| Photoactivatible or "caged" oligonucleotides have their function temporarily blocked by use of a chemical moiety that can be removed by light. The use of irradiation as an orthogonal trigger allows for high spatial and temporal control of oligonucleotide function. We have developed new techniques for caging these molecules using photocleavable strand breaks to create DNA and RNA that break apart upon photolysis. "RNA bandages" are composed of two 6-12-mer 2'-OMe RNA strands complementary to an mRNA target that are joined by a photocleavable linker. These tandem oligonucleotides typically exhibit much higher affinity for the mRNA than the individual strands. An RNA bandage with binding arms of different lengths and a 4-base gap blocked translation in vitro of GFP mRNA; subsequent near-UV irradiation restored translation. This provides a general method of photomodulating hybridization for a variety of oligonucleotide-based technologies. DNAzymes incorporating photocleavable spacers were switched "on" or "off' with light. A DNAzyme with two photocleavable spacers, in a binding arm and the catalytic core, lost the ability to hydrolyze RNA upon UV photolysis. Conversely, a circular DNAzyme attached to a self-complementary strand via two photocleavable spacers remained "off' until UV irradiation restored activity. New hybrid bandage-hairpins are being developed for caging antisense oligonucleotides with a FRET pair as a fluorescent reporter of hybridization. |
