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Developing a DNA pull-down assay to identify Pt(II) bound DNA Sequences
1
Guevara,
2
Sutton
2
DeRose
Maria V.
Emily
and Victoria J.
1Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL 32611-6550
2Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, 97403-1253
1. Abstract
3. Azide-Modified Complexes and Click Chemistry
Platinum (II)-based drugs such as Cisplatin are widely used in chemotherapy treatments but not well
understood. Pt(II) drugs are known to bind DNA, however, specific Pt(II) targets in vivo have not been
studied. The DeRose lab uses click chemistry to study the behavior of platinum(II) compounds in
cells. Reactive azide-modified platinum(II) compounds have been synthesized, allowing us to attach
biotin for affinity pull-down. Our research attempts to demonstrate the effectiveness of a DNA pulldown assay in order to locate platinum(II) bound DNA sequences in cells. Results show that the assay
has been able to successfully isolate a platinated DNA hairpin from a mixture with an unplatinated
single stranded oligonucleotide. Now a new question has been formulated: Will the pull-down assay
work with more complex DNA structures? We expect that we will not see interference in the pulldown binding process even when in a background of larger fragments of double stranded DNA. The
long-term goal of our project is to apply the pull-down assay to isolate DNA from cells treated with
the platinum(II) reagents. Understanding the modes of action platinum(II)-based drugs with different
biomolecules is crucial for the development of selective cancer cell oriented drugs in order to deliver
more efficient and safer anticancer medications.
The DeRose Lab has focused on developing methods to identify potential Pt(II) targets in the cell,
which can provide insight into the mechanisms of action of platinum(II)-based drugs.
5. Results
In order to answer these questions, the DeRose Lab has synthesized platinum(II)-based compounds
with clickable reactive handles. These reactive azide-modified molecules allow us to apply click
chemistry in order to attach structures such as biotin or fluorophores for post-treatment analysis
such as pull-down assays.
AZIDE-MODIFIED PLATINUM(II) COMPLEXES
Cis-[Pt(2-azido1,3-propanediamine)Cl2]
Picazoplatin
2. Background and Significance
Regina’s Platinum
1,2-platin
Input
Mixture of clicked
hairpin and DNA
fragments
Elution
Supernatant
Clicked hairpin
DNA fragments
(700bp-25bp)
Traces of DNA
fragments
Wash #1,2,3
CLICK CHEMISTRY
Platinum(II)-based chemotherapy drugs are used for cancer
treatments such as ovarian, testicular, cervical, bladder and non-smallcell lung cancer.1-2 Despite the extensive usage and high rate of
success of platinum(II)-based drugs, platinum’s interaction with
specific biomolecules in the cell remains unclear.
Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction is an example of a click reaction.
The azide and alkyne are unobtrusive groups that do not interfere with the function of their
respective groups.4 This bioorthogonal reaction allows us to explore various biological applications,
including the delivery of safer drugs.
Platinum(II)-based drugs have been previously known for their ability to bind DNA. One
proposed mechanism of action consists of platinum binding DNA, inducing a structural change
that inhibits the DNA replication and causes apoptosis. However, platinum’s preference to bind
specific DNA regions has not been studied.
Platinum
Alkyne
Triazoles
Azide
Input
Targets2
4. Pull-Down Assay
-DNA
-RNA
-Proteins
-Mitochondria
-Ribosomes
Platinum binds to 2 adjacent
guanines of genomic DNA.
1
CISPLATIN
Cisplatin and its derivatives such as carboplatin and oxaliplatin are Pt(II) therapeutics used in
treatments of 50%-70% of cancer patients.3
6. Conclusion and Future Approach
Platination of DNA hairpin with azide
modified Pt(II) complex
G
G
Regina’s Platinum
5’-GG-3’ (Hairpin)
2
• Our pull-down assay was able to isolate platinum bound DNA hairpin from a mixture of large
fragments of double stranded DNA. No traces of double stranded DNA fragments are observed in the
elution solution.
Click Reaction
• However, it is noticed that the clicked hairpin is running higher than the samples used as standard.
• Our future directions are focus on improving the pull-down assay as well as to be able to replicate our
assay with S. cerevisiae for future identification of platinated DNA sequences.
Cu (I)
(a)
Carboplatin
(1989)
Cisplatin
(1978)
B
Oxaliplatin
(1996)
The efficacy of Pt(II)-based drugs is limited due to drug resistance, nephrotoxicity, neurotoxicity,
cardiotoxicity,
ototoxicity, emetogenesis, among others.1
(c)
(b)
SIDE EFFECTS
Nephrotoxicity |
Neurotoxicity
| Cardiotoxicity
|
Wash #1,2,3
In order to accomplish our goal, we treat synthetic DNA hairpin with an azide-modified platinum(II)
complex, to later selectively pull-down platinum bound DNA hairpin from a background of large
fragments of double stranded DNA. Unbound supernatant is washed off, and the bound DNA is eluted
off the beads.
Common coordination
of
cisplatin on DNA:
• 1, 2-intra-strand crosslink
• Protein-DNA crosslink
In the case of Cisplatin, around 10% of Pt
agglomerates within genomic DNA.2
Elution
Supernatant
Platinum’s interaction with specific regions of DNA in vivo is still not well understood. Therefore, our
research attempts to demonstrate the effectiveness of a DNA pull-down assay to locate platinum(II)
bound DNA sequences in cells. Results obtain from this assay will provide information of platinum
behavior with DNA in an in vitro environment.
Ototoxicity
Mixture of clicked
hairpin and DNA
fragments
Clicked hairpin
DNA fragments
(1500 bp-100bp)
Traces of DNA
fragments
+
G
G
“Click”
G
G
7. Acknowledgements
B = Biotin
3
Platinated Hairpin
Selective pull-down of
platinum bound DNA with
streptavidin beads
(1) NSF REU Site Program in Molecular Biosciences at the University of Oregon: NSF DBI/BIO 1460735)
(2) DeRose Lab
(3) SPUR Program
Clicked Hairpin
Fragmented
DNA
G
G
Magnetic
Streptavidin
Beads
8. Sources
(1) Wang, D. and Lippard, S. (2005) Cellular Processing of Platinum Antincancer drugs. Nature Reviews Drug Discovery 4., 307-320.
(2) Wirth, R., Whit,e J., Moghaddam, A., Ginzburg, A., Zakharov, L., Haley, M., and DeRose, V. (2015). Azide vs Alkyne Functionalization in Pt(II)
Complexes for Post-treatment Click Modification: Solid-State Structure, Fluorescent Labeling, and Cellular Fate. Journal of the American
Chemical Society 137 (48).
(3) Dyson, P.J., and Sava, G. (2006) Metal-based antitumor drugs in the post genomic era. Dalton Trans., 1929-1933.
(4) Kolb, H.C.; Sharpless, B.K. (2003). "The growing impact of click chemistry on drug discovery". Drug Discov Today 8 (24): 1128–1137