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/dn
a-wranglers