Tapping-mode AFM analysis of λ-DNA complexed with activated and non-activated cisplatin. Jaya P. Shrestha, Eric J. Voss, Department of Chemistry, Southern Illinois University Edwardsville. Edwardsville, Illinois, 62026. Project Overview Tapping-mode atomic force microscopy (AFM) is a powerful tool for the analysis of biomolecules on surfaces. We are interested in observing changes in the confirmation of DNA upon coordination with metal complexes. Surfaces suitable for the immobilization of DNA were prepared by vapor phase deposition of 3-aminopropyltriethoxy silane (APTES) on freshly cleaved mica in the presence of N,N-diisopropylethylamine (DIPEA). AFM images in air of λ-DNA alone were compared to AFM images of λ-DNA complexed with activated and non-activated cisplatin. Once DNA complexes with cisplatin, it develops microloops. The extent of looping and formation of globules depends on the concentration DNA, the concentration of cisplatin, whether the cisplatin has been activated by reaction with silver nitrate, and the incubation time. Figure 9: 2.5 µm x 2.5 µm AFM image of a λ DNA complexed with cisplatin for 20 minutes. Figure 10: 1 µm x 1 µm AFM image of a λ DNA complexed with cisplatin for 1 hour. Figure 4: Stretched λ DNA Figure 3: λ DNA Mesh Formation. Non-activated Cisplatin Complex With DNA 5 µL of λ-DNA was incubated with 45 µL of 77µM of cisplatin stock solution. 1µL of incubated solution was taken out in different time interval ( 20 min, 1 hr, 2 hr, 24 hr.) and diluted with 49 µL of distilled water. The diluted suspension was deposited on mica surface for 2 minutes. Conclusion and Discussion APTES modified mica surface was successfully prepared following literature. Several trial experiments were carried out to optimize conditions for DNA deposition and for tapping mode AFM in the air. It was observed that both activated and non-activated cisplatin were able to bind with DNA and form a condensed globule. The activated cisplatin were found to be more effective than the non- activated cisplatin. The activated cisplatin only took 1 hour for the complete globulization while condensation only started after 1 hour for the non-activated cisplatin. The DNA condensation with non-activated cisplatin (Fig.1) showed variations in DNA structure which consists of some very condensed form, some linear DNA and some fragmented DNA. It was thought that non-activated cisplatin binds with DNA after loosing chlorine atom which is an active form. However, not all cisplatin are activated at the same time which causes variation in the DNA condensation. A complete golbulization was observe for both activated and non-activated cisplatin at 24 hours.. APTES Mica Surface Modification Vapor phase method was chosen. A desiccator was cleaned with methanol. Two micro tubes were taken; cut the caps and were labeled for APTES and DIPEA. These caps were attached on the plastic disc inside the desiccator. The desiccator was vacuum for 20 s and filled with argon. A freshly cleaved mica was attached in the desiccator lid. Then 20 µL of APTES and 60 µL DIPEA were added in the respective caps. The desiccator was once again vacuumed for 20 s and purged with argon. The modification process was allowed for 1 hour; the Cap containing APTES was removed carefully and left it for overnight. Figure 6: 2 µm x 2 µm AFM image of a λ DNA complexed with cisplatin for 1 hour. Figure 5: 2 µm x 2 µm AFM image of a λ DNA complexed with cisplatin for 20 minutes. Figure 1: Mica surface modification mechanism [1]. Figure 2: Vapor phase reaction set up References Liu, Z.; LI, Z.; Zhou, H.; Wei, G.; Song, Y.; Wang, L. J.Microscopy 2005, 218, 233–239. Hou, X.M; Zhang, X.H.; Wei, K.J.; Ji, C.; Dou, S.X.; Wang, W.C.; Li, M.; Wang, P.Y. Nucleic Acid Research 2009, 37, 1400-1410. Xaio, Zhanwen.; Xu, M.; Sagisaka, K.; Fujita, D. Thin Solid Films 2003, 438-439, 114-117. Liu, Z.; Tana, S.; Zua, Y.; Fua, Y.; Menga, R.; Xinga, Z. Micron 2010, 41, 833-839. Onoa, G.B.; Moreno, V. I.J. of Pharmaceutics 2002, 245, 55-65. DNA Sample Preparation The frozen λ-DNA was defreezed at 40 °C in a water bath for 5 minutes. 2.5 µL λ-DNA was taken into a microtube and 47.5 µL of sterilized distilled water was added. The resulting suspension was mixed very gently. The suspension was deposited on APTES modified mica surface for 2 minutes. Figure 7 : 1 µm x 1 µm AFM image of a λ DNA complexed with cisplatin for 2 hours. Figure 8: : 1 µm x 1 µm AFM image of a λ DNA complexed with cisplatin for 24 hours. Acknowledgements Dr. Erick J Voss Dr. Chin-Chuan Wei Dr. The Graduate School, Southern Illinois University Edwardsville Department of Chemistry, Southern Illinois University Edwardsville National Science Foundation DUE-0633186 DNA Mesh & Stretched DNA APTES modified mica surface were placed at an angle of ~90°. 50 µL of λ-DNA solution was added on the upper part of the inclined mica surface and allowed to flow down on the surface. Micropipette was used to reabsorb the droplet once it reached the bottom of the inclined mica surface. and added again on the upper part of the surface. This process was repeated at different time intervals for 2 minutes. Activated Cisplatin The cisplatin solution was activated with silver nitrate solution for over night. The silver chloride precipitation was separated by centrifuge (2×10 minutes) and finally filtered through milipore filter. Then followed the same process as non activated cisplatin for incubation and deposition.