1. Spectroscopic Analysis: Deoxyribonucleosides and DNA Maria I. Lambousis with Dr. Milan Balaz Department of Chemistry University of Wyoming UW Honors Program April 24, 2010

2. Abstract Chirality - primary role in nature - immense importance in chemical processes of all living organisms, since most biomolecules (DNA, proteins, sugars) are chiral DNA used as a study molecule - arguably the most important biomolecule Highly desirable to gain a good understanding of DNA structure and properties DNA built of chiral nucleosides - stereochemistry of these DNA building blocks influences the DNA secondary structure

3. Objectives Explore the chirality of DNA ▫ Central  Axial Understand the various DNA conformations Examine DNA binding and recognition

4. Chirality Molecule that cannot be superimposed on its mirror image ▫ Ability to rotate polarized light  Measured by circular dichroism spectroscopy Enantiomers ▫ A molecule and its non-superimposable mirror image ▫ Differ in interaction with polarized light while sharing chemical and physical properties

5. DNA Deoxyribonucleosides ▫ Nitrogenous base + deoxyribose sugar B-DNA ▫ Most common form ▫ Right-handed helical twist Z-DNA ▫ Left-handed helical twist

6. B  Z DNA Transition The various DNA forms are interchangeable ▫ environmental conditions (high salt conc.), special sequences (alternating purines and pyrimidines), and addition of substituents Various mechanisms have been proposed to explain how the transition occurs Too many contradictions exist to be definitive

7. Porphyrins Aromatic organic compounds Heme = porphyrin + iron Chlorophyll = = porphyrin + magnesium ZnT4 - zinc derivative of meso-tetrakis (4-N-methylpyridyl)porphyrin

8. Nucleoside Enantiomers UV-vis and CD D and L enantiomers Thymidine Cytidine Guanosine Adenosine

9. Thymidine D enantiomer

10. UV-vis Absorption vs. Concentration

14. 6-mer 5 ′-CGCGCG ▫ Short sequence ▫ 50 mM buffer Melting Curve ▫ Stability B- and Z-DNA ▫ NaCl

15. 6-mer

16. 6-mer B to Z Transition Using NaCl and NiCl 2

17. 24-mer Part 1 24-mer sequence ▫ 5 ′-(dCdG) 12 ▫ 50 mM buffer Inducing Z-DNA ▫ NaCl

18. 24-mer Part 1

19. B-Z Comparison of 6-mer and 24-mer

20. 24-mer Part 2 Try 1 mM buffer and EtOH 80 % EtOH Stability overnight

21. 24-mer Part 2

22. 24-mer Part 2 - Stability

23. ZnT4 alone...

24. ZnT4 Titration - 1 Z-DNA Signal between 400nm and 600 nm ? ▫ Yes: DNA imprint chirality ▫ No: unorganized porphyrin complex

25. Z-DNA with Porphyrin

26. ZnT4 Titration - 2 B DNA Without EtOH ▫ Signal ? With about 40% EtOH ▫ Expected same as w/o ethanol

27. B-DNA with Porphyrin

28. 17  M ZnT4 Titration of 24-mer

29. Summary

30. Conclusion Nucleosides provided spectra like those previously reported by Voelter et al. B- and Z-DNA CD spectra indicated the two forms, while having opposite helical twists, are not enantiomers. 24-mer can be a scaffold for non-chiral ZnT4 with some differentiation between B- and Z-DNA

31. Acknowledgements University of Wyoming, College of Arts and Sciences Summer Independent Study Award – 2009 Dr. Milan Balaz Alessandro D’Urso

32. References Shells Accessed: January 2009. DNA forms: Principles of Biochemistry. Nelson, D.L.; Cox, M.M.; Lehninger. 5 th ed. 2008. B->Z: Fuertes, M.A.; Cepeda, V.; Alonso, A.; Pérez, J.M. Chemical Reviews. 2006, 106 (6), 2045-2064. Nucleosides: W. Voelter, R. Records, E. Bunnenberg, C. Djerassi, J. Am. Chem. Soc. 1968, 90:22, 6163-6170.

33. End THANK YOU