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Understanding the Elasticity of DNA and Cooperative Binding of Proteins and Drugs

This project aims to investigate the relationship between DNA elasticity and cooperative binding of proteins and drugs. Using optical tweezers, the persistence and contour length of single DNA molecules will be measured. The interaction between DNA and molecules of pharmaceutical interest, such as beta-cyclodextrin, will also be studied. Experimental results and theoretical models will be used to understand the Hill cooperativity in biochemical reactions. The project is supported by Fapemig, CNPq, Pronex-Facepe, and INCFx.

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Understanding the Elasticity of DNA and Cooperative Binding of Proteins and Drugs

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  1. Instituto de Física da Universidade de São Paulo, São Carlos “Projeto Café com Física” Relação entre elasticidade de DNA e a ligaçãocooperativa de proteínas e fármacos Oscar Nassif Mesquita Departamento de Física, Universidade Federal de Minas Gerais, Belo Horizonte Trabalho em colaboração com: Lívia Siman Gomes (Doutoranda, Física - UFMG) Ismael S. Silva Carrasco (Mestrando, Física - UFV) Prof. Jafferson K. L. da Silva (Física - UFMG) Prof. Ricardo S. Schor (Física – UFMG) Profa. Mônica C. de Oliveira (Farmácia – UFMG) Prof. Márcio Santos Rocha (Física – UFV) Agênciasfinanciadoras: Fapemig, CNPq, Pronex-Facepe, INCFx-Instituto Nacional de Fluidos Complexos e Aplicações

  2. Outline • Stretchingsingle DNA moleculeswithopticaltweezers: measurementofthepersistencelengthandcontourlength. • Studyoftheinteractionbetween DNA andmoleculesofpharmaceuticalinterest. • Interactionbetween DNA and beta-cyclodextrin: non-monotonicflexibility. • HU-DNA interaction: previousexampleof non-monotonicflexibility. • Hill cooperativity in biochemicalreactions. • Ourtwo-sites quencheddisordermodeltoexplain non-monotonicflexibilities. • Resultsanddiscussion. • Conclusions.

  3. An optical tweezers is just a light beam trapping some material (A. Ashkin example)

  4. Single Molecule Experiments Schematic set-up of optical tweezers Optical tweezers is an invention of A. Ashkin in 1970, Phys. Rev. Lett. 24, 156 (1970) Complete theory of optical tweezers for dielectric spheres by Maia Neto and Nussenzveig (Europhys. Lett, 50, 70C2 (2000)), and Mazolli, Maia Neto and Nussenzveig (Proc. R. Soc. Lond. A 459, 3021 (2003)), named Mie-Debye (MD) theory. Viana, Rocha, Mesquita, Mazolli, Maia Neto, and Nussenzveig, APL (2006), and PRE (2007).

  5. Set – up at UFMG

  6. Brownianmotionof a microsphere in a harmonicpotential Langevin equation: Position correlation function satisfies the Langevin equation: Neglecting inertia and using the equipartition theorem

  7. Time autocorrelationfunctionofback-scatteredintensityfluctuationsof a trappedbead Intensityback-scattering profile From the time autocorrelation function we obtain the tweezers´ stiffness for motion perpendicular and parallel to the incident direction.

  8. Tweezerscalibrationwithvideo-imaging =-ln (probability)

  9. Entropicelasticityof a single DNA molecule DNA and RNA stretching experiments First experiment byCarlos Bustamante and co-workers Science (1992) Nucleotides Adenine, Guanine, Cytosine, Tymine

  10. Stretching DNA : entropic elasticity Aisthepolymerpersistencelength A = bendingrigidity/thermalenergy

  11. Marko and Siggia expression for the entropic force, where A is the persistence length, z is the end-to-end distance and L is the contour length of the polymer. Viana, Freire & Mesquita, PRE 65, 041921 (2002)

