Force-modulated FRET for resolving biomolecular motion and bonding

The project will develop a new technique to measure the strength of macromolecular interactions in biology with higher precision than current methods. The new tool consists of a unique combination of using ultrasound to rupture the interactions and fluorescence microscopy for detection of the rupture. For example, one process that will be monitored in unparalleled detail is that of protein synthesis by the ribosome.

The ribosome is a complex macromolecular machine that "reads" information transcribed from DNA and manufactures its product accordingly. Yet just how it steps along the "message" to read the instructions, and sometime slips out of frame to manufacture an alternate product, are not fully understood. A dynamic picture of the process, combined with what we now know about the structure of the machine, will greatly enhance our understanding how this machine functions, and misfunctions.

Further applications to understand how proteins distinguish among cells surfaces to recognize a distinct target are envisioned. The research activities will generate a wealth of training opportunities for graduate students, undergraduate students, and high school students. In particular, students in underrepresented groups will be encouraged to participate.

In addition, the research will enhance education via the development of a new course at the graduate level that focuses on modern physical, chemical, and biological techniques.

The unique imaging technique, termed as force-modulated fluorescent resonance energy transfer (fmFRET) microscopy, will provide sub-nanometer motion resolution for nucleic acids and sub-piconewton force resolution for non-covalent bonds. The fmFRET technique will integrate the concept of force spectroscopy with FRET detection for the first time: by applying acoustic radiation force with sub-piconewton resolution, the macromolecular interactions will be precisely resolved based on their dissociation forces and detected as fluorescence signal.

Because nucleic acid duplexes of different lengths can be resolved by their dissociation forces, this technique will be able to precisely distinguish the positions of nucleic acids in functioning macromolecular complexes with sub-nucleotide resolution, which cannot be achieved by any other techniques. From the point of view of force spectroscopy, fluorescence detection will be faster, more sensitive, and easier to implement than magnetic detection.

Furthermore, the new technique will be more precise and robust than optical imaging of microparticles that relies solely on software fitting methods.

This work is funded by Molecular Biophysics (Molecular and Cellular Biosciences, BIO) and Chemical Measurement and Imaging (CHE) programs.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.