A sequence-specific threading tetra-intercalator with an

extremely slow dissociation rate constant

Article by: Garn F. Holman, Maha Zewail-Foote, Amy Rhoden Smith, Kenneth A. Johnson, and Brent L. Iverson

Presentation by: Maggie Palmer and Joy Uyanwune

“If you think of DNA as a spiral staircase imagine sliding something between the steps. That’s what our molecule does. It can be visualized as binding to DNA in the same way a snake might climb a ladder.” ~Brent L. Iverson

Photo/quote source: http://web5.cns.utexas.edu/news/2012/02/dna-wranglers/

What is a threading Intercalator?

• A molecule that is capable of binding and inserting itself into a DNA sequence.

• Sequence specific binding – 5’ – GATAAGTACTTATC – 3’

http://sketchfu.com/drawing/1919089-snake-and-ladder-

Why is this important?

• Molecules that can bind to DNA can interfere and possibly control transcription, repair and replication.

• Possible cure for chronic diseases

Photo source: http://www.nature.com/nchem/journal/v3/n11/full/nchem.1173.html

Tetra-intercalator 3

• Designed with a minor-major-minor groove binding linkers

• Tetra-intercalator 3 was designed using studies done previously on 1 and 2.

• -βAla3-Lys- is the minor groove binding linker taken from bisintercalator 2.

• Adipic acid with 2 L-Lysine residues are part of the major groove binding linker. – Causes C2 symmetry that simplifies the NMF

• 14 Basepair binding site. • Pieced together by connecting together NDI units

How to make a tetra-intercalator

• Naphthalenetetracarboxylic diimide (NDI) molecules are chained together with resin beads.

• Reactions are ran directly on the resin beads until the molecule is large enough to cleave.

• “The larger molecule is composed of little pieces that bind to short segments of DNA, kind of life the way Legos fit together” ~Amy Rhoden-Smith

http://io9.com/5885072/dna+tangling-molecule-could-revolutionize-treatments-for-cancer-and-hiv

Napthalenetetracarboxylic diimide

• NDI units can be pieced together in different ways

• The different arrangements then must be scanned in order to find the sequences the new arrangements will select for

http://www.chemspider.com/Chemical-Structure.24771033.html

Picture source: Lee et.al

Slowed Dissociation Rate

• 3 will have a slow dissociation from its preferred DNA binding site.

• In order for 3 to fully dissociate from DNA it the individual units will have to dissociate in an ordered fashion before rebinding occurs.

Photo source: http://mysam.net/wiki/index.php/%ED%8A%B9%EC%88%98%EA%B8%B0%EB%8A%A5:NatureNews?reply=4&page=1

EXPERIMENT SECTION

Threading bisintercalators 1&2

• figure 1a picture here • figure 1b picture here.

Tetra-Intercalator 3

• Figure 1c

Threading Polyintercalator of 3

• Show extremely slow dissociation from its preferred DNA sequence.

• Association is suspected to be rapid result of electrostatic interactions.

DNase/Footprinting

• Figure 2

Dissociation Kinetics

Nonspecific Dissociation Kinetic

• Stopped Flow Method

Association rate measurements

• 1H NMR spectroscopy,

• Gel mobility shift assay and

• stopped-flow ultraviolet–visible spectroscopy.

Stopped Flow Ultraviolet-visible Spectroscopy

• Figure 5

Conclusion

• Tetra-intercalator 3 has a slow dissociation rate.

• The half life of tetra-intercalator 3 was found to be16 days.

• Tetra-intercalator 3 also shows a rapid association rate to its target specific sequence driven by electrostatic attraction.

• Suspected to have the same threading topology on long pieces of DNA.

Work Cited

• Holman, Garen G., Zewail-Foote, Maha; Smith, Amy Rhoden; Johnson, Kenneth A.; & Iverson, Brent L. 2001. A Sequence-specific threading teta-intercalator with an extremely slow dissociation rate constant. Nature Chemistry. 3; 875-881

• Lee, J.; Guelev, V.; Sorey, S.;Hoffman, D.W. &Iverson, B.L.2004 NMR Structural analysis of a modular threading tetraintercalator bound to DNA. J. Am. Chem. Soc, 126; 14036-14042

Work Cited Cont.

• Oppenheimer, Daniel. 2012 DNA- Targeting Molecule has Potential to Help Treat Genetic Disease and HIV. Online at. http://web5.cns.utexas.edu/news/2012/02/dna-wranglers