Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography.
J Chromatogr A
; 1218(33): 5609-17, 2011 Aug 19.
Article
em En
| MEDLINE
| ID: mdl-21737080
ABSTRACT
Small interfering RNAs (siRNA) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. siRNA are typically formed by annealing of two complementary single stranded oligoribonucleotides. Compared to purity determination of non-hybridized single strands by denaturing chromatographic methods, characterization of the hybridized duplex is challenging. Here we are reporting a non-denaturing ion pairing-reversed phase (IP-RP) chromatography method capable of separating optimal duplex (full-length single strands only) from non-optimal duplex variants (containing shortmers, longmers and 2',5'-isomers) using ultraviolet- and mass spectrometric detection. The impact of different annealing conditions on siRNA composition was investigated. Optimized annealing conditions lead to a significant increase in optimal duplex, while total duplex content remained constant. The non-denaturing method reported herein showed high mass spectrometric sensitivity and superior separation efficiencies compared to other IP-RP buffer systems. The method is useful for in-process control and release testing of therapeutic double stranded nucleic acids such as siRNA.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromatografia Líquida de Alta Pressão
/
RNA Interferente Pequeno
/
Cromatografia de Fase Reversa
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article