project 3: ru – dna binding today’s topics: 1. macromolecules 2. macromolecular interactions 3....

Project 3:Ru – DNA Binding

Today’s topics:1. Macromolecules2. Macromolecular Interactions3. Ru-DNA Project4. Team Assignments5. Experiments

Macromolecules: DNA and Proteins

http://www.lclark.edu/~bkbaxter/200lecture/lecture_images/1_22_peptidebond.jpg

Pyrimidines : C and TPurines: A and G

www.solarnavigator.net www.wikipedia.org

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

Protein-Protein interactions

Molecular Biology of The Cell, 4th Edition (2002)

Protein-Nucleic Acid: Replication

Molecular Biology of The Cell, 4th Edition (2002)

DNA Polymerase

Molecular Biology of The Cell, 4th Edition (2002)

Protein-Small Molecule

Molecular Biology of The Cell, 4th Edition (2002)

Hydrogen bonds and ionic interactions formed between protein and cyclic AMP

SerineArginineprotien backboneGlutamic AcidThreonineSerine

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

Modes of Binding

• Green: surface binding

• Yellow: intercalation• Red: groove binding

• Intercalators push apart base pairs– Increase helix length

– Induce structural changes

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/

Known Intercalators

Have planar aromatic cyclic structures that can “stack”

Ethidium Bromide Dipyridophenazine (dppz)

N

Ru

N

NN

N

N

N

N

N

Ru

N

NN

N

N

N

N

N

Ru

N

NN

N

N

N

NN

Ru

N

NN

N

N

N

N

N

Ru

N

NN

N

N

N

N

[Ru(bpy)2(L-pterin)]2+ [Ru(bpy)2(L-diamino)]2+

[Ru(bpy)2(L-amino)]2+

[Ru(bpy)2(L-allox)]2+ [Ru(bpy)2(L-Me2allox)]2+

NH

NH

O

O

N

N

NH2

N

NH

O

NH2

N

N

O

O

CH3

CH3

N

N

NH2

NH2

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?

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

Viscometry• DNA helix can be distorted

and lengthened upon intercalation

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

=(t-t0)/t0 = viscosityt = flow time (seconds)t0 = flow time of buffer alone (seconds)0 = viscosity of DNA alone

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

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 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

DNA Cleavage

DNA Cleavage

FluorescenceMolecular Light SwitchFluorescenceMolecular Light Switch

ViscosityViscosity

ThermalDenaturation

ThermalDenaturation

DNA Cleavage

DNA Cleavage

FluorescenceMolecular Light SwitchFluorescenceMolecular Light Switch

ViscosityViscosity

ThermalDenaturation

ThermalDenaturation

DNABinding

Ru-DNA Project Schedule

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

DNA Cleavage

Weeks 2 & 3: Lucy & Kaylee,Yuan & Amanda

Week 4: Anna & June,Steph & Kathy,

Liz & Allison

DNA Cleavage

Weeks 2 & 3: Lucy & Kaylee,Yuan & Amanda

Week 4: Anna & June,Steph & Kathy,

Liz & Allison

FluorescenceMolecular Light SwitchWeeks 2 & 3: Liz & Allison

Week 4:TBA

FluorescenceMolecular Light SwitchWeeks 2 & 3: Liz & Allison

Week 4:TBA

ViscosityWeeks 2 & 3: Steph & Kathy

Week 4:Yuan & Amanda

ViscosityWeeks 2 & 3: Steph & Kathy

Week 4:Yuan & Amanda

ThermalDenaturationWeeks 2 & 3: Anna & June

Week 4:Lucy & Kaylee,

ThermalDenaturationWeeks 2 & 3: Anna & June

Week 4:Lucy & Kaylee,

DNA CleavageEveryone!

DNA CleavageEveryone!

FluorescenceMolecular Light Switch

Liz & Allison

FluorescenceMolecular Light Switch

Liz & Allison

ViscositySteph & Kathy

Yuan & Amanda

ViscositySteph & Kathy

Yuan & Amanda

ThermalDenaturation

Anna & June, Lucy & Kaylee

ThermalDenaturation

Anna & June, Lucy & Kaylee

ResultsOf DNABinding

Week 1 (tomorrow): Buffer, Solution Prep

Goals:

1. Make appropriate buffers for your experiment2. Make Calf Thymus DNA solution3. Make Ru solutions

Week 1 (tomorrow): sequence

1. Make appropriate buffer A or B– If needed, dilute provided buffer to assigned

concentration– Add mass of NaCl to assigned concentration

2. Make Calf Thymus DNA solution– Mass out solid DNA, add to buffer and sonicate to

dissolve (will take ~1.5-2 hours)3. Practice Pipettor Technique4. Analyze [DNA] using Abs. at 260nm and extinction

coefficient to determine DNA soln concentration5. Make Ru solutions

– Mass out solid Ru compounds and add buffer to make assigned concentration solutions