Comparison of In-Vitro and In-Vivo DNA Hybridization Kinetics Using 3D Single-Molecule Tracking Method

While single-molecule detection enables direct characterization of annealing/melting kinetics of nucleic acids (NA) without the need for synchronization of molecular states, the current experiments are mostly performed on a surface or inside a trap rather than in a native cellular context. Here we demonstrate an integrated 3D single-molecule tracking (3D-SMT) and fluorescence lifetime measurement method that can follow individual DNA molecules diffusing inside a mammalian cell and observe multiple annealing and melting events on the same molecules. With sub-diffraction-limit spatial resolution in molecular tracking and 15 ms temporal resolution in lifetime monitoring, we evaluated and compared the in-vitro and in-vivo hybridization kinetics of three 8-bp duplex DNAmodelswith different GC content and DNA modification. From the kon-koff kinetics map, we could see the ranking of kon was 87.5% GC > 37.5% GC > 87.5% PS-GC while koff was 37.5% GC > 87.5% PS-GC > 87.5% GC for both in-vivo and in-vitro studies (here PS stands for phosphorothioate modification). However, the in-vivokon of 87.5% GC strand 108-fold was higher than its in-vitro value (492 vs. 4.56∗106 M−1s−1). The in-vivo association constant (Ka = kon/koff) was also higher than its in-vitro value (163-fold increase for 87.5% GC strand, 72-fold increase for 37.5% GC strand, and 13-fold increase for 87.5% PS-GC strand). These findings indicated a generally accelerated DNA hybridization in vivo, which might result from the molecular crowding effect and existence of recombination mediator proteins. This is the first time that DNA hybridization kinetics were measuredin live cells using the 3D-SMT method. The kinetics data acquired can be used to generate new models that can predict in-vivo hybridization kinetics from sequence, study the molecular crowding inside cells, and probe the cellular development and transition states.