Efficient Splinted Ligation of Synthetic RNA Using RNA Ligase

Abstract

RNA ligation allows the creation of large RNA molecules from smaller pieces. This can be useful in a number of contexts: to generate molecules that are larger than can be directly synthesized; to incorporate site-specific changes or RNA modifications within a large RNA in order to facilitate functional and structural studies; to isotopically label segments of large RNAs for NMR structural studies; and to construct libraries of mutant RNAs in which one region is extensively mutagenized or modified. The impediment to widespread use of RNA ligation is the low and variable efficiency of standard ligation strategies, which frequently preclude joining more than two pieces of RNA together.We describe a method using RNA ligase (Rligation), rather than DNA ligase (Dligation), in a splint-mediated ligation reaction that joins RNA molecules with high efficiency. RNA ligase recognizes single-stranded RNA ends, which are held in proximity to one another by the splint. Monitoring the reaction is easily accomplished by denaturing gel electrophoresis and ethidium bromide staining. Using this technique, it is possible to generate a wide range of modified RNAs from synthetic oligoribonucleotides.