Tethered Particle Motion Experiments as a Gateway to Biophysics ResearchObinna A. Ukogu , Adam D. Smith , Robert D. Schwab and Ashley R. Carter

Department of Physics, Amherst College, Amherst, MA 01002

Tethered particle motion (TPM) is a technique used to study the mechanical properties of biological molecules like DNA. Setting up a TPM assay involves attaching one end of a DNA molecule to a surface and the other end to a small bead (1 µm in diameter). Changes in the bead's position with time (due to displacement by Brownian motion) are then tracked using video microscopy and used to infer the length of the DNA. If a condensing agent is added, one can measure the rate of DNA condensation as the length decreases. In this lab, our goal is to use TPM to experimentally determine how long it takes to completely condense a strand of DNA in the presence of a condensing agent at a chosen concentration. Possible condensing agents include protamine, hexamminecobalt(III), spermidine, and spermine. Here, we will measure the condensation rate for protamine. Decreasing the concentration of the condensing agent will allow for open-ended research on the formation of the condensed structure. Com-bining this laboratory with the laboratories described in “Using Tethered Particle Motion Exper-iments in Statistical Mechanics or Biophysics Labs” will offer a gateway to biophysics research in the undergraduate laboratory.

Abstract

Goals

Laboratory Open-ended Research

Conclusion

Before Protamine After Protamine

● Track Beads

● Measure Condensation rate

• To provide an interdisciplinary laboratory in biophysics

• To provide a small project that will help introduce undergraduates to a research project

Acknowledgments: This work was supported by the Carter lab and Amherst College.

Intro to Tethered Particle Motion and DNA Folding

We used tethered particle motion to measure the rate of DNA condensation by protamine. Students performing this lab will learn an interdisciplinary approach to biophysics by combining skills learned in biochemistry, microscopy, and computer vision. This laboratory could serve as small research project laboratory in a biophysics class and should be preceded by an intoductory laboratory on tethered particle motion (see “Using Tethered Particle Motion Experi-ments Statistical Mecyhanics or Biophysics Labs” by Adam D. Smith). Successful completion of this lab will leave students with a profound sense of satisfaction.

For further research:

• Students can measure the rate of condensation with different condensing agents and compare their results. They should try Spermidine and hexacobalammine(vi).

• Students can measure the rate of condensation with protamine at different concentrations.

• Students can investigate how protamine forms DNA toroids by (1) Directly measuring DNA folding of protamine, (2) Observing intermediates between folding states, and (3) Studying the folding & unfolding dynamics of toroids

● Image Beads

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● Prepare Sample

t5 t5

Variables:Condensation Time (T) = Condensation End - Condensatiion Start = 270 s - 235 s = 35 s Length (L) = Length of DNA strand = 1023 nmR = L/T = 1023/35 ~ 29 nm/s

Questions for Students:1) How would you calculate the rate of condensation, R, in basepairs per second?2) Why is our rate not a very good one? What is the length of the DNA strand after condensation?

Goal: Measure the rate of DNA condensation by protamine using TPM.Time Required: 3 hours to prepare assay, 1 hour to take data and analyze it

Free Particle

Tethered Particle

Stuck ParticleDNA

Cover slip

Figure 1. Differences between free particle motion and tethered particle motion (TPM). In TPM [1], a particle is tethered to a cover slip via a DNA molecule (top) and does not undergo dif-fusion like a free particle or stay in place like a stuck particle. While the mean squared displace-ment (MSD) of the free particle in one dimension (<x2>) increases with time (t), the stuck parti-cle has a negligible MSD, and a tethered particle has a constant MSD that is given by the length DNA tether.

Figure 2. We can use TPM to measure DNA folding. When a DNA molecule (top) is ex-posed to particular condensing protein, like protamine (blue dots), the DNA will fold, in this case, into loops. The motion of the tethered particle will decrease as the DNA folds, allowing us to measure a folding rate.

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References:[1] Schafer, Dorothy A., Gelles, Jeff, Sheetz, Michael P., Landick, Robert. (1991). Transcription by single molecules of RNA polymerase observed by light microscopy. Nature, 352.[2] Catipovic, Marco A., Tyler, Paul M., Trapani, Josef G., Carter, Ashley R. (2012). Improving the quantification of Brownian motion. American Journal of Physics, 81. [3 Beausang, J.F., Zurla, C., Finzi, L., Sullivan, L. & Nelson, P.C. Elementary simulation of tethered Brownian motion. American Journal of Physics, 75.

Goal: To provide advanced projects of varying levels of difficulty fro undergraduates who have comleted the lab detailed in this poster.

Figure 3. Single 663 nm DNA Trace with Protamine. After the introduction of protamine (oc-curs during the green rectangle), the amplitude of bead movement decreases. The numbers above the trace indicate the different levels of bead movement, where (1) corresponds the highest standard deviation of bead position after the introduction of protamine. Data where flowis present is not shown (covered by “green flow” box).

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