Stemless self-quenching reporter molecules identify target sequences in mRNA.
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.
Gifford
Lida K
LK
Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, Philadelphia, 19104, USA.
Jordan
David
D
Pattanayak
Vikram
V
Vernovsky
Kathy
K
Do
Bao T
BT
Gewirtz
Alan M
AM
Lu
Ponzy
P
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006224:Cricetinae; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry*; D020598:Proto-Oncogene Proteins c-myb / Q000037:antagonists & inhibitors / Q000235:genetics; D016372:RNA, Antisense / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D034741:RNA, Small Interfering / Q000737:chemistry*
D015345:Oligonucleotide Probes; D020598:Proto-Oncogene Proteins c-myb; D016372:RNA, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering