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Metal Binding to Modified tRNA Bases and Nucleosides: Implications for Pharmacology

This presentation explores the role of metal ions in tRNA modifications, particularly S4U and S2U, which impact tRNA folding and binding sites. Utilizing methods like UV/VIS spectroscopy, LC/MS, and NMR, we analyze the binding interactions and stoichiometry of mercury complexes with modified nucleosides. The results highlight the limitations of these methods for certain metals like zinc and cadmium and outline future directions for studying effective ligands. Our findings could have significant medical applications in pharmacology and biochemistry.

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Metal Binding to Modified tRNA Bases and Nucleosides: Implications for Pharmacology

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  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

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

  17. 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

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

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