Single Molecule Protein Folding with DNA Origami in Optical Tweezers
Carlos Bustamante, Professor
QB3: Quantitative Biomedical Research
Applications for Fall 2025 are closed for this project.
Optical tweezers are a method to exert force on individual molecules and directly measure forces generated during their biochemical reactions.[1] Molecules are tethered between double strand DNA (dsDNA) handles to micron-sized beads that are held in optical traps. The flexibility of these handles limits the resolution of optical tweezers. DNA origami is a nanoengineering method of assembling DNA into highly structured constructs. Replacing dsDNA handles with DNA origami generates higher spatial resolution in optical tweezers.[2-3] However, applications have been limited.[4] I seek to establish DNA origami as the next-generation method of optical tweezers. Students will help establish proof of concept that origami reveals hidden dynamics in two biological contexts where poor signal-to-noise (SNR) restricts biophysical insight: nucleosome unwrapping and protein folding.
Many proteins with highly conserved structural elements contain just a few secondary structures and generate small extension changes upon folding. Additionally, most naturally-occurring proteins are unstable and fold in regimes where the signal is masked by a high background. By increasing spatial resolution, DNA origami will illuminate the folding mechanism of representative proteins and suggest folding patterns for a wide number of proteins that share the studied structural elements.
[1] Bustamante, C. et al. Optical tweezers in single-molecule biophysics. Nature Reviews 2021. https://doi.org/10.1038/s43586-021-00021-6
[2] Pfitzner, E., Wachauf, C., Kilchherr, F. et al. Rigid DNA beams for high-resolution single-molecule mechanics. DNA Nanotechnology 2013. https://doi.org/10.1002/anie.201302727
[3] Shaw, A., Satija, R. et al. Rigid DNA nanotube tethers suppress high frequency noise in dual-trap optical tweezers systems. bioRxiv 2021. https://doi.org/10.1101/2021.11.15.468716
[4] Zhang, H., Canari-Chumpitaz, C., et al. DNA origami–enhanced force spectroscopy and AlphaFold structural analyses reveal the folding landscape of calcium-binding proteins. Science Advances 2025. https://doi.org/10.1126/sciadv.adv1962
Role: ● Guided literature review and research proposal (~3 weeks)
○ Students will learn how to read scientific papers.
○ Students will learn the physical principles underlying optical tweezers and DNA origami and be able to explain them to a scientific audience. They will also learn what has been previously studied about single molecule protein folding and nucleosome unwrapping.
○ Students will learn how to write a one-page research proposal in a scientific voice.
● Training on optical tweezers (~5 weeks)
○ Students will learn to operate and troubleshoot optical tweezers, as well as collect consistent high-resolution data
○ Students will learn to run MATLAB code to analyze single-molecule data
● Present to lab members (1 week each semester)
○ Students will learn how to convey background information, present data and methodology to an informed audience, and receive scientific feedback from specialists.
● Protein design, expression, and purification (~4 weeks)
○ Students will learn to biochemically modify proteins to facilitate characterization of folding pathways, use computational tools to assess structures, interpret molecular dynamics simulations of protein folding, recombinantly express and purify proteins from E. Coli, and perform fast performance liquid chromatography (FPLC).
● Collect and analyze single molecule data with DNA origami on optical tweezers (~4 weeks)
○ Students will learn how to identify trends in single molecule data, rigorously quantify biological processes with statistics, and assess hypothesized models.
● Design a research poster (1 week)
○ Students will learn how to format a research poster and present data and methodology to an uninformed audience.
Qualifications: No specific coursework required, although mechanics, optics, and thermodynamics are helpful.
● Engage critically with the research project
● Attend lab on a previously agreed-upon schedule (at least 10 hours per week)
● Communicate absences in advance
● Be responsive to feedback from graduate mentor
● Attend weekly research cluster meetings
● Schedule permitting, attend lab meetings and journal club
Day-to-day supervisor for this project: Shantanu Kadam, Graduate Student
Hours: 9-11 hrs
Related website: https://bustamante.berkeley.edu