Project 3:Ru DNA BindingTodays topics:1. Macromolecules2. Macromolecular Interactions3. Ru-DNA Project4. Team Assignments5. Experiments

Macromolecules: DNA and Proteinshttp://www.lclark.edu/~bkbaxter/200lecture/lecture_images/1_22_peptidebond.jpgPyrimidines : C and TPurines: A and G

www.solarnavigator.net www.wikipedia.org

Types of InteractionsProtein - ProteinSubunits make up functional proteinProtein Nucleic AcidReplication, DNA repair, Transcription, TranslationProtein Small moleculeATP-dependent enzymesNucleic Acid Small moleculePharmaceuticals

Protein-Protein interactionsMolecular Biology of The Cell, 4th Edition (2002)

Protein-Nucleic Acid: ReplicationMolecular Biology of The Cell, 4th Edition (2002)

DNA PolymeraseMolecular Biology of The Cell, 4th Edition (2002)

Protein-Small MoleculeMolecular Biology of The Cell, 4th Edition (2002)Hydrogen bonds and ionic interactions formed between protein and cyclic AMPSerineArginineprotien backboneGlutamic AcidThreonineSerine

Nucleic Acid-Small Molecule: CisplatinCis-platin binds covalently to Guanine basesBends DNA by 35-40o

Bent DNA mimics binding site for High Mobility Group (HMG) proteins100x greater affinity

HMG proteins increase cisplatin cytotoxicity by binding onto DNA adducts and obstructing DNA repair. +http://pubs.acs.org/cen/coverstory/83/8325/8325cisplatin.html

Modes of BindingGreen: surface bindingYellow: intercalationRed: groove binding

Intercalators push apart base pairsIncrease helix lengthInduce structural changes

Why is intercalation important?Pharmaceutical applicationsCancer chemotherapyDaunomycin and adriamycinAntibiotics

Causes buckle and prevents replication by interfering with DNA-protein interaction

http://www.jonathanpmiller.com/intercalation/

Known IntercalatorsHave planar aromatic cyclic structures that can stackEthidium BromideDipyridophenazine (dppz)

DNA-Binding Experiments: Overview

Molecular Light SwitchViscometry: argued best method for demonstrating intercalationThermal DenaturationPhotocleavage

Do our Ru compounds intercalate DNA or bind in some other way?

Molecular Light Switch RuDPPZ RuDPPZ+DNA RuDAP+DNA RuDAP

Inherent fluorescence of compound quenched in aqueous bufferWhen bound to DNA, helix shields from solvent quenchingDemonstrate by obtaining emission spectra with fluorimeter instrument

ViscometryDNA helix can be distorted and lengthened upon intercalation

Lengthening increases viscosity of DNA solution, which can be monitored with a viscometer

h=(t-t0)/t0h = viscosityt = flow time (seconds)t0 = flow time of buffer alone (seconds)h0 = viscosity of DNA alone

Thermal DenaturationAs double stranded DNA is heated, it is denatured to single strandedMelting temperature defined as the inflection point

Intercalated molecules stabilize the helix, requiring a larger temperature to denatureRuDppz can shift melting temperature from 64.5 to 80 oC

Measured by recording absorbance at 260 nm

Photocleavage: what is it?An intercalated Ru compound excited by UV light triggersa reaction that can cut the phosphate backbone of DNA

Monitor using Electrophoresis

Why Study DNA Cleavage?Activated photochemicallyRxn not initiated without irradiationTherapeutic agentsActivated in vivo by laserSelective excitation of photocleaverSensitive to light longer than 300nmNucleic acids and proteins transparentLimited side reactions

DNA CleavageFluorescenceMolecular Light SwitchViscosityThermalDenaturation

DNABinding

Ru-DNA Project Schedule

Week 1 April 2 Buffer, Solution PrepWeek 2 April 9First assigned techniqueWeek 3 April 16First assigned technique (repeat)Week 4 April 23Groups rotate: second assigned techniqueWeek 5 April 30 Class presentation and discussion of results

DNA CleavageWeeks 2 & 3: Lucy & Kaylee,Yuan & AmandaWeek 4: Anna & June,Steph & Kathy,Liz & Allison

FluorescenceMolecular Light SwitchWeeks 2 & 3: Liz & AllisonWeek 4:TBA

ViscosityWeeks 2 & 3: Steph & KathyWeek 4:Yuan & Amanda

ThermalDenaturationWeeks 2 & 3: Anna & JuneWeek 4:Lucy & Kaylee,

Week 1 (tomorrow): Buffer, Solution Prep

Goals:Make appropriate buffers for your experimentMake Calf Thymus DNA solutionMake Ru solutions

Week 1 (tomorrow): sequenceMake appropriate buffer A or BIf needed, dilute provided buffer to assigned concentrationAdd mass of NaCl to assigned concentrationMake Calf Thymus DNA solutionMass out solid DNA, add to buffer and sonicate to dissolve (will take ~1.5-2 hours)Practice Pipettor Technique4. Analyze [DNA] using Abs. at 260nm and extinction coefficient to determine DNA soln concentration5. Make Ru solutionsMass out solid Ru compounds and add buffer to make assigned concentration solutions

*While our work was in progress, Gao and coworkers reported the synthesis and some DNA binding studies of the Ruthenium bisbipyridine alloxazine and dimethyl alloxazine compounds.