Analysis of 2'-OMe and 2'-MOE Modified Phosphorothioated Oligo-RNA by LC/UV/MS (VN-50 2D)

RNAs are composed of ribose (a pentacarbonate), phosphoric acid, and base. Phosphorothioated oligonucleic acids are often used in nucleic acid drugs, because they are more stable than regular oligonucleic acids. Further modification of ribose increases the binding affinity even more.
In this application, four compounds were analyzed by LC/UV/MS using HILICpak VN-50 2D, a polymer-based HILIC column. They were (i) synthetic phosphorothioated oligo-RNA with all ribose modified with 2'-MOE (2’-Methoxyethyl),  (ii) synthetic phosphorothioated oligo-RNA with 5-bases at both ends of ribose modified with 2'-MOE, (iii) synthetic phosphorothioated oligo-RNA with all ribose modified with 2'-OMe (2'-O-Methyl), and (iv) synthetic phosphorothioated oligo-RNA without ribose modification.
Under HILIC mode, the higher the hydrophilicity, the stronger the retention becomes. Among synthetic phosphorothioated oligo-RNA, one with more 2’-MOE modification has lower hydrophilicity. Thus, 2’-MOE modified ones are less retained than non-modified ones (compare peaks 1, 2, and 5). On the other hand, 2’-OME has higher hydrophilicity than 2’-MOE. Thus, even all ribose are modified with 2’-OME, its retention is not affected so much and elutes at about the same time as the one partially modified with 2’-MOE (compare peaks 2 and 3).  
The application developed here does not require a use of ion-pairing reagent nor highly concentrated salt in the eluent. Therefore, it is suitable for LC/MS analysis of oligonucleotides.

Analysis of Various Oligonucleotides by LC/UV/MS (VN-50 2D)

Sample : 0.2 mg/mL each (in H2O), 1 μL
1. Synthesized phosphorothioated oligo-2'-MOE-RNA 20mer(cartridge purified),

2. Synthesized phosphorothioated oligo-2'-MOE(1-5/16-20)-RNA 20mer(cartridge purified),

3. Synthesized phosphorothioated oligo-2'-OMe-RNA 20mer(cartridge purified),

4. Synthesized phosphorothioated oligo-RNA 20mer(cartridge purified),

* means phosphorothioated position

Column       : Shodex HILICpak VN-50 2D (2.0 mm I.D. x 150 mm)
Eluent       : (A)50 mM HCOONH4 aq. (pH9.8)/(B) CH3CN
               Linear gradient ;
               (B %) 64 to 57 % (0 to 10 min), 57 % (10 to 20 min), 
                     57 to 64 % (20 to 20.01 min), 64 % (20.01 to 25 min)
Flow rate    : 0.3 mL/min
Detector     : UV (260 nm) (small cell volume), ESI-MS (SIM Negative)
Column temp. : 40 °C

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