A single natural RNA modification can destabilize a U•A-T-rich RNA•DNA-DNA triple helix.

Recent studies suggest noncoding RNAs interact with genomic DNA, forming RNA•DNA-DNA triple helices, as a mechanism to regulate transcription. One way cells could regulate the formation of these triple helices is through RNA modifications. With over 140 naturally occurring RNA modifications, we hypothesize that some modifications stabilize RNA•DNA-DNA triple helices while others destabilize them. Here, we focus on a pyrimidine-motif triple helix composed of canonical U•A-T and C•G-C base triples. We employed electrophoretic mobility shift assays and microscale thermophoresis to examine how 11 different RNA modifications at a single position in an RNA•DNA-DNA triple helix affect stability: 5-methylcytidine (m 5 C), 5-methyluridine (m 5 U or rT), 3-methyluridine (m 3 U), pseudouridine (Ψ), 4-thiouridine (s 4 U), N 6 -methyladenosine (m 6 A), inosine (I), and each nucleobase with 2'- O -methylation (Nm). Compared to the unmodified U•A-T base triple, some modifications have no significant change in stability (Um•A-T), some have ∼2.5-fold decreases in stability (m 5 U•A-T, Ψ•A-T, and s 4 U•A-T), and some completely disrupt triple helix formation (m 3 U•A-T). To identify potential biological examples of RNA•DNA-DNA triple helices controlled by an RNA modification, we searched RMVar, a database for RNA modifications mapped at single-nucleotide resolution, for lncRNAs containing an RNA modification within a pyrimidine-rich sequence. Using electrophoretic mobility shift assays, the binding of DNA-DNA to a 22-mer segment of human lncRNA Al157886.1 was destabilized by ∼1.7-fold with the substitution of m 5 C at known m 5 C sites. Therefore, the formation and stability of cellular RNA•DNA-DNA triple helices could be influenced by RNA modifications.