Figure 2.
DNA hairpin adapter ligation. (A) DNA hairpin adapter for LOTTE-seq. The 5ʹ-end of the TGGN overhang is phosphorylated for ligation, the base-paired 3ʹ-end for blocking unwanted side reactions. RT primer binding site is indicated. (B) Adapter ligation catalysed by T4 DNA ligase, T4 RNA ligase 1 and T4 RNA ligase 2 (truncated KQ). Hairpin adapter was incubated with radioactively labelled in vitro transcribed yeast tRNAPhe with and without CCA-end. Only T4 DNA ligase fuses the tRNA with 3ʹ-CCA-end to the adapter hairpin at high selectivity, while RNA ligases 1 and 2 show considerable amounts of side reaction products with the transcript lacking a CCA-end (indicated by asterisks *). T4 RNA ligase 1 shows an additional high molecular weight product migrating in the upper part of the gel, probably resulting from the ligation of two tRNA molecules (**). The panel shows a prolonged exposure of the gels in order to visualize any unspecific ligation side reaction products. In a subsequent PCR-based amplification, such products will represent a considerable unwanted part of the sequence reads. (C) T4 DNA ligase-catalysed hairpin adapter ligation on tRNA transcripts with different 3ʹ-ends. Only the tRNA with a complete 3ʹ-CCA end was accepted for ligation, indicating a high specificity of the adapter ligation for mature tRNA 3ʹ-ends. When the tRNAs with different 3ʹ-ends were pooled, T4 DNA ligase exclusively selects the mature tRNA with CCA-end for ligation.