Concise Affinity-Based Purification of Ligated mRNA for Structure-Activity Relationship Studies of Nucleosugar Modification Patterns.

Abstract

Position-specific nucleoside sugar modifications have been shown to improve the translational activity and stability of chemically synthesized mRNA. For pharmaceutical applications of chemically modified mRNAs, a rapid purification methodology is imperative to identify the optimal modification pattern. However, while the chemical synthesis of mRNAs can be accomplished by splint ligation of oligonucleotide fragments, the current purification method for ligated mRNAs based on denaturing polyacrylamide gel electrophoresis tends to be time consuming. In this study, we developed a two-step affinity purification method for rapid sample preparation. In this method, ligated mRNA is captured by oligo dT magnetic beads and streptavidin magnetic beads with 3'-biotinylated oligo DNA, which are complementary to the 3'-poly(A) and 5' terminal sequences of the target mRNA, respectively. Therefore, the target mRNA can be isolated from a complex mixture of splint ligations. Using this method, six sugar-modified mRNAs were simultaneously purified, and the translational activities of these mRNAs were evaluated immediately after purification. The results demonstrate that this methodology is suitable for the rapid preparation of various chemically synthesized mRNAs to identify their optimal modification patterns.