Single -stranded nucleic acid conformation in context

The design of oligonucleotides for gene silencing requires a rational method for identifying hybridization accessible sequences within the target RNA. We describe a mapping strategy employing self-quenching reporter molecules to locate these structures in the target mRNA molecule. The self-quenching reporter molecules have a 5′ fluorophore, a sequence of 20–30 bases, and a quenching moiety on the 3′ end. Without the target mRNA, the non-hybridized state of the self-quenching reporter molecule does not emit photons because of either a designed stem-loop structure or simple fluorescein-quencher collisional interaction. The fluorescein-quencher distance is increased by double helix formation to the target mRNA; fluorescence is detected when excited by light of the appropriate wavelength. We propose that complementary sequences separated by about 20 bases offer targets for antisense oligodeoxynucleotide hybridization because they would form stem-loop structures in the RNA sequence. As a result of our experiences in targeting these potential stem-loop structures within mRNA, we developed stemless SQRMs. We show that the original design constraint of a base paired stem is not needed both in vitro and 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 are presented showing that the sequences found in this manner can be targeted to reduce c-Myb protein synthesis in vitro and the SQRMs can be used for real-time in vivo assays. Our experiments using these stemless SQRMs to target the Tetrahymena Group I intron ribozyme indicate that target accessibility is governed by tertiary structure more than expected