Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
A broadly applicable, robust, and rapid method for complete sequence confirmation of highly modified oligonucleotides containing a mixture of 2'-deoxy, 2'-fluoro, 2'-o-methyl, abasic and ribonucleotides is presented. The passenger (sense) and guide (antisense) strands from synthetic short interfering RNA duplexes (siRNA) were digested individually using both 5'- and 3'-exonucleases and the resulting ladders were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Conditions for enzymatic digestion and MALDI-TOF mass analysis were investigated and optimized, and the digestion pattern and sequence coverage of each strand was discussed. Complete sequence confirmation for the antisense strands of four synthetic RNA duplexes was obtained, whereas a three-base sequence gap in the 5'-end was observed for all four sense strands. A general strategy is proposed for routine sequence confirmation of highly modified oligonucleotides, and the potential for complete automation of the method is also discussed.
Gao
Hong
H
Department of Analytical Development and Commercialization, Merck & Co. Inc., 126 E Lincoln Ave, Rahway, NJ 076065, USA. hong_gao2@merck.com
Liu
Yong
Y
Rumley
Megan
M
Yuan
Huimin
H
Mao
Bing
B
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D005092:Exonucleases / Q000737:chemistry* / Q000378:metabolism; D043264:Phosphodiesterase I / Q000737:chemistry* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
D034741:RNA, Small Interfering; D005092:Exonucleases; C022650:spleen exonuclease; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom