Dreyer, Geoffrey B. and Dervan, Peter B. (1985) Sequence-specific cleavage of single-stranded DNA: Oligodeoxynucleotide-EDTA·Fe(II). Proceedings of the National Academy of Sciences of the United States of America, 82 (4). pp. 968-972. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:DREpnas85
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The synthesis of a DNA hybridization probe 19 nucleotides in length, equipped with the metal chelator EDTA at C-5 of thymidine in position 10 (indicated by T*) is described. DNA-EDTA 1 has the sequence 5'-T-A-A-C-G-C-A-G-T*-C-A-G-G-C-A-C-C-G-T-3', which is complementary to a 19-nucleotide sequence in the plasmid pBR322. In the presence of Fe(II), O2, and dithiothreitol, DNA-EDTA 1 affords specific cleavage (25 degrees C, pH 7.4, 60 min) at its complementary sequence in a heat-denatured 167-base-pair restriction fragment. Cleavage occurs over a range of 16 nucleotides at the site of hybridization of 1, presumably due to a diffusible reactive species. No other cleavage sites are observed in the 167-base-pair restriction fragment. The procedure used to synthesize DNA-EDTA probes is based on the incorporation of a thymidine modified at C-5 with the triethyl ester of EDTA. By using routine phosphoramidite procedures, thymidine-EDTA can be incorporated into oligodeoxynucleotides of any desired length and sequence. Because the efficiency of the DNA cleavage reaction is dependent on the addition of both Fe(II) and reducing agent (dithiothreitol), the initiation of the cleavage reaction can be controlled. These DNA-EDTA-Fe(II) probes should be useful for the sequence-specific cleavage of single-stranded DNA (and most likely RNA) under mild conditions.
|Additional Information:||Copyright © 1985 by the National Academy of Sciences. We are grateful to S. C. Schultz for instruction in phosphoramidite oligonucleotide methods and to Dr. J. H. Richards for the use of equipment. We are grateful to the National Institutes of Health for grant support (GM-27681). G.B.D. was the recipient of a Damon Runyon-Walter Winchell Cancer Fund Postdoctoral Fellowship (DRG-687). This is contribution no. 7079 from the Division of Chemistry and Chemical Engineering, California Institute of Technology. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||modified nucleosides, DNA probes, oxidative cleavage of DNA|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||05 Jan 2006|
|Last Modified:||26 Dec 2012 08:43|
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