We have developed novel methods for the preparation of multimicromole quantities of extremely pure, uniquely photoadducted psoralen-DNA cross-links, furan-side monoadducts and pyrone-side monoadducts. Psoralen cross-linked and furan-side monoadducted DNA were produced by employing high intensity argon ion and krypton ion lasers as light sources. Pyrone-side monoadducts were prepared by base-catalyzed photoreversal of psoralen cross-links. The various psoralen-adducted DNA oligomers were efficiently purified by high performance liquid chromatography. These methods have permitted us to synthesize 4 μmol each of a self-complementary 8-mer d(GCGTACGC) 4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen (HMT) furan-side monoadduct and HMT cross-link. Preliminary nuclear magnetic resonance (NMR) data on the HMT cross-linked 8-mer d(GCGTACGC) have been obtained which confirmed the presence of the diadducted psoralen at the unique 5′TpA3′ site. NMR data obtained from the 8-mer furan-side monoadduct revealed that the psoralen molecule is intercalated into the DNA double helix. Preliminary crystals of 8-mer cross-linked DNA molecule have been grown. Conditions for the growth of X-ray diffraction-quality crystals and the further analysis of these crystals are now in progress.
|Number of pages||15|
|Journal||Journal of Photochemistry and Photobiology, B: Biology|
|State||Published - Jun 30 1992|
Bibliographical noteFunding Information:
We are thankful to Dr. David Cook for advice and suggestionsd uring the course of the experimentsa nd the laser training course and Mr. David Koh for oligonucleotide synthesis. We thank Professor R. Mathies and Dr. J. Ames for their help with the use of the krypton laser. The various suggestions and help of the members of the Hearst Laboratory are also appreciated. This work was supported by the Director, Office of Energy Research, Office of General Life Sciences,M olecular Biology Division (J.E.H.) and Office of Energy Research, Office of Health and Environmental Research (D.E.W.) of the US Department of Energy under Contract DE-ACO3-76SF00098 and National Institute of Health Grant GM 41911t o J.E.H. S.S.S. was supported by Grant NIEHS 07075-11 from United States Public Health Service. Instrumentation was provided by US Department of Energy Grant DE FGOS-86ER75281 and National Science Foundation Grants DMB86-09035 and BBS87-20134.
- DNA photoadducts
- high performance liquid chromatography
- nuclear magnetic resonance.
ASJC Scopus subject areas
- Radiological and Ultrasound Technology
- Radiology Nuclear Medicine and imaging