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Binding and conformational analysis of phosphoramidate-restriction enzyme interactions
Julie B King1, Lori M Bowen, Cynthia M Dupureur
1Department of Chemistry & Biochemistry, University of Missouri-St. Louis, St. Louis, Missouri 63121, USA.
Biochemistry
|June 30, 2004
Summary
Phosphoramidate modified oligonucleotides resist PvuII endonuclease cleavage. These modified DNA molecules show significantly enhanced binding affinity to the enzyme, especially without metal ions, indicating potential therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Medicinal Chemistry
Background:
- Phosphoramidates are modified deoxyoligonucleotides with nitrogen replacing the phosphodiester linkage's 3'-oxygen.
- These modifications enhance stability against nuclease activity, presenting mechanistic and therapeutic potential.
- Limited data exists on how single phosphoramidate substitutions impact protein-oligonucleotide interactions' thermodynamics and structure.
Purpose of the Study:
- Investigate the effects of single phosphoramidate substitutions on protein-oligonucleotide interactions.
- Characterize the interaction of PvuII endonuclease with phosphoramidate-modified oligonucleotides.
- Determine the impact on DNA cleavage, binding affinity, and enzyme conformation.
Main Methods:
- Synthesis of phosphoramidate-substituted oligonucleotide duplexes.
- Enzymatic cleavage assays using PvuII endonuclease.
- Isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) for binding affinity (Kd) determination.
- (31)P Nuclear Magnetic Resonance ((31)P NMR) spectroscopy.
- (1)H-(15)N Heteronuclear Single Quantum Coherence (HSQC) analysis.
Main Results:
- Phosphoramidate-containing duplexes were resistant to PvuII endonuclease cleavage.
- PvuII cleaved the native strand in a heteroduplex at a rate comparable to native substrate.
- Ca(II)-stimulated PvuII binding to phosphoramidate oligonucleotides was similar to native duplexes (Kd ~200 pM).
- Mg(II)-bound Kd values were weaker (~10 nM).
- Under metal-free conditions, enzyme affinity for phosphoramidate duplexes was ~50-fold higher (Kd ~5 nM vs. 240 nM).
- (31)P NMR showed increased chemical shift dispersion in free phosphoramidate duplexes.
- (1)H-(15)N HSQC indicated comparable enzyme conformations with native and modified duplexes.
Conclusions:
- Single phosphoramidate substitutions confer resistance to PvuII endonuclease cleavage.
- These modifications significantly enhance enzyme binding affinity, particularly under metal-free conditions.
- The observed effects are attributed to local conformational adjustments propagating from the O-->N substitution.