Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Anno: 2014

Autori: Monico C., Belcastro G., Vanzi F., Pavone FS., Capitanio M.

Affiliazione autori: Univ Florence, LENS European Lab Nonlinear Spect, Florence, Italy; Univ Oxford, Chem Res Lab, Oxford, England; Univ Florence, Dept Biol, Florence, Italy; Univ Florence, Dept Phys & Astron, Florence, Italy; CNR, Natl Inst Opt, Rome, Italy; Int Ctr Computat Neurophoton, Rome, Italy.

Abstract: The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Giornale/Rivista: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS

Volume: (90)      Da Pagina: e51446-1  A: e51446-8

Maggiori informazioni: We thank Gijs Wuite, Erwin J.G. Peterman, and Peter Gross for help with the microfluidics and Alessia Tempestini for help with sample preparation. This research was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no 284464 and from the Italian Ministry for Education, University and Research FIRB 2011 RBAP11X42L006, Futuro in Ricerca 2013 RBFR13V4M2, and in the framework of the Flagship Project NANOMAX.
Parole chiavi: Bioengineering; Issue 90; Single molecule biophysics; Optical tweezers; fluorescence microscopy; DNA binding proteins; lactose repressor; microfluidics
DOI: 10.3791/51446

Citazioni: 5
dati da “WEB OF SCIENCE” (of Thomson Reuters) aggiornati al: 2025-05-18
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