Single Molecule Mechano-analytical Chemistry

The discovery of optical trapping and invention of laser tweezers in the 1980s offer an unprecedented opportunity to investigate chemical and biochemical processes at the single molecular level. Click here for the mechanism of the laser trapping. Unlike bulk assays where ensemble average information is obtained, single molecule experiments can reveal properties of subpopulations or energetics of sub-trajectories. Laser tweezers have another unique capacity to measure or apply forces from femto- to hundreds of pico-Newtons. These forces are in the same range as those generated by most protein motors, or those needed to alter the rate or fate of many biochemical reactions. As a result, biological systems and processes have been studied using laser tweezers. However, there is a lag of laser-tweezers applications in chemistry. This can be attributed to several reasons. First, tiny amount of the material contained inside a trapped object is difficult to be characterized by many traditional detection methods, such as UV-vis and IR. Second, to build a strong optical trap, objectives with short working distance are often used. This leaves rather constrained space to incorporate other detection methods. Our lab explores unique capabilities of the laser tweezers, i.e., force detection in the range of picoNewtons and spatial measurement down to Angstroms, to follow chemical and biochemical interactions, such as the binding events between receptors and ligands. In addition, highly sensitive mechanochemical biosensors are developed in our lab by combination of laser tweezers and microfluidics.
To perform a laser-tweezers experiment, we grab a non-B DNA structure using two optically trapped particles (see the diagram above). By moving one of the particles away from the other, the structure is mechanically unfolded (see below). Notice the double-stranded DNA (dsDNA) serves as a handle to isolate the non-B DNA structure from the particle surface. Click here for typical force-extension (F-X) curves collected in the lab.
Since 2009, we pioneered the research on the mechanical stabilities of non-B DNA structures, G-quadruplex and i-motif in particular. Since the nineteen fifties, DNA has been known to serve as a storage for genetic codes only. Corresponding to this sole physiological role is the predominant right-handed double helix conformation (a.k.a. B DNA) in vivo. This dogma has not been seriously challenged until people found non-B DNA conformations exist in vivo. Now, it has been recognized that the sites to host these non-B DNA structures are throughout the human genome, most of which are located in the vast non-coding regions. In vivo evidences have confirmed the precence of G-quadruplex structures in cells and pointed out that these structures will function as regulatory elements to control important cellular processes such as transcription and replication. Our studies have so far indicated that the mechanical strength of G-quadruplex or i-motif are comparable to the load force of DNA or RNA polymerase, which are key enzymes for DNA replication and RNA transcription, respectively. These provide a strong support, from mechanical perspective, that the formation of non-B DNA structures may regulate replication or transcription processes.
In 2012, we demonstrated that DNA structures can partition into conformations with and without tertiary interactions. This leads to parallel folding/unfolding pathways, a phenomenon previously only seen in proteins and RNA structures. These observations firmly establish the presence of tertiary DNA conformations.
In 2013, we examined mechanical and kinetical properties of human telomeric RNA G-quadruplex. We discovered the presence of an intermediate that likely assumes RNA G-triplex conformation.
In 2014, we started to investigate protein structures at the single molecular level. See Publications for our most recent progress in this field.