Synthesis of Site‐Specifically Phosphate‐Caged siRNAs and Evaluation of Their RNAi Activity and Stability

Abstract

A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site-specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate-caged siRNA and the single-stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from the internally caged siRNA was observed, irrespective of the presence of Mg(2+). Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate-caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate-caged siRNA or single-stranded caged RNA prefers to form π-π stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.

Photo‐Tethers for the (Multi‐)Cyclic, Conformational Caging of Long Oligonucleotides

“…Photolabile groups (“caging groups”) are widely used in biochemical research to trigger oligonucleotide activity . Versatile applications including photoregulation of gene expression with antisense approaches, DNAzymes and ribozymes, small interfering RNAs (siRNA) and microRNAs (miRNA) have been demonstrated. Major improvements were recently made on the development of new caging groups, which now allow for one‐photon activation at higher wavelength (>500 nm) or two‐photon activation with pulsed IR laser light .…”

Optimal Destabilization of DNA Double Strands by Single‐Nucleobase Caging

“…The NPBY and DEACBYs creenings (Figure 7) reveal am elting temperature plateauw here-within the error-the destabilization of the duplex depends only weakly on the specific bulky triazole residues.T ounderstand how our caged DNA design interferesw ith the native duplex we proceeded with molecular dynamics (MD) simulations of the native DNA, dT DPMTC -caged (S)-or (R)-2f and dT DEACM -caged DNA variants.T hese caged nucleobases are sterically more demanding than the dC NPPM cages discussed above in the NMR to MD structurec omparison, for [48] The stability of the caged DNA was investigated in five independents imulations per cage (cumulative 1 ms/cage). Overall, the DNA retained its helical structure in all simulations (Figure 9a).…”

Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

“…Considering DNAzymes, their catalytic activity is inhibited until photoirradiation releases the native DNAzyme [ 77 ]. In phosphate-caged siRNAs, chemical derivatization of phosphates either in the phosphodiester backbone [ 72 ] or at a terminal phosphate [ 73 – 74 ] of ON was performed following two different approaches: a) post-functionalization of ON with a suitable reagent, which generally is a diazo derivative bearing a photoresponsive moiety, or b) incorporation of an appropriate photocaged phosphoramidite during the solid-supported ON synthesis [ 73 , 78 ]. The advantage of the first approach is that the functionalization results from a reaction with available unmodified ONs, while the second approach first requires the synthesis of a modified unit followed by its incorporation into ON during solid-phase synthesis.…”