HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 31, 28991-28998, August 3, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/31/28991    most recent M103409200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Puri, N. Articles by Seidman, M. M. Search for Related Content PubMed PubMed Citation Articles by Puri, N. Articles by Seidman, M. M. Social Bookmarking                      What's this?

Targeted Gene Knockout by 2'-O-Aminoethyl Modified Triplex Forming Oligonucleotides^*

Nitin Puri, Alokes Majumdar, Bernard Cuenoud^§, Francois Natt^¶, Pierre Martin^¶, Andre Boyd, Paul S. Miller, and Michael M. Seidman^**

From the  NIA, National Institutes of Health, Baltimore, Maryland 21224, the ^§ Novartis Horsham Research Centre, Horsham, West Sussex, RH12 4AB United Kingdom, ^¶ Novartis Pharma, Ltd., 4002 Basel, Switzerland, and the  Department of Biochemistry and Molecular Biology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205

Triplex forming oligonucleotides (TFOs) are of interest because of their potential for facile gene targeting. However, the failure of TFOs to bind target sequences at physiological pH and Mg²⁺ concentration has limited their biological applications. Recently, pyrimidine TFOs with 2'-O-aminoethyl (AE) substitutions were shown to have enhanced kinetics and stability of triplex formation (Cuenoud, B., Casset, F., Husken, D., Natt, F., Wolf, R. M., Altmann, K. H., Martin, P., and Moser H. E. (1998) Angew. Chem. Int. Ed. 37, 1288-1291). We have prepared psoralen-linked TFOs with varying amounts of the AE-modified residues, and have characterized them in biochemical assays in vitro, and in stability and HPRT gene knockout assays in vivo. The AE TFOs showed higher affinity for the target in vitro than a TFO with uniform 2'-OMe substitution, with relatively little loss of affinity when the assay was performed in reduced Mg²⁺. Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. M. KALISH and P. M. GLAZER Targeted Genome Modification via Triple Helix Formation Ann. N.Y. Acad. Sci., November 1, 2005; 1058(1): 151 - 161. [Abstract] [Full Text] [PDF]

				S. Richards, S.-T. Liu, A. Majumdar, J.-L. Liu, R. S. Nairn, M. Bernier, V. Maher, and M. M. Seidman Triplex targeted genomic crosslinks enter separable deletion and base substitution pathways Nucleic Acids Res., September 25, 2005; 33(17): 5382 - 5393. [Abstract] [Full Text] [PDF]

				A. MAJUMDAR, N. PURI, N. McCOLLUM, S. RICHARDS, B. CUENOUD, P. MILLER, and M. M. SEIDMAN Gene Targeting by Triple Helix-Forming Oligonucleotides Ann. N.Y. Acad. Sci., December 1, 2003; 1002(1): 141 - 153. [Abstract] [Full Text] [PDF]

				R. A. Cassidy, N. Puri, and P. S. Miller Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides Nucleic Acids Res., July 15, 2003; 31(14): 4099 - 4108. [Abstract] [Full Text] [PDF]

				A. Majumdar, N. Puri, B. Cuenoud, F. Natt, P. Martin, A. Khorlin, N. Dyatkina, A. J. George, P. S. Miller, and M. M. Seidman Cell Cycle Modulation of Gene Targeting by a Triple Helix-forming Oligonucleotide J. Biol. Chem., March 21, 2003; 278(13): 11072 - 11077. [Abstract] [Full Text] [PDF]

				F. Nagatsugi, S. Sasaki, P. S. Miller, and M. M. Seidman Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog Nucleic Acids Res., March 15, 2003; 31(6): e31 - e31. [Abstract] [Full Text] [PDF]

				M. P. Knauert and P. M. Glazer Triplex forming oligonucleotides: sequence-specific tools for gene targeting Hum. Mol. Genet., October 1, 2001; 10(20): 2243 - 2251. [Abstract] [Full Text] [PDF]