Abstract
The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead’s random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Similar content being viewed by others
References
J.F. Beausang and P.C. Nelson, Diffusive hidden Markov model characterization of DNA looping dynamics in tethered particle experiments, Phys. Biol., 4 (2007), pp. 205–219.
J.F. Beausang, C. Zurla, C. Manzo, D. Dunlap, L. Finzi, and P.C. Nelson, DNA looping kinetics analyzed using diffusive hidden Markov model, Biophys. J., 92 (2007), pp. L64–6.
S. Blumberg, A. Gajraj, M.W. Pennington, and J. Meiners, Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy, Biophys. J. (2005), pp. 1272–1281.
L. Czapla, D. Swigon, and W.K. Olson, Sequence-dependent effects in the cyclization of short DNA, Journal of Chemical Theory and Computation, 2 (2006), pp. 685–695.
L. Finzi and J. Gelles, Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules, Science, 267 (1995), pp. 378–80.
J. Gelles, B. Schnapp, and M. Sheetz, Tracking kinesin-driven movements with nanometre-scale precision, Nature, 331 (1988), pp. 450–453.
L. Han, H.G. Garcia, S. Blumberg, K.B. Towles, J.F. Beausang, P.C. Nelson, and R. Phillips, Concentration and length dependence of DNA looping in transcriptional regulation. Submitted; available at http://arxiv.org/abs/0806.1860, 2008.
P.C. Nelson, Colloidal particle motion as a diagnostic of DNA conformational transitions, Curr. Op. Colloid Intef. Sci., 12 (2007), pp. 307–313.
P.C. Nelson, C. Zurla, D. Brogioli, J.F. Beausang, L. Finzi, and D. Dunlap, Tethered particle motion as a diagnostic of DNA tether length, J. Phys. Chem. B, 110 (2006), pp. 17260–17267.
D. Normanno, F. Vanzi, and F. Pavone, Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping, Nucl. Acids Res., 36 (2008), pp. 2505–2513.
N. Pouget, C. Dennis, C. Turlan, M. Grigoriev, M. Chandler, and L. Salome, Single-particle tracking for DNA tether length monitoring, Nucl. Acids Res., 32 (2004), pp. e73–(1–7).
D.A. Schafer, J. Gelles, M.P. Sheetz, and R. Landick, Transcription by single molecules of RNA polymerase observed by light microscopy, Nature, 352 (1991), pp. 444–8.
D.E. Segall, P.C. Nelson, and R. Phillips, Volume-exclusion effects in tethered-particle experiments: Bead size matters, Phys. Rev. Lett., 96 (2006), pp. 088306–(1–4).
M. Singh-Zocchi, S. Dixit, V. Ivanov, and G. Zocchi, Single-molecule detection of DNA hybridization, Proc. Natl. Acad. Sci. USA, 100 (2003), pp. 7605–10.
S. Tolic-Norrelykke, M. Rasmussen, F. Pavone, K. Berg-Sorensen, and L. Oddershede, Stepwise bending of DNA by a single TATA-box binding protein, Biophys. J., 90 (2006), pp. 3694–703.
K. Towles, J.F. Beausang, H.G. Garcia, R. Phillips, and P.C. Nelson, First-principles calculation of DNA looping in tethered particle experiments. Submitted; available at http://arxiv.org/abs/0806.1551, 2008.
B. van den Broek, F. Vanzi, D. Normanno, F.S. Pavone, and G.J. Wuite, Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI, Nucleic acids research, 34 (2006), pp. 167–74.
A.M. van Oijen, P.C. Blainey, D.J. Crampton, C.C. Richardson, T. Ellenberger, and X.S. Xie, Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder, Science, 301 (2003), pp. 1235–8.
F. Vanzi, C. Broggio, L. Sacconi, and F.S. Pavone, Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion, Nucl. Acids Res., 34 (2006), pp. 3409–20.
F. Vanzi, S. Vladimirov, C.R. Knudsen, Y.E. Goldman, and B.S. Cooperman, Protein synthesis by single ribosomes, RNA, 9 (2003), pp. 1174–9.
M.D. Wang, H. Yin, R. Landick, J. Gelles, and S.M. Block, Stretching DNA with optical tweezers, Biophys. J., 72 (1997), pp. 1335–1346.
O.K. Wong, M. Guthold, D.A. Erie, and J. Gelles, Interconvertable lactose repressor-DNA looped complexes revealed by single-molecule experiments, 2008. PLoS Biology, in press.
H. Yin, R. Landick, and J. Gelles, Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule, Biophys. J., 67 (1994), pp. 2468–2478.
C. Zurla, A. Franzini, G. Galli, D. Dunlap, D.E.A. Lewis, S. Adhya, and L. Finzi, Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda, J. Phys.: Condens. Matter, 18 (2006), pp. S225–S234.
C. Zurla, T. Samuely, G. Bertoni, F. Valle, G. Dietler, L. Finzi, and D.D. Dunlap, Integration host factor alters LacI-induced DNA looping, Biophys. Chem., 128 (2007), pp. 245–52.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Springer Science+Business Media, LLC
About this paper
Cite this paper
Han, L., Lui, B.H., Blumberg, S., Beausang, J.F., Nelson, P.C., Phillips, R. (2009). Calibration of Tethered Particle Motion Experiments. In: Benham, C., Harvey, S., Olson, W., Sumners, D., Swigon, D. (eds) Mathematics of DNA Structure, Function and Interactions. The IMA Volumes in Mathematics and its Applications, vol 150. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-0670-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-4419-0670-0_6
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4419-0669-4
Online ISBN: 978-1-4419-0670-0
eBook Packages: Mathematics and StatisticsMathematics and Statistics (R0)