A Two-Photon-Photocleavable Linker for Triggering Light-Induced Strand Breaks in Oligonucleotides

Abstract

We synthesized a two-photon-sensitive photocleavable linker based on the 7-diethylaminocoumarin structure and introduced it successfully into DNA strands. First, we demonstrated the inducibility of strand scissions upon irradiation at 365 nm. To verify and visualize the two-photon activity, we used a fluorescence assay based on a DNA strand displacement immobilized in a hydrogel. Additionally, we investigated its use in a new class of DNA decoys that are able to catch and release nuclear factor κB (NF-κB) by using light as an external trigger signal. In cell culture we were able to show the regulation of NF-κB-controlled transcription of green fluorescent protein.

DePEGylation strategies to increase cancer nanomedicine efficacy

“…175 Furthermore, advances in fibre optic technologies (to deliver UV light deep within tissue), 176 the development of photolabile chemical bonds sensitive to longer wavelength light 177 and the optimization of photosensitive chemical functionality to minimize light exposure, will only further the clinical applicability of light. One promising development has been photolabile chemical groups sensitive to two photon light, 178 to not only increasing tissue penetration (>1 cm) of light and minimising induced photocytotoxicity but, by restricting light activation to the focal point of two photon beams, enabling activation volumes in patients of <1 femtolitre. In this vein, we and others have also shown it is possible to cleave o-Nb groups using 2-photon light.…”

CRISPRoff enables spatio-temporal control of CRISPR editing

“…We have successfully demonstrated this technology in multiple human cell lines across multiple genomic loci and expect this technology to be turn-key ready with any CRIPSR-based application currently using synthetic or to replace in vitro transcribed sgRNAs. While, at the moment, CRISPRoff is limited in in vivo applications, due to the low penetrance of UV light through tissues, we are excited by the possibility of further chemistries extending the range of photocleavable molecules such as with two-photon cleavage systems 27,28 . Further, because CRISPRoff makes modifications to the backbone of the sgRNA, it can be compatible with other technologies, such as sgRNA modifications to activate gene editing 25 , or Cas9 modifications to enhance on-target specificity 29,30 .…”

Efficient Synthesis of Light‐Triggered Circular Antisense Oligonucleotides Targeting Cellular Protein Expression

“…Light‐controlled modulation of gene expression through RNA targeting has been intensely investigated over the past decade. In recent studies, exemplar caged oligonucleotides have been developed, including DNA decoy, RNA, siRNA, antagomir, and analogues such as PNA and morpholino, and have been tested for direct gene knockdown, transcription regulation, and gene editing. RNA targeting, in general, involves binding a complementary oligo to a selected target sequence through highly specific base pairing, which can interrupt downstream processes either by steric hindrance or degradation of the RNA target.…”

“…[8a] Progress has also been made in generating photolinkers that can be activated at longer wavelengths, with potential for lower phototoxicity and deeper tissue penetration. [2, 23b, 25] …”