Surface-Immobilized pH-sensitive DNA Triplexes

Dynamic DNA machines leverage base-pairing specificity and environmental sensitivity to reversibly switch between conformational states. A prominent example is pH-sensitive DNA nanoswitches, actuated by proton-mediated Hoogsteen interactions in triplex-forming domains. To date, studies of pH-sensitive DNA nanoswitches have largely focused on DNA machines that are freely diffusing in the solution phase. For many applications such as biosensing and DNA data processing and archival, it is advantageous to integrate these dynamic DNA machines with solid-state devices, requiring immobilization on surfaces. This thesis explores the dynamics of pH-sensitive DNA triplexes immobilized as dense 2D monolayers (∼ 10^12 molecules/cm^2) on gold using thiol chemistry. Switching dynamics on-surface was monitored using quartz crystal microbalance with dissipation (QCM-D), while complementary single-molecule Förster resonance energy transfer (smFRET) studies confirmed solution-phase pH-responsivity. In solution, a 5-base loop triplex showed reversible switching with a pKa of 7.83 ± 0.03 (SD) and thermodynamic analysis indicated enthalpy-driven triplex formation. Following surface-immobilization, QCM-D confirmed that the DNA triplex switch retained functionality, switching between conformations with a pKa of 8.02 ± 0.03 (SD) and that the switching was repeatable and reversible over 20 pH cycles. Kinetic analysis revealed pH-induced opening of the immobilised triplex switch followed a first order exponential process while closing was a second-order exponential, in agreement with previous literature, with time constants of 20–90 s. Variations in loop length (from five bases-5B to twelve bases- 12B) showed that longer loops modified both the pKa and the kinetics of triplex closing, with the 12B construct exhibiting faster, first-order kinetics. The kinetics of loop opening were also shown to be influenced by the presence of mismatches within the triplex forming domain, with mismatches at the extremities resulting in faster opening rates. Finally, loop-complementary strands were seen to bind selectively to open triplexes, preventing reclosure and highlighting competition between Hoogsteen and Watson–Crick interactions. This study advances understanding of immobilized DNA triplex dynamics and supports the development of hybrid bioelectronic devices for sensing, logic, and data storage.