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

5. Types of Interactions Protein - Protein Protein – Nucleic Acid
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
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. +

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

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

14. 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.

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

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 Therapeutic agents
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. Molecular Light Switch
Thermal
Denaturation
DNA
Cleavage
Fluorescence
Molecular Light Switch
Viscosity

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

27. Week 1 (tomorrow): Buffer, Solution Prep Goals:
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