitive DNA probe was designed efficiently and economically. Product inhibition effect which significantly limited the sensitivity of existing probes was eliminated by decreasing the key interactions between DNA probe products and enzyme. Experimental mechanism studies showed the existence of intramolecular hairpin structure in DNA probes is important for the recognition of APE1 and elimination of product inhibition, which is in consistent with the simulations. The obtained fluorescent DNA nanoprobe (Nanoprobe N) showed a high sensitivity for APE1 with the detection limit as low as 0.5 U/L (∼0.018 pM), and the Nanoprobe N could effectively respond to the variation of APE1 within cells and distinguish cancer cells from normal cells. This work not only demonstrated the effectiveness of molecular simulations in probe design, but also provided a reliable platform for accurate imaging of APE1 and effectors screening at single-cell level.