Published online 17 February 2005
Methods Online |
Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules
1 Department of Chemistry, School of Arts and Sciences 231 South 34th Street, Philadelphia, PA 19104, USA 2 Division of Hematology/Oncology, Department of Medicine, School of Medicine, University of Pennsylvania Room 713, BRB II/III 421 Curie Boulevard, Philadelphia, PA 19104, USA
*To whom correspondence should be addressed. Tel: +1 215 898 4499; Fax: +1 215 573 2078; Email: gewirtz{at}mail.med.upenn.edu
Received January 18, 2005. Accepted January 19, 2005.
We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Pattanayak, L. K. Gifford, P. Lu, and A. M. Gewirtz Observed versus predicted structure of fluorescent self-quenching reporter molecules (SQRM): Caveats with respect to the use of "stem-loop" oligonucleotides as probes for mRNA folding RNA, April 1, 2008; 14(4): 657 - 665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Lundblad, G. Xiao, J.-h. Ko, and S. Altman Rapid selection of accessible and cleavable sites in RNA by Escherichia coli RNase P and random external guide sequences PNAS, February 19, 2008; 105(7): 2354 - 2357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kalota, L. Karabon, C. R. Swider, E. Viazovkina, M. Elzagheid, M. J. Damha, and A. M. Gewirtz 2'-Deoxy-2'-fluoro-{beta}-D-arabinonucleic acid (2'F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing Nucleic Acids Res., January 18, 2006; 34(2): 451 - 461. [Abstract] [Full Text] [PDF]
|
|