Detection and relative quantification of siRNA double strands by MALDI mass spectrometry.
While MALDI-MS is widely accepted for quality control of synthetic oligonucleotides, this method has been regarded as not applicable for a control of the purity and correct annealing of double strands. The results presented here show that the double-strand intensities measured by MALDI-MS maintain and reflect the solution conditions. Using a single-stranded RNA as internal standard, the double-strand intensity can be determined by measuring the intensity ratio of the single strands to the standard under "native" conditions and after denaturation with formic acid. For siRNAs with fully matched 20-21 base pairs, relative intensities of the double strands are between 94 and 97.2%. The stability determined by MALDI-MS for different RNA duplexes correlates well with calculated T m values and the content of G-C pairs. Furthermore, the quantification method enables one to determine an excess of one single strand and the contribution of duplex formation by truncated strands. The results show that MALDI-MS is a fast and reliable method for quality control of synthetic siRNA.
Bahr
Ute
U
Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, University of Frankfurt, 60438 Frankfurt, Germany. bahr@pharmchem.uni-frankfurt.de
Aygün
Hüseyin
H
Karas
Michael
M
D012330:RNA, Double-Stranded / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000032:analysis* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering