Development of Novel Template Mediated Strategies in the Chemical Synthesis of Lariat and Branched RNA

In the past twenty years, nucleic acid lariats and branched molecules have been the subject of a wide range of investigation in both chemical and biological communities since their discovery as intermediates in the RNA splicing mechanism.  Similar to branched RNA, lariats possess 2', 5'and 3', 5'- phosphodiester bonds at the branch site as a result of the formation of a loop or lasso type structure.  The structural uniqueness of such lariats has presented challenges in the development of efficient synthetic methodologies.  Limitations such as expensive reagents, synthetic complexity of modified monomers, and restrictions in the overall structural diversity of lariat species has led to the proposal of a novel synthetic approach.  This approach involves the template directed chemical ligation via cyanogen bromide of RNA asymmetric lariat and branched molecules.  More specifically, a DNA or RNA template can be employed as a “pre-organization” tool to facilitate the connectivity of a 2'-hydroxyl to a 5'-phosphate group at the branching point center.  Their synthesis and characterization by PAGE, HPLC, MALDI-TOF MS and RNA debranching enzymatic assays will be presented.  Thus, such a strategy offers a divergent synthetic route for the production of lariats possessing chemical diversity in sequence, length and branch point structure.

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Figure 1.  Asymmetric Lariat and Branched Molecules