Binding and conformational analysis of phosphoramidate-restriction enzyme interactions

Biochemistry
Julie B KingC M Dupureur

Abstract

Phosphoramidates are modified deoxyoligonucleotides that feature nitrogen in place of the 3'-oxygen of a phosphodiester linkage. Noted for stability against nuclease activity, these linkages are of both mechanistic and therapeutic interest. While a number of studies characterizing the properties of oligonucleotides composed entirely of phosphoramidate linkages have been published, little is known about how singly substituted phosphoramidate substitutions affect the thermodynamics and structure of protein-oligonucleotide interactions. We chose to investigate these interactions with PvuII endonuclease, the DNA binding behavior of which is well-characterized. Oligonucleotide duplexes containing a phosphoramidate substitution at the scissile phosphates were resistant to cleavage by the enzyme, even after extended incubations. However, the enzyme was able to cleave the native strand in a native:phosphoramidate heteroduplex at a rate comparable to that observed with the native substrate. Ca(II)-stimulated PvuII binding for a phosphoramidate-substituted oligonucleotide is comparable to that of the native duplex (K(d) approximately 200 pM). K(d) values obtained in the presence of Mg(II) are somewhat weaker (K(d) approximately 10 nM). U...Continue Reading

References

May 30, 1997·Journal of Molecular Biology·L E EnglerL Jen-Jacobson
May 15, 1997·European Journal of Biochemistry·A Pingoud, A Jeltsch
Nov 5, 1997·The Journal of Biological Chemistry·H G NastriP D Riggs
Jul 25, 2000·Journal of Molecular Biology·J R Horton, X Cheng
Apr 16, 2003·European Journal of Biochemistry·Jens Kurreck

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Citations

Aug 11, 2006·Nucleosides, Nucleotides & Nucleic Acids·Cynthia M Dupureur
Jul 5, 2011·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Grigorios A Papadakos, Cynthia M Dupureur
Feb 22, 2012·Analytical Biochemistry·Cynthia M DupureurGaofei He
Jan 24, 2009·Archives of Biochemistry and Biophysics·Fuqian Xie, Cynthia M Dupureur

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