Optimizing splinted ligation of highly structured small RNAs

Abstract

The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73mer tRNA, the other a 49mer synthetic ribozyme. A stable LCC could be isolated and turned over with > 95% ligation efficiency. In conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies.

Keywords

• microsurgery
• T4-DNA ligase
• splint
• ligation
• hybridization
• RNA structure
• FRET
• SEC, size exclusion chromatography
• FRET, fluorescence resonance energy transfer
• LCC, ligation-competent-complex
• ARI, acceptor-RNA-intermediate
• DRI, donor-RNA-intermediate
• NHS, N-hydroxy-succinimide
• tR, retention time
• SEC, size exclusion chromatography
• T4- PNK, T4 polynucleotide kinase