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

Junji Iwahara, D. Eric Anderson, Elizabeth C. Murphy, G. Marius Clore

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

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 (̃0.25 Å 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 9 to 35 Å, to be probed.

Original languageEnglish (US)
Pages (from-to)6634-6635
Number of pages2
JournalJournal of the American Chemical Society
Volume125
Issue number22
DOIs
StatePublished - Jun 4 2003
Externally publishedYes

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Ethylenediaminetetraacetic acid
Protein Binding
Edetic Acid
Thymidine
DNA
Ions
Oligonucleotides
Metals
Metal ions
Bearings (structural)
DNA Cleavage
Dithiothreitol
DNA-Binding Proteins
deoxythymidine-EDTA
Protons
Mass Spectrometry
High Pressure Liquid Chromatography
Electrons
Mass spectrometry
Proteins

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

EDTA-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to DNA by intermolecular paramagnetic relaxation enhancement. / Iwahara, Junji; Anderson, D. Eric; Murphy, Elizabeth C.; Clore, G. Marius.

In: Journal of the American Chemical Society, Vol. 125, No. 22, 04.06.2003, p. 6634-6635.

Research output: Contribution to journalArticle

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abstract = "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 (̃0.25 {\AA} 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 9 to 35 {\AA}, to be probed.",
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