1. Developing a DNA pull-down assay to identify Pt(II) bound DNA Sequences Maria V. Guevara, 1 Emily Sutton 2 and Victoria J. DeRose 2 1 Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL 32611-6550 2 Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, 97403-1253 (a) (b) (c) 5. Results 6. Conclusion and Future Approach 7. Acknowledgements 1. Abstract 2. Background and Significance 3. Azide-Modified Complexes and Click Chemistry 8. Sources 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 pull- down 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 pull- down 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. Platinum(II)-based chemotherapy drugs are used for cancer treatments such as ovarian, testicular, cervical, bladder and non-small- cell 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. Cisplatin and its derivatives such as carboplatin and oxaliplatin are Pt(II) therapeutics used in treatments of 50%-70% of cancer patients. 3 Cisplatin (1978) Carboplatin (1989) Oxaliplatin (1996) CISPLATIN SIDE EFFECTS Nephrotoxicity | Neurotoxicity | Cardiotoxicity | Ototoxicity The efficacy of Pt(II)-based drugs is limited due to drug resistance, nephrotoxicity, neurotoxicity, cardiotoxicity, ototoxicity, emetogenesis, among others. 1 -DNA -RNA -Proteins -Mitochondria -Ribosomes Platinum Targets 2 In the case of Cisplatin, around 10% of Pt agglomerates within genomic DNA. 2 Platinum binds to 2 adjacent guanines of genomic DNA. Common coordination of cisplatin on DNA: 1, 2-intra-strand crosslink Protein-DNA crosslink 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. 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. Picazoplatin Cis-[Pt(2-azido- 1,3-propanediamine)Cl 2 ] 1,2-platin Regina’s Platinum AZIDE-MODIFIED PLATINUM(II) COMPLEXES CLICK CHEMISTRY 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. Azide Alkyne Triazoles 4. Pull-Down Assay 1 Platination of DNA hairpin with azide modified Pt(II) complex Click Reaction 2 Fragmented DNA Magnetic Streptavidin Beads Selective pull-down of platinum bound DNA with streptavidin beads 3 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. 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. (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 (1)NSF REU Site Program in Molecular Biosciences at the University of Oregon: NSF DBI/BIO 1460735) (2)DeRose Lab (3)SPUR Program B B = Biotin B B Regina’s Platinum 5’-GG-3’ (Hairpin) G G B B + “Click” Cu (I) Platinated Hairpin Clicked Hairpin G G G G G G 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. 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. 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. InputMixture of clicked hairpin and DNA fragments ElutionClicked hairpin SupernatantDNA fragments (1500 bp-100bp) Wash #1,2,3Traces of DNA fragments Input Elution Supernatant Wash #1 Wash #2Wash #3 Fragmented DNA Clicked Hairpin Input Elution Supernatant Wash #1 Wash #2 Wash #3 Fragmented DNA Clicked Hairpin InputMixture of clicked hairpin and DNA fragments ElutionClicked hairpin SupernatantDNA fragments (700bp-25bp) Wash #1,2,3Traces of DNA fragments G G G G