Proximal disruptor aided ligation (ProDAL) of kilobase-long RNAs

Abstract

ABSTRACTRNA with site-specific modification is a useful tool for RNA biology studies. However, generating kilobase (kb) -long RNA with internal modification at a site distant from RNA termini remains challenging. Here we report an enhanced splint ligation technique, proximal disruptor aided ligation (ProDAL), which allows adequate efficiency toward this purpose. The key to our approach is using multiple DNA oligonucleotides, ‘proximal disruptors’, to target the RNA substrate sequence next to the ligation site. The binding of disruptors helps to free the ligation site from intramolecular RNA basepairing, and consequently promotes more efficient formation of the pre-ligation complex and a higher overall ligation yield. We used naturally occurring 1.0 kb renilla and 1.9 kb firefly luciferase mRNA sequences to test the efficacy of our approach. ProDAL yielded 9–14% efficiency for the ligation between two RNA substrates, both of which were between 414 and 1313 nucleotides (nt) long. ProDAL also allowed similarly high efficiency for generating kb-long RNA with site-specific internal modification by a simple three-part ligation between two long RNA substrates and a modification-carrying RNA oligonucleotide. In comparison, classical splint ligation yielded a significantly lower efficiency of 0–2% in all cases. We expect that ProDAL will benefit studies involving kb-long RNAs, including translation, long non-coding RNAs, RNA splicing and modification, and large ribonucleoprotein complexes.