While single-molecule detection enables direct characterization of annealing/melting kinetics of nucleic acids (NA) without the need for synchronization of molecular states, all current experiments are performed on a surface or inside a trap. As the conditions for these single-molecule experiments are not physiologically relevant, there has been speculation that the hybridization kinetics are different inside cells. 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 DNA models with different GC content and DNA modification. Our results indicated a generally accelerated DNA hybridization kinetics kon in live cells (up to 108-fold), while the extent of acceleration was quite different among the three model DNA. Interestingly, we found phosphorothioate-modified strands can be thermodynamically less stable in the cellular environment than we expected, which cannot be predicted by high-resolution melting analysis. This information is critical to the use of modified nucleic acids in disease treatment, despite the fact that they are resistant to the nuclease degradation. This is the first time that DNA hybridization kinetics were measured in 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.

Measuring DNA hybridization kinetics in live cells using a time-resolved 3D single-molecule tracking method

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.