Optochemical control of RNA interference in mammalian cells

Short interfering RNAs (siRNAs) and microRNAs (nniRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules

Regulation of Transcription through Light-Activation and Light-Deactivation of Triplex-Forming Oligonucleotides in Mammalian Cells

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

Cellular Delivery and Photochemical Activation of Antisense Agents through a Nucleobase Caging Strategy

Antisense oligonucleotides are powerful tools to regulate gene expression in cells and model organisms. However, a transfection or microinjection is typically needed for efficient delivery of the antisense agent. We report the conjugation of multiple HIV TAT peptides to a hairpin-protected antisense agent through a light-cleavable nucleobase caging group. This conjugation allows for the facile delivery of the antisense agent without a transfection reagent, and photochemical activation offers precise control over gene expression. The developed approach is highly modular, as demonstrated by the conjugation of folic acid to the caged antisense agent. This enabled targeted cell delivery through cell-surface folate receptors followed by photochemical triggering of antisense activity. Importantly, the presented strategy delivers native oligonucleotides after light-activation, devoid of any delivery functionalities or modifications that could otherwise impair their antisense activity

Nuclear PTEN tumor-suppressor functions through maintaining heterochromatin structure

<p>The tumor suppressor, PTEN, is one of the most commonly mutated genes in cancer. Recently, PTEN has been shown to localize in the nucleus and is required to maintain genomic stability. Here, we show that nuclear PTEN, independent of its phosphatase activity, is essential for maintaining heterochromatin structure. Depletion of PTEN leads to loss of heterochromatic foci, decreased chromatin compaction, overexpression of heterochromatic genes, and reduced protein stability of heterochromatin protein 1 α. We found that the C-terminus of PTEN is required to maintain heterochromatin structure. Additionally, cancer-associated PTEN mutants lost their tumor-suppressor function when their heterochromatin structure was compromised. We propose that this novel role of PTEN accounts for its function in guarding genomic stability and suppressing tumor development.</p