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.