Sequential Gene Silencing Using Wavelength‐Selective Caged Morpholino Oligonucleotides

Abstract

Sequential gene silencing in zebrafish embryos has been achieved using caged morpholino oligonucleotides with spectrally distinct triggers. Using these optochemical tools, the genetic interactions that dynamically regulate mesoderm patterning have been examined.

Development of Green/Red-Absorbing Chromophores Based on a Coumarin Scaffold That Are Useful as Caging Groups

“…The fluorinated arylacetonitrile derivatives, 2-(2-fluoro-4-nitrophenyl)acetonitrile (14) and 2-(2,6-difluoro-4-nitrophenyl)acetonitrile (15) were synthesized in two steps from 1,2-difluoro-4-nitrobenzene and 1,2,3-trifluoro-4-nitrobenzene, respectively. Reaction with ethyl cyanoacetate and K 2 CO 3 in the presence of a catalytic amount of KI provided the corresponding alkylated intermediates (17 and 18).…”

“…The aqueous layer was washed with ethyl acetate (50 mL) two times. Finally, the combined organic layers were washed with brine (50 mL), dried over anhydrous (14). The published method with some modifications was followed to synthesize compound 14.…”

“…8,13 This modification has also been incorporated in coumarin-caged cyclic morpholino oligonucleotides by Deiters, Chen and collaborators for sequential gene silencing, 14 as well as by us in a caged cyclic RGD peptide for applications in photocontrolled targeted therapies.…”

The concept of photochemical enzyme models – State of the art

“…Such optochemical tools can also be successfully employed for indirectly influencing more complex intracellular processes. For example gene expression controlled by riboswitches [180] can be manipulated using light as an external trigger [181] and sequential gene silencing becomes possible by introducing caged oligonucleotides [182] which has possible implications for the development of future therapeutic agents such as antisense drugs [183]. In addition, new techniques for bio-orthogonal ligation based on the photocleavage of protecting groups are now available to carry out selective and spatiallly resolved labelling of biomolecules in their native setting even within living organisms [184].…”

Caged oligonucleotides for studying biological systems

“…Expanding photoactivation wavelengths into the visible will reduce phototoxicity, increase depth penetration of the excitation source, and allow for the application of multiple, orthogonally-caged constructs that can be simultaneously delivered and then sequentially photoactivated with high spatiotemporal resolution. The Deiters and Chen labs recently advanced this idea by developing a caged MO using a red-shifted coumarin derivative, [7-(diethylamino)coumarin-4-yl]-methyl (DEACM), which is responsive to 470 nm light [46]. The DEACM linker was reacted with 5′-amine, 3′-disulfide functionalized MOs in order to cyclize them.…”