Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne-modified photolabile nucleosides into DNA sequences, followed by a Cu -catalyzed alkyne-azide cycloaddition with bis-azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full-length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo-tether strategy presented here provides a robust and versatile tool for the light-activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.
Photolabile protecting groups are widely used to trigger oligonucleotide activity. The ON/OFF-amplitude is a critical parameter. An experimental setup has been developed to identify protecting group derivatives with superior caging properties. Bulky rests are attached to the cage moiety via Cu-catalyzed azide-alkyne cycloaddition post-synthetically on DNA. Interestingly, the decrease in melting temperature upon introducing o-nitrobenzyl-caged (NPBY-) and diethylaminocoumarin-cages (DEACM-) in DNA duplexes reaches a limiting value. NMR spectroscopy was used to characterize individual base-pair stabilities and determine experimental structures of a selected number of photocaged DNA molecules. The experimental structures agree well with structures predicted by MD simulations. Combined, the structural data indicate that once a sterically demanding group is added to generate a tri-substituted carbon, the sterically less demanding cage moiety points towards the neighboring nucleoside and the bulkier substituents remain in the major groove.
Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne‐modified photolabile nucleosides into DNA sequences, followed by a CuI‐catalyzed alkyne–azide cycloaddition with bis‐azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full‐length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo‐tether strategy presented here provides a robust and versatile tool for the light‐activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.
Caged compounds are light-sensitive molecules with temporarily inactivated biological function. The active compound is released upon irradiation, in which exact spatial and temporal control is accomplished. Beyond this inherently irreversible concept of triggering, the idea of multi-wavelength uncaging provides experiments with more complexity. This unit describes the syntheses of protected nucleoside phosphoramidites of caged dT(NpHP) [pHP = (p-hydroxyphenacyl)], dT(DEACM) {DEACM = [(7-diethylaminocoumarin-4-yl)methyl]} or a dC(NDBF) {NDBF = [1-(3-nitrodibenzofuran-1-yl)ethyl]} modification on the nucleobase, their incorporation in oligonucleotides, characterization, and their wavelength-selective uncaging up to four levels.
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