Project 3: Ru – DNA Binding

1. Project 3:Ru – DNA Binding Today’s topics: 1. Macromolecules 2. Macromolecular Interactions 3. Ru-DNA Project 4. Team Assignments 5. Experiments

2. Macromolecules: DNA and Proteins Pyrimidines : C and T Purines: A and G http://www.lclark.edu/~bkbaxter/200lecture/lecture_images/1_22_peptidebond.jpg

3. www.wikipedia.org www.solarnavigator.net

5. Types of Interactions • Protein - Protein • Subunits make up functional protein • Protein – Nucleic Acid • Replication, DNA repair, Transcription, Translation • Protein – Small molecule • ATP-dependent enzymes • Nucleic Acid – Small molecule • Pharmaceuticals

6. Protein-Protein interactions Molecular Biology of The Cell, 4th Edition (2002)

7. Protein-Nucleic Acid: Replication Molecular Biology of The Cell, 4th Edition (2002)

8. DNA Polymerase Molecular Biology of The Cell, 4th Edition (2002)

9. Protein-Small Molecule Serine Arginine protien backbone Glutamic Acid Threonine Serine Hydrogen bonds and ionic interactions formed between protein and cyclic AMP Molecular Biology of The Cell, 4th Edition (2002)

10. Nucleic Acid-Small Molecule: Cisplatin • Cis-platin binds covalently to Guanine bases • Bends DNA by 35-40o • Bent DNA mimics binding site for High Mobility Group (HMG) proteins • 100x 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

11. Modes of Binding • Green: surface binding • Yellow: intercalation • Red: groove binding • Intercalators push apart base pairs • Increase helix length • Induce structural changes

12. Why is intercalation important? Pharmaceutical applications • Cancer chemotherapy • Daunomycin and adriamycin • Antibiotics • Causes “buckle” and prevents replication by interfering with DNA-protein interaction http://www.jonathanpmiller.com/intercalation/

13. Known Intercalators Have planar aromatic cyclic structures that can “stack” Ethidium Bromide Dipyridophenazine (dppz)

15. DNA-Binding Experiments: Overview • Molecular “Light Switch” • Viscometry: argued best method for demonstrating intercalation • Thermal Denaturation • Photocleavage Do our Ru compounds intercalate DNA or bind in some other way?

16. Molecular Light Switch RuDPPZ RuDPPZ+DNA RuDAP+DNA RuDAP • Inherent fluorescence of compound quenched in aqueous buffer • When bound to DNA, helix shields from solvent quenching • Demonstrate by obtaining emission spectra with fluorimeter instrument

17. Viscometry • DNA helix can be distorted and lengthened upon intercalation • Lengthening increases viscosity of DNA solution, which can be monitored with a viscometer h=(t-t0)/t0 h = viscosity t = flow time (seconds) t0 = flow time of buffer alone (seconds) h0 = viscosity of DNA alone

18. Thermal Denaturation • As double stranded DNA is heated, it is denatured to single stranded • Melting temperature defined as the inflection point • Intercalated molecules stabilize the helix, requiring a larger temperature to denature • RuDppz can shift melting temperature from 64.5 to 80 oC • Measured by recording absorbance at 260 nm

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

20. Monitor using Electrophoresis

21. Why Study DNA Cleavage? • Activated photochemically • Rxn not initiated without irradiation • Therapeutic agents • Activated in vivo by laser • Selective excitation of photocleaver • Sensitive to light longer than 300nm • Nucleic acids and proteins transparent • Limited side reactions

22. Thermal Denaturation DNA Cleavage Fluorescence Molecular Light Switch Viscosity

23. Thermal Denaturation DNA Cleavage Fluorescence Molecular Light Switch Viscosity DNA Binding

24. Ru-DNA Project Schedule Week 1 – April 2 Buffer, Solution Prep Week 2 – April 9 First assigned technique Week 3 – April 16 First assigned technique (repeat) Week 4 – April 23 Groups rotate: second assigned technique Week 5 – April 30 Class presentation and discussion of results

25. Thermal Denaturation Weeks 2 & 3: Anna & June Week 4:Lucy & Kaylee, DNA Cleavage Weeks 2 & 3: Lucy & Kaylee, Yuan & Amanda Week 4: Anna & June, Steph & Kathy, Liz & Allison Fluorescence Molecular Light Switch Weeks 2 & 3: Liz & Allison Week 4:TBA Viscosity Weeks 2 & 3: Steph & Kathy Week 4:Yuan & Amanda

26. Thermal Denaturation Anna & June, Lucy & Kaylee DNA Cleavage Everyone! Results Of DNA Binding Fluorescence Molecular Light Switch Liz & Allison Viscosity Steph & Kathy Yuan & Amanda

27. Week 1 (tomorrow): Buffer, Solution PrepGoals: • Make appropriate buffers for your experiment • Make Calf Thymus DNA solution • Make Ru solutions

28. Week 1 (tomorrow): sequence • Make appropriate buffer A or B • If needed, dilute provided buffer to assigned concentration • Add mass of NaCl to assigned concentration • Make Calf Thymus DNA solution • Mass out solid DNA, add to buffer and sonicate to dissolve (will take ~1.5-2 hours) • Practice Pipettor Technique 4. Analyze [DNA]using Abs. at 260nm and extinction coefficient to determine DNA soln concentration 5. Make Ru solutions • Mass out solid Ru compounds and add buffer to make assigned concentration solutions