MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity.
Synthetic siRNA duplexes are used widely as reagents for silencing of mRNA targets in cells and are being developed for in vivo use. Serum stability is a major concern if siRNA is to be used for therapeutic delivery within blood circulation. We have developed the use of MALDI-TOF mass spectrometry as a rapid and convenient analytical tool to identify the most vulnerable sites within siRNA to serum degradation. Using this approach, we found that one siRNA duplex (Dh3) with UpA sequences close to one end was particularly vulnerable to rapid cleavage. This produced a fragment of mass consistent with the presence of a 2',3'-cyclic phosphate that was slowly hydrolysed to a 2'-(3'-)phosphate on extended incubation. Substitution of these sites with 2'-O-methyl U residues prevented cleavage and confirmed that the major pathway for initial degradation is via cleavage by an RNAse A-like activity. Mass spectral analysis was used to follow the serum degradation of siRNA over more prolonged periods to show the accumulation of many fragments, almost all showing cleavage following pyrimidine nucleoside residues. Overall, the MALDI-TOF mass spectral analysis technique should prove useful for preliminary screening of the serum stability of siRNA duplexes and for identification of the most vulnerable cleavage sites.
Turner
John J
JJ
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, UKCB2 2QH.
Jones
Simon W
SW
Moschos
Sterghios A
SA
Lindsay
Mark A
MA
Gait
Michael J
MJ
D001483:Base Sequence; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry*; D044967:Serum / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D034741:RNA, Small Interfering; D012260:Ribonucleases