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Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases

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Abstract

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A16). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.

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Acknowledgments

We gratefully acknowledge the NIH (AI 30988) and Kimeragen, Inc. for the support of this research. H.L. is grateful to the Department of Chemistry and Biochemistry, University of Notre Dame for financial support in the form of a Reilly fellowship. C.A.F. is grateful to the Department of Chemistry and Biochemistry, University of Notre Dame for financial support in the form of a Podrasnik Fellowship. We appreciate the use of the Bioscience Core facility for the solid phase DNA synthesis, Mass Spectrometry and NMR facilities at the Department of Chemistry and Biochemistry, University of Notre Dame. We are particularly indebted to Prof. P. Huber for assistance with electrophoresis studies and helpful discussions. We also thank Ms. Maureen Metcalf for assistance with the manuscript.

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  1. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556, USA

    Marvin J. Miller, Hui Li & Catherine A. Foss

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  1. Marvin J. Miller
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  2. Hui Li
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  3. Catherine A. Foss
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Correspondence to Marvin J. Miller.

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Miller, M.J., Li, H. & Foss, C.A. Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases. Biometals 22, 491–510 (2009). https://doi.org/10.1007/s10534-009-9206-7

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  • DOI: https://doi.org/10.1007/s10534-009-9206-7

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