PRESENTATION OUTLINE

1. METAL BINDING TO MODIFIED BASES AND NUCLEOSIDESIskra MuhamedagicNorth Carolina Agricultural and Technical State University

2. PRESENTATION OUTLINE • INTRODUCTION -tRNA and importance of metals in tRNA -Pharmacological/medical applications of modified nucleosides • METHODS -UV/VIS and Job’s plot method -LC/MS -1D/2D NMR -Restrained Molecular Dynamics of U-S4U-U oligonucleotide • RESULTS AND DISCUSSION -UV spectra, Job’s plots, and LC/MS data discussion -1D NMR of monomers -1D/2D NMR and RMD of U-S4U-U • CONCLUSIONS AND FUTURE WORK

3. INTRODUCTION • tRNA is polynucleotide chain of 75 to 90 units long that folds into native L shape • Most common modifications are S4U and S2U at positions 8 and 34, respectively • S4U controls tRNA folding, S2U provides binding site for metal ions

4. Torsion Angle        0 1 2 3 4 Atoms involved (n-1)O3’PO5’C5’ PO5’C5’C4’ O5’C5’C4’C3’ C5’C4’C3’O3’ C4’C3’O3’P C3’O3’PO5’(n+1) O4’C1’N1C2 C4’O4’C1’C2’ O4’C1’C2’C3’ C1’C2’C3’C4’ C2’C3’C4’O4’ C3’C4’O4’C1’ Bases and nucleosides S4U • modifications can take place • in sugar or base • C1’―N1 is b-glycosyl bond • there are five metal binding sites nucleotide unit S2U

5. 3E 2E 32T 3T2 Sugar puckering and base’s orientation • relative to sugar base can adopt anti or syn orientation • S2U puckers into C3’-endo/anti form; S4U prefers C2’-endo/syn conformation anti • sugar can pucker into C3’-endo or C2’-endo form with either twist or envelope conformation syn

6. METHODS • UV/VIS (Ultraviolet/ Visible Spectroscopy) -absorbance was monitored in 200 – 400 nm range -for S2U and 2TU, lmax = 272 nm -for S4U and 4TU, lmax = 332 nm -binding stoichiometry was determined by modified Job’s plot

7. LC/MS (Liquid Chromatography/ Mass Spectroscopy) -1 mM solutions of ligands and mercury acetate were prepared in deionized H20 and in 50% H20/50% ACN -binding stoichiometry and charge is obtained from m/z ratio • NMR (Nuclear Magnetic Resonance) -NMR data (1D and 2D DQFCOSY and ROESY) were acquired on 500 MHz DRX spectrometer at 25°C at The School of Pharmacy, University of Connecticut or at North Carolina State University, Department of Chemistryby Dr. Mufeed Basti

8. Restrained Molecular Dynamics (RMD) of U-S4U-U -energy minimization and molecular dynamics was performed using Discover (Accelrys) -distance constraints generated from ROESY NMR data were used in molecular dynamics simulation -force constant: 1000 kcal mol-1 deg-2 at 298 K

9. RESULTS AND DISCUSSION • UV spectra of ligands and complexes

10. Job’s plots and Kequilibrium

11. LC/MS data analysis

12. 1D NMR analysis of S2U and S4U S2U and S2U-Hg -C3’-endo -N3 involvement

13. Proposed model for Hg-ligand complexes

14. 1D NMR of U-S4U-U U1 U3

15. 2D NMR of U-S4U-U

17. RMD and U-S4U-U-A-stacking of bases-observed equilibrium between C3’-endo/ C2’-endo and anti/syn

18. CONCLUSIONS AND FUTURE WORK • UV/VIS in combination with the Job’s plot method can be used to calculate the binding constant and stoichiometry of binding of mercury to modified nucleosides and bases • method is not suitable for zinc and cadmium complexes because of lower affinity of ligand to metal as well as the formation of multiple types of complexes • future studies are directed towards finding effective ligands for zinc and cadmium metal ions

19. THANK YOU My advisors Dr. Mufeed Basti and Robert Gdanitz Dr. Nadja Cech from UNC-Greensboro