Regulation of Transcription
through Light-Activation
and Light-Deactivation of Triplex-Forming Oligonucleotides in Mammalian
Cells
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Abstract
Triplex-forming oligonucleotides (TFOs) are efficient
tools to
regulate gene expression through the inhibition of transcription.
Here, nucleobase-caging technology was applied to the temporal regulation
of transcription through light-activated TFOs. Through site-specific
incorporation of caged thymidine nucleotides, the TFO:DNA triplex
formation is blocked, rendering the TFO inactive. However, after a
brief UV irradiation, the caging groups are removed, activating the
TFO and leading to the inhibition of transcription. Furthermore, the
synthesis and site-specific incorporation of caged deoxycytidine nucleotides
within TFO inhibitor sequences was developed, allowing for the light-deactivation
of TFO function and thus photochemical activation of gene expression.
After UV-induced removal of the caging groups, the TFO forms a DNA
dumbbell structure, rendering it inactive, releasing it from the DNA,
and activating transcription. These are the first examples of light-regulated
TFOs and their application in the photochemical activation and deactivation
of gene expression. In addition, hairpin loop structures were found
to significantly increase the efficacy of phosphodiester DNA-based
TFOs in tissue culture