EDTA-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to DNA by intermolecular paramagnetic relaxation enhancement

J Am Chem Soc. 2003 Jun 4;125(22):6634-5. doi: 10.1021/ja034488q.


EDTA-derivatized deoxythymidine (dT-EDTA), incorporated into DNA and complexed to Fe2+ in the presence of dithiothreitol, is a widely used reagent for sequence-specific cleavage of duplex DNA. Using HPLC/electrospray mass spectrometry, we show that cleavage is specific to Fe2+, and no cleavage occurs when DNA-EDTA is complexed to other metal ions such as Ca2+, Mn2+, and Fe3+ even after many days. Because dT-EDTA can be incorporated at any desired position of a synthetic oligonucleotide, DNA-EDTA is ideally suited for the measurement of intermolecular paramagnetic relaxation enhancement effects between a paramagnetic ion chelated to DNA-EDTA and a bound protein. Measurements on the SRY/DNA-EDTA complex using two double-stranded oligonucleotides bearing dT-EDTA at opposite ends of the sequence indicate that intermolecular 1HN-T2 enhancement by chelated Mn2+ can be used to readily ascertain the polarity of protein binding to DNA and to derive quantitative long-range distance information for structure refinement. In the case of the SRY-DNA complex, excellent agreement between observed and calculated 1HN-T2 paramagnetic relaxation enhancement data can be achieved with insignificant shifts in the atomic coordinates ( approximately 0.25 A for all heavy atoms) while simultaneously satisfying all other experimental restraints. A unique feature of DNA-EDTA is that the relaxation enhancement effect can be tuned by judicious choice of the paramagnetic metal ion, thereby permitting a wide range of long-range intermolecular electron-proton distances, ranging from approximately 9 to 35 A, to be probed.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Calcium / chemistry
  • Chelating Agents / chemistry
  • DNA / chemistry
  • DNA / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Edetic Acid / analogs & derivatives*
  • Edetic Acid / chemistry
  • Manganese / chemistry
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Nuclear Proteins*
  • Nucleic Acid Conformation
  • Protein Binding
  • Sex-Determining Region Y Protein
  • Thymidine / analogs & derivatives*
  • Thymidine / chemistry
  • Transcription Factors*


  • Chelating Agents
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Sex-Determining Region Y Protein
  • Transcription Factors
  • Manganese
  • DNA
  • Edetic Acid
  • Calcium
  • Thymidine