Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups.
Diazo-based precursors of photolabile groups have been used extensively for modifying nucleic acids, with the intention of toggling biological processes with light. These processes include transcription, translation and RNA interference. In these cases, the photolabile groups have been typically depicted as modifying the phosphate backbone of RNA and DNA. In this work we find that these diazo-based reagents in fact react very poorly with backbone phosphates. Instead, they show a remarkable specificity for terminal phosphates and very modest modification of the nucleobases. Furthermore, the photo deprotection of these terminal modifications is shown to be much more facile than nucleobase modified sites. In this study we have characterized this regiospecificity using RNA duplexes and model nucleotides, analyzed using LC/MS/MS. We have also applied this understanding of the regio-specificity to our technique of light activated RNA interference (LARI). We examined 27-mer double-stranded precursors of siRNA ('dsRNA'), and have modified them using the photo-cleavable di-methoxy nitro phenyl ethyl group (DMNPE) group. By incorporating terminal phosphates in the dsRNA, we are able to guide DMNPE to react at these terminal locations. These modified dsRNA duplexes show superior performance to our previously described DMNPE-modified siRNA, with the range of expression that can be toggled by light increasing by a factor of two.
Shah
Samit
S
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64110, USA.
Jain
Piyush K
PK
Kala
Ashish
A
Karunakaran
Dipu
D
Friedman
Simon H
SH
D001391:Azo Compounds / Q000737:chemistry* / Q000528:radiation effects; D002853:Chromatography, Liquid; D015226:Dinucleoside Phosphates; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008027:Light; D010766:Phosphorylation; D034622:RNA Interference / Q000528:radiation effects*; D012330:RNA, Double-Stranded / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014542:Uridine Monophosphate
D001391:Azo Compounds; D015226:Dinucleoside Phosphates; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; C026519:uridine monophosphate-uridine; D014542:Uridine Monophosphate