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Professor and Howard Hughes Medical Institute Investigator
We are primarily a single molecule biophysics lab based in the Department of Physics at Cornell University. Our current research focuses on the motion, dynamics, mechanisms, and regulation of molecular motors that translocate along DNA during the replication and transcription of DNA. To work with biological motors at the single molecule level, we develop and utilize state-of-the-art (and often one-of-a-kind) instruments and novel techniques. Here, we highlight a few novel experimental approaches that we have recently developed.
Single molecule mechanical manipulations of biological molecules; high-resolution optical trapping and detection; single molecule fluorescence imaging and detection; nanophotonics and lab-on-a-chip; molecular motor mechanisms; biopolymer kinetics and dynamics; protein-DNA interactions during transcription and replication; modeling of diffusion, kinetics, and dynamics of biomolecules.
- Biochemistry, Molecular and Cell Biology
- Laboratory of Atomic and Solid State Physics (LASSP)
- Nanobiotechnology Center (NBTC)
- Weill Institute for Cell and Molecular Biology Advisory Committee
Angular Optical Trapping
Our lab pioneered an angular optical trapping technique for simultaneous torque and force generation and detection (La Porta et al., Physical Review Letters, 2004; Deufel et al., Nature Methods, 2007). When a birefringent particle is trapped in a polarized laser beam, rotation of the laser polarization induces rotation of the particle, while torque exerted on the particle is detected as a change in the polarization of the trapping beam. This technique allows the control and detection of the torque of a biological molecule attached to the particle and has opened new dimensions for applications of optical trapping techniques. We have used this technique to determine torque during DNA supercoiling (Forth et al., Physical Review Letters, 2008; Sheinin et al., Physical Review Letters, 2011) and during a Holliday junction migration (Forth et al., Biophysical Journal, 2011). More recently, we have directly measured the torque generated by the RNA polymerase motor during transcription (Ma et al., Science, 2013). These measurements establish RNA polymerase as a powerful torsional motor and offer new insights into how DNA supercoiling regulates transcription.
Protein-DNA interactions dictate gene expression and replication. Although several techniques are able to detect the location of an interaction, few are able to measure its strength, a critical determinant of DNA accessibility. Our lab developed the powerful and versatile DNA unzipping method to measure protein-DNA interactions (Koch et al., Biophysical Journal, 2002; Koch and Wang, Physical Review Letters, 2003; Shundrovsky et al., Nature Structural and Molecular Biology, 2006; Hall et al., Nature Structural and Molecular Biology, 2009). Using an optical trap, we mechanically separate a double-stranded DNA (dsDNA) with bound proteins into two single strands. As the unzipping fork reaches a bound protein, the unzipping force increases dramatically, and then reduces suddenly as the interaction is disrupted. Typically, multiple interactions are detected for a given protein, and the unzipping method maps the locations of these interactions to near single-base-pair precision, while also providing a quantitative measure of their strengths. A variation of this approach also has also allowed us to gain mechanistic insights into a helicase motor that carries out DNA strand separation activities (Johnson et al., Cell, 2007; Sun et al., Nature, 2011).
Optical trapping techniques have proven to be powerful experimental tools, and in the past three decades, they have transformed many areas of biochemistry and molecular biology. Yet these techniques are typically restricted to a specialized group of individuals who spend many years constructing a single instrument, and require repeated measurements, one molecule at a time. In order to make these techniques accessible to a broader scientific community, a new generation of optical trapping instruments is essential. A ‘plug-and-play’ instrument, capable of manipulation with both high resolution and high throughput, would potentially revolutionize the single molecule field. In the first steps towards this goal, we demonstrate dynamic optical trapping control of nanoparticles by a nanophotonic standing-wave array trap (nSWAT) (Soltani et al., Nature Nanotechnology, 2014). This novel electro-optofluidic platform uses photonic interference functionalities to establish an array of stable, three-dimensional on-chip optical traps at the antinodes of a standing-wave evanescent field on a nanophotonic waveguide. The nSWAT contains integrated electric microheaters that enable precision trap repositioning at high speeds, ultimately allowing for the sorting and manipulation of individual DNA molecules. This controllable trapping device has the potential to achieve high-throughput precision measurements on chip.
Ryan Badman, Lucy Brennan, Jessie Killian, Jaeyoon Lee, Ming Li, Seong ha Park, and Guillermo Vargas
James Baker, Tung Le, Jun Lin, Chuang Tan, Yi Yang, and Fan Ye
Dr. James Inman
Dr. Shanna Fellman