Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups.
Nucleic Acids Res
; 37(13): 4508-17, 2009 Jul.
Article
en En
| MEDLINE
| ID: mdl-19477960
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Compuestos Azo
/
ARN Bicatenario
/
ARN Interferente Pequeño
/
Interferencia de ARN
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2009
Tipo del documento:
Article
País de afiliación:
Estados Unidos