Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (338K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (61)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Scherr, M.
Right arrow Articles by Rossi, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scherr, M.
Right arrow Articles by Rossi, J. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, Vol 26, Issue 22 5079-5085, Copyright © 1998 by Oxford University Press

ARTICLES

Rapid determination and quantitation of the accessibility to native RNAs by antisense oligodeoxynucleotides in murine cell extracts

M Scherr and JJ Rossi
Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3011, USA.

A major concern for antisense experiments is the prediction of effective oligonucleotide binding sites. We have developed a system to carry out oligodeoxyribonucleotide-RNA and ribozyme-RNA binding experiments in cell extracts to create a protein environment known to directly influence the structure of the mRNA. In these experiments the native, endogenous mRNA is probed using oligodeoxyribonucleotides (ODNs) to identify RNase H-accessible sites. The resulting RNase H- mediated cleavages in the cell extracts were quantified using RT-PCR with fluorescein and rhodaminetagged primers to generate fluorescent products that are analyzed and quantified on an automated DNA sequencer. As a model substrate for testing this system, we have targeted the murine DNA methyltransferase (MTase) mRNA. An ODN binding site in native MTase mRNA was identified that was cleaved by endogenous RNase H with an efficiency of 85% in the extracts. The ODN that was most effective in the cell extracts was also found to provide the best activity in vivo , resulting in a 75-85% reduction of the MTase mRNA. These data support the use of cell extracts and native transcripts to identify antisense and perhaps ribozyme target sites.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J BiochemHome page
M. Shiota, M. Sano, M. Miyagishi, and K. Taira
Ribozymes: Applications to Functional Analysis and Gene Discovery
J. Biochem., August 1, 2004; 136(2): 133 - 147.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. Zippo, A. De Robertis, M. Bardelli, F. Galvagni, and S. Oliviero
Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro
Blood, June 15, 2004; 103(12): 4536 - 4544.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
D. Husken, F. Asselbergs, B. Kinzel, F. Natt, J. Weiler, P. Martin, R. Haner, and J. Hall
mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity
Nucleic Acids Res., September 1, 2003; 31(17): e102 - e102.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
J. J. Gao, V. Diesl, T. Wittmann, D. C. Morrison, J. L. Ryan, S. N. Vogel, and M. T. Follettie
Regulation of gene expression in mouse macrophages stimulated with bacterial CpG-DNA and lipopolysaccharide
J. Leukoc. Biol., December 1, 2002; 72(6): 1234 - 1245.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
R. M. Smith, C. M. Walton, C. H. Wu, and G. Y. Wu
Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3'-Terminal Sequences
J. Virol., August 28, 2002; 76(19): 9563 - 9574.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. M. Luger, S. G. O'Brien, J. Ratajczak, M. Z. Ratajczak, R. Mick, E. A. Stadtmauer, P. C. Nowell, J. M. Goldman, and A. M. Gewirtz
Oligodeoxynucleotide-mediated inhibition of c-myb gene expression in autografted bone marrow: a pilot study
Blood, February 15, 2002; 99(4): 1150 - 1158.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
N. S. LEE, B. SUN, R. WILLIAMSON, N. GUNKEL, P. M. SALVATERRA, and J. J. ROSSI
Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes
FASEB J, November 1, 2001; 15(13): 2390 - 2400.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
A. A. Mir, T. J. Lockett, and P. Hendry
Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers
Nucleic Acids Res., May 1, 2001; 29(9): 1906 - 1914.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Amarzguioui, G. Brede, E. Babaie, M. Grotli, B. Sproat, and H. Prydz
Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes
Nucleic Acids Res., November 1, 2000; 28(21): 4113 - 4124.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
K.-Y. Jen and A. M. Gewirtz
Suppression of Gene Expression by Targeted Disruption of Messenger RNA: Available Options and Current Strategies
Stem Cells, September 1, 2000; 18(5): 307 - 319.
[Abstract] [Full Text]

Home page
 Nucleic Acids ResHome page
M. Scherr, J. J. Rossi, G. Sczakiel, and V. Patzel
RNA accessibility prediction: a theoretical approach is consistent with experimental studies in cell extracts
Nucleic Acids Res., July 1, 2000; 28(13): 2455 - 2461.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. Wrzesinski, M. Legiewicz, and J. Ciesiolka
Mapping of accessible sites for oligonucleotide hybridization on hepatitis delta virus ribozymes
Nucleic Acids Res., April 15, 2000; 28(8): 1785 - 1793.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.