Figure 3: Ligation of DNA oligos to both ends of RNA substrate using a DNA splint. FAM fluorescence (attachment to 5’ end) is illustrated indicated in green, TAMRA (attachment to 3’ end) is indicated in red. When both adapters are ligated, the band becomes yellow and shifts further up.
With the current focus in biotechnology on RNA therapeutics, synthesis and analysis, there are multiple potential applications for producing chimeric RNA-DNA oligonucleotides. For example, chimeric guide DNA-RNA for CRISPR editing or synthesis of DNA-RNA chimeras for specific cutting of RNA with RNase H [15]. The novel DNA-RNA activity of R2D may also find a use in RNA analysis, e.g. for capturing microRNA or novel approaches to DNA adapter ligation of RNA. Additionally, such approaches can be used for RNA enrichment or RNA depletion workflows where there is a need to keep the 3’ end of the RNA unoccupied. Finally, we envision that R2D ligase may be used for barcoding of RNA sequencing libraries prior to sample multiplexing. This could remove the need for cDNA synthesis prior to barcode attachment and facilitates multiplexing of direct RNA sequencing workflows.