Enzymes involved in many fundamental cellular processes, such as DNA replication, RNA transcription, and protein translation, are motor proteins that apply a load force during catalytic reactions.  Interaction of these motor proteins with secondary structures in nucleic acid templates can therefore be understood from a mechanical perspective.  To serve as an effective mechanical blocker to motor proteins, the mechanical stability of these secondary structures, which include G-quadruplex and i-motifs, must be strong enough to withstand the load force of motor proteins.  Optical tweezers provide a convenient tool to evaluate the mechanic stability of nucleic acid structures.  Since 2009, we have evaluated mechanical stabilities of hairpins, G-quadruplexes, and i-motifs in DNA, and G-quadruplexes in RNA.  Under physiologically relevant conditions, such as in double stranded DNA templates or in crowded solutions, mechanical stabilities of G-quadruplexes are higher than stall forces of polymerases.  Mechanical stability of a structure is dependent on unfolding angles.  Due to the limitation in current molecular biology strategies that process nucleic acid templates along the 5'-3' direction, most of mechanical unfolding has been confined into the same direction, which may not necessarily be a physiologically relevant angle. Assisted with click chemistry modifications, we expanded the unfolding into other geometries by introducing two unfolding handles at specific residues inside a G-quadruplex forming sequence.  Statistical analyses allowed retrieving kinetic, thermodynamic, and mechanical information of nucleic acid structures along desired trajectories.  In the presence of G-quadruplex specific ligands, such information demonstrates significant difference, suggesting that the interaction between small molecules and G-quadruplexes not only changes the kinetic and thermodynamic properties, but also varies the mechanical stability of G-quadruplexes.

Dissection of individual G-quadruplex structures with optical scalpels

247th National Meeting (2014)

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