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Single Molecule Studies of DNA Mechanics with Optical Tweezers

Single Molecule Studies of DNA Mechanics with Optical Tweezers

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Single Molecule Studies of DNA Mechanics with Optical Tweezers

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  1. Single Molecule Studies of DNA Mechanics with Optical Tweezers Mustafa Yorulmaz Koç University, Material Science and Engineering

  2. Outline • Motivation • Methods • Models • Aims • Basics of optical tweezers • Physics beyond optical trapping • Experimental part • Force-extension curves • Results • Conclusions Koç University, Material Science and Engineering

  3. Motivation • Physical and chemical studies of DNA were performed in bulk previously and measurements were averaged which made it difficult to resolve the time-dependent stresses and strains. • Now, it is possible to make direct measurements of mechanical properties of DNA. • The nature of interactions between proteins and DNA can be investigated with this process. • Knowledge of DNA’s stretching and twisting properties leads to knowledge of DNA replication and transcription. Koç University, Material Science and Engineering

  4. Techniques • There are two techniques in general that are used to study the mechanical properties of DNA; • Optical tweezers • Apply and sense forces on micron-sized dielectric particles in an aqueous environment • Atomic force microscopy • Force extension curve can be obtained through the use of a tip at the end of a flexible cantilever of known force constant Koç University, Material Science and Engineering

  5. Models • In order to understand the physics of DNA, two models were used; • Freely jointed chain (FJC) model • The molecule is made up of rigid segment • Worm-like chain (WLC) model • The molecule is treated like a flexible rod of length L curves smoothly • The stiffer the chain, the longer the persistence length Koç University, Material Science and Engineering

  6. Aims • The force-extension (F-x) regimes of DNA were studied to investigate the elastic behaviors of DNA • Experimental F-x relationships were fit to the entropic elasticity theories based on FJC and WLC models • Persistence lengths which characterize the elastic behavior of DNA were obtained through these fits. Koç University, Material Science and Engineering

  7. Basics of optical tweezers • Optical Tweezers are examples of optical trapping which is a subfield of laser physics. • They are used to trap and manipulate particles (in range from mm to nm) and also to measure the forces (in range from 1 to 100 pN) on these particles • A strongly focused laser beam is used for trapping the particles. • The particles that are of interest has to show dielectric property. Y. Kimuraab and P. R. BiancoAnalyst, 2006, 131, 868-874 * http://www.physics.uq.edu.au/lp/tweezers/papers/nieminen2001jqsrt70/ Koç University, Material Science and Engineering

  8. Physics beyond optical trapping • Trapping occurs due to radiation pressure of laser which results by the momentum change of light. • There are two forces that let the particle to be hold in trap: • Scattering force: due to reflection of light (to push object) • Gradient force: due to refraction of light (to pull object) • nparticle>nmediumso that Fgradient>Fscattering • Using high NA objectives and high power lasers maximizes the trapping force. Schematic diagram showing the force on a dielectric sphere due to both reflection and refraction of two rays of light. Koç University, Material Science and Engineering

  9. Illustrative examples from our laboratory: Manipulation of microspheres Thanks to Ahmet Faruk Coşkun Koç University, Material Science and Engineering

  10. Experimental part • Two ends of DNA were attached to dielectric spheres. The dual beam optical tweezer is used for stretching the DNA http://www.the-scientist.com/article/display/15545/ • Two ends of DNA were attached to the trapped dielectric sphere and glass substrate One end of the DNA is attached to the glass substrate and the other end is attached to the dielectric particle which is trapped by the optical tweezer M. D. Wang et al., Biophys. J. 72, 1335 Koç University, Material Science and Engineering

  11. Force-extension curves F-x curve of DNA which is stretched between two dielectric particles is shown. We observe the overstretching over 60 pN F-x curve for l-phage dsDNA. The data are fit to WLC models and FJC model. Persistence length P=53 nm. Bustamente, C., Bryant Z., Smith S. B., Nature 421 423-427 Bustamante, C., S. B. Smith, J. Liphardt, and D. Smith, 2000, Curr. Opin. Struct. Biol. 10, 279 Koç University, Material Science and Engineering

  12. Force-extension curve Data of F-x of a single DNA were fit to WLC and FJC model. Contour length of the DNA is Lo=~1314 nm and persistence length is found as Lp= ~43 nm for WLC models and Lp=~3.94 nm for FJC model M. D. Wang et al., Biophys. J. 72, 1335 Koç University, Material Science and Engineering

  13. Results • Persistence lengths are calculated under different buffer conditions of 10mM Na+ and Mg2+ or spermidine3+ to be ~0.47 nm and ~0.40 nm respectively: Entropic elasticity. • There was no further decrease in persistence length: Intrinsic elasticity. • DNA behaves like an ideal entropic springs for low-to-moderate extensions beyond their rest length. • Overstretching of DNA was observed above extension forces for >6o pN. Koç University, Material Science and Engineering

  14. Conclusions • Use of optical tweezers is very useful for determining the mechanical properties of single molecules of DNA. • This relationship informs us about the biological interactions. Koç University, Material Science and Engineering

  15. Thank you for your listening.. Questions?? Koç University, Material Science and Engineering

  16. Mechanical Properties can be obtained Bending rigidity of the polymer represented as an elastic rod Energy required to bend a segment. It reveals information about interactions between protein and DNA as DNA is bending protein convert some part of its binding energy into mechanical work. Koç University, Material Science and Engineering