Mechanistic Insights of miRNA Hybridization with ssDNA Self Assembled Monolayers on Gold Electrodes: An Electrochemical and Computational Study.

This work studies the hybridization dynamics of microRNAs with surface-immobilized complementary ssDNA probes. The ssDNA with a terminal thiol group was designed to allow it to form a self-assembled monolayer (SAM) on a Au electrode with low density of coverage to promote flexible ssDNA surface conformations. We characterized the hybridization of immobilized ssDNA with miRNA-4484, a biomarker for various diseases via multiple electrochemical methods including voltammetry, impedance spectroscopy, and open circuit potential. Hybridization events are monitored via electrochemical readouts of the Methylene Blue (MB) labeled ssDNA. For ssDNA without a MB label, an external redox probe, namely Fe(CN)6 3-/Fe(CN)6 4- was used for electrochemical measurements. Experimental results show that the MB label significantly reduced the surface coverage of the ssDNA compared to those without a MB label. The kinetics of the hybridization events in MB-labeled ssDNA show the concentration dependent heterogeneous binding kinetics. Computational studies revealed that the full 20-bp duplex of DNA-miRNA is thermodynamically favorable but kinetically slow to form. Two major kinetic trapping structures are identified, which contain bent DNA strands and may explain the nonlinear behavior and the two-step mechanism observed by our experimental results: miRNA is initially kinetically trapped and then transitions to the 20-bp duplex, as thermodynamic equilibrium is reached given sufficient time. This work provides the fundamental understanding of DNA-miRNA hybridization reactions that are important for designing electrochemical DNA biosensors for miRNA detection.