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Molecular Modeling of Nanoscale Bioassemblies for Clinical Laboratory Diagnostics

Molecular Modeling of Nanoscale Bioassemblies for Clinical Laboratory Diagnostics. City of Hope National Medical Center Scott F. Roalofs with Dr. Steven S. Smith. Overview. Purpose Introduction to Bionanotechnology FRET Modification of Bioassemblies Molecular Models Conclusions

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Molecular Modeling of Nanoscale Bioassemblies for Clinical Laboratory Diagnostics

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  1. Molecular Modeling of Nanoscale Bioassemblies for Clinical Laboratory Diagnostics City of Hope National Medical Center Scott F. Roalofs with Dr. Steven S. Smith

  2. Overview • Purpose • Introduction to Bionanotechnology • FRET • Modification of Bioassemblies • Molecular Models • Conclusions • Acknowledgements

  3. Purpose • To design and develop a tool that would allow rapid diagnose of tumor cells from a biopsy • My specific part: Modeling of bioassemblies using Insight II

  4. Fluorescent Molecules Ni LinkerStructure Y-Junction Tumor Cell

  5. Bionanotechnology • Uses molecular biology, chemistry and physics to link molecules into complex assemblies • Must be under 100nm in its largest dimension • Artificial and require some assembly outside of a living system

  6. FRET • Förster resonance energy transfer • Fluorescent donor is excited at its specific excitation wavelength • Dipole-dipole interactions: • Excited state is non-radioactively transferred to an acceptor • Acceptor returns to the electronic ground state

  7. Changing Target Specificity • DNA methyltransferase HhaI has the ability to be targeted to specific recognition sites in the Y-Junction • Covalently trapped on duplex DNA using 5’-fluorocytosine • Bioassemblies can be modified with various targeting and detection chemistries

  8. Y-Junction Structure

  9. Fluorine Fluorine Fluorine Fluorine Fluorine

  10. Sulfur Linker Structure 3’ oligomer IDA 5’ oligomer

  11. Sulfur Linker 17,234 kcal/mol

  12. Previous work • Horsey et al., Chem Comm, 2002, 1950-51. • Compared chelated vs. non-chelated Sulfur Linker complex • 15°C difference in Tm: chelated vs. non-chelated Sulfur Linker complex • Smith lab has been unable to reproduce these results

  13. 6 Linker Structure 3’ ologomer IDA 5’ oligomer

  14. 6 Linker 17,249 kcal/mol

  15. Linker Modifications • Linker structures were modified • Previous models identified possible unwanted interactions with Ni • Synthesis protocol was modified • Removed (CH2)3 OH • Replaced with a Cytosine base

  16. Modified Linker

  17. Conclusions • Models are visually appealing for use in presenting one’s ideas • Molecular modeling provides 3D visual renderings of hand drawn structures • Models may identify questionable structure flaws that may not have be noticeable on a hand drawn structure

  18. Acknowledgements • Dr. Steven S. Smith • Jarrod Clark • Katarzyna Lamparska-Kupsik • SoCalBSI • NIH

  19. Questions ?

  20. FRET www.olympusfluoview.com/applications/fretinfo.html

  21. IDA=iminodiacetic acid

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