Stemless self-quenching reporter molecules identify target sequences in mRNA |
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Authors: | Gifford Lida K Jordan David Pattanayak Vikram Vernovsky Kathy Do Bao T Gewirtz Alan M Lu Ponzy |
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Affiliation: | Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, Philadelphia, 19104, USA. |
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Abstract: | The design of oligonucleotides for gene silencing requires a rational method for identifying hybridization-accessible sequences within the target RNA. To this end, we have developed stem-loop self-quenching reporter molecules (SQRMs) as probes for such sequence. SQRMs have a 5' fluorophore, a quenching moiety on the 3' end, an intervening sequence that forms an approximately 5-basepaired stem, and a loop sequence of approximately 20-30 bases. We have previously described a mapping strategy employing SQRMs to locate stem-loop structures in the target mRNA molecule. We now show that the original design constraint of a basepaired stem is not needed, either in vitro or in vivo. We propose that stemless probes possess sufficient signal-to-noise for use in vivo and that this ratio is an indication of hybridization of the probe to its target. Data showing that these SQRMs can specifically target and reduce c-Myb protein synthesis and can be used for real-time in vivo assays are presented. |
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Keywords: | Fluorescence imaging Molecular beacons Antisense Targeting |
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