  12. DNA/Ethidium Bromide Fit to the neighbor exclusion model

  13. DNA-psoralen interaction Persistence length with and without UV light Relative increase of contour length

  14. ab initio DFT calculations Psoralen-DNA fragment with five base CG pairs and two intercalated psoralens obtained from our ab initio DFT calculations. DNA-psoralen: Single-molecule experiments and first principles calculations, APL (2009) M. S. Rocha, A. D. Lúcio, S. S. Alexandre, R. W. Nunes, and O. N. Mesquita

  15. Cyclodextrins are used for condensing DNA intosmalllipidvesicles for gene therapy

  16. CD-DNA persistencelengthmeasuredwithopticaltweezers Blue squares – cationic CD Redcircles – neutral CD

  17. HU-DNA persistencelengthmeasuredwithmagnetictweezers (continuous curve is a guidetotheeye) total HU concentration (nM) J. van Noort et al., PNAS 101 (18), 6969 (2004)

  18. HU-DNA model for bindingand DNA structuralchanges Sagi et al., J. Mol. Biol., 341, 419 (2004) smallerpersistencelength largerpersistencelength HU dimmers (spheres) bindcooperatively (bound-clusters with 4 or 5 HU molecules as measuredby FRET) andcompacts DNA atlowproteinconcentration, each HU dimmerintroducing a small local bend. At high HU concentrations, compactationby HU isreversed, andtheproteinappearstoform a complexwithhelicalstructurewith DNA.

  19. A mechanismofinteractionof CD and DNA with a flipping-out DNA base M. A. Spiesand R. L. Schowen, J. Am. Chem. Soc. 124, 14049 (2002)

  20. Hill cooperativity nligandsbindsimultaneouslytothesubstrate (bound-cluster) L for ligandand S for substrate Mass-actionlaw: Fraction ofligandsbound: isthedissociationconstant; for Hill exponent n < 1 negative cooperativity n = 1 non-cooperativity n > 1 positive cooperativity = 40

  21. Two-sites quencheddisordermodel Assumption 1: When a bound-cluster bindsto DNA it decreasesthepersistencelengthfromthebare DNA valueto; iftwobound-clusters becomenearest-neighborstheystiffenthe DNA, resulting in a largerpersistencelength. Assumption 2: The bound-clusters havethesameaveragesizeof n molecules, cannot move alongthe DNA (quencheddisorder), and are randomlydistributedalongthe DNA. As oneincreasestheligandconcentration in solution, thenumberof clusters increasesproportionally, butnottheirsize. a) Two sites empty, , have probability ; b) One site emptyandtheotheroccupied, have probability c) Two sites occupied, have probability Resultingequation for themodel ,, freeadjustableparameters

  22. Solving Hill equationiteratively Equationhas a single-fixed point solution Experimentally weknowthe total ligandconcentrationbutnotthefreeligandconcentration. Since then, a) zeroth-ordersolution: b) first-ordersolution: Iterativesolutionpossibleif , then

  23. Cationic CD-DNA interaction Fit usingourmodelwithfirst-order Hill equation

  24. HU-DNA interaction Fit usingourmodelwitha zeroth-ordem Hill equation Data from J. van Noort et al., PNAS 101 (18), 6969 (2004)

  25. Conclusions • Wecanstudy DNA interactionswithligandsbymeasuringthepersistencelengthandcontourlengthofthe complexes formed, usingopticaltweezers in single-moleculeassays. • Interactionbetween DNA andbeta-cyclodextrinandbetweenHU-DNA cause non-monotonicpersistencelengthbehavior, indicatingthat for lowligandconcentrationthecomplexformedis more flexibleand for higherconcentrations more rigid. • Wepropose a two-sites quencheddisorderstatisticalmodeltogetherwith Hill cooperativity, whichprovides a modelfunctionwhichfitsverywellboth sets of data. Ourmodelpredictsthatthebindingkineticsismediatedbysizestabilizedbound-clusters. Withthequantitativeparametersobtainedwewereabletopropose a microscopicphysicalmechanism for the CD-DNA cooperativebinding. • Therefore, from a single mechanicalmeasurementwecanobtaintheelasticparametersrelatedtostructuralchangesofthe DNA moleculecausedbytheligands, togetherwiththechemicalparametersofthebindingreaction.

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