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Nucleic Acids Research 2005 33(21):6700-6711; doi:10.1093/nar/gki968
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Published online 27 November 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells

Huafeng Fang, Xuan Yue, Xiaoxu Li and John-Stephen Taylor*

Department of Chemistry, Washington University St Louis, MO 63130, USA

*To whom correspondence should be addressed. Tel: +1 314 935 6721; Fax: +1 314 935 4481; Email: taylor{at}wustl.edu

Received October 12, 2005. Revised November 2, 2005. Accepted November 2, 2005.

We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders.


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H. Fang and J.-S. Taylor
Serial analysis of mutation spectra (SAMS): a new approach for the determination of mutation spectra of site-specific DNA damage and their sequence dependence
Nucleic Acids Res., October 1, 2008; 36(18): 6004 - 6012.
[Abstract] [Full Text] [PDF]


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