METAL BINDING TO MODIFIED BASES AND NUCLEOSIDES Iskra Muhamedagic North Carolina Agricultural and Technical State University

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METAL BINDING TO MODIFIED BASES AND
NUCLEOSIDESIskra MuhamedagicNorth Carolina
Agricultural and Technical State University
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PRESENTATION OUTLINE

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

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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

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Bases and nucleosides

• modifications can take place
• in sugar or base
• C1?N1 is b-glycosyl bond
• there are five metal binding sites

S4U
nucleotide unit
S2U
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Sugar puckering and bases orientation

• relative to sugar base can adopt anti or syn
  orientation
• S2U puckers into C3-endo/anti form S4U prefers
  C2-endo/syn conformation

• sugar can pucker into
• C3-endo or C2-endo
• form with either twist or envelope conformation

anti
syn
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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 Jobs plot

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• 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 25C at
  The School of Pharmacy, University of Connecticut
  or at North Carolina State University, Department
  of Chemistry by Dr. Mufeed Basti

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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
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RESULTS AND DISCUSSION

• UV spectra of ligands and complexes

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Jobs plots and Kequilibrium
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LC/MS data analysis
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1D NMR analysis of S2U and S4U
S2U and S2U-Hg -C3-endo -N3 involvement
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Proposed model for Hg-ligand complexes
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1D NMR of U-S4U-U
U1
U3
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2D NMR of U-S4U-U
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RMD and U-S4U-U-A-stacking of bases-observed
equilibrium between C3-endo/ C2-endo and
anti/syn
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CONCLUSIONS AND FUTURE WORK

• UV/VIS in combination with the Jobs 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

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THANK YOU

• My advisors Dr. Mufeed Basti and Robert Gdanitz
• Dr. Nadja Cech from UNC-Greensboro