High-resolution Capillary Electrophoresis Separation of Large RNA under Non-aqueous Conditions

Large RNAs, such as, mRNAs have great potential as promising candidates for new drugs as well as
vaccines. Therefore, development of highly efficient analytical methods for large RNAs is critical to support
new drug development and manufacture. Size separation analysis is one of the most critical characterization
attributes of large RNA. However, the current resolution of separation of large RNAs is limited even using
capillary electrophoresis (CE), which is one of the most efficient separation methods. This paper presents a
capillary gel electrophoresis (CGE) method for separating large RNA molecules (200 nt to 6000 nt) by
length, with non-aqueous gel that consisting of high molecular polymers and formamide for the first time.
The separation conditions were optimized for RNAs with approximate 2000 nt. Under the optimized
conditions, the separation resolution is increased by almost 3 times for 1000 nt and 2000 nt RNAs as
compared to those using aqueous gel. The impact of polymer type, molecular weight of the polymer and
polymer concentration to the separation was studied and optimized. The separation mechanism was also
investigated using logarithmic plot, and the results provide guidance for optimization of separation
conditions for RNAs with different sizes.