RNA Modifications Destabilize a Pyrimidine‐Motif RNA●DNA‐DNA Triple Helix

Abstract

The formation of pyrimidine‐motif RNA●DNA‐DNA (R●D‐D) triple helices (or triplexes), in which ‘●’ and ‘‐’ represent Hoogsteen and Watson‐Crick interactions, has been proposed for over 60 years, but the stability of these structures with RNA modifications has yet to be studied. Eleven RNA modifications were chosen for this study based on their presence in human transcripts and their effects on human health: 5‐methylcytidine (m5C), 5‐methyluridine (m5U), pseudouridine (Ψ), 2ʹ‐O‐methyladenosine (Am), 2ʹ‐O‐methylcytidine (Cm), 2ʹ‐O‐methylguanosine (Gm), 2ʹ‐O‐methyluridine (Um), 3‐methyluridine (m3U), 4‐thiouridine (s4U), inosine (I), and N6‐methyladenosine (m6A). Several of these had been previously found to stabilize or destabilize other nucleic acid duplex and triplex structures. Using both native gel‐shift assays and microscale thermophoresis, the relative stability of a single modified position in a pyrimidine‐motif R●D‐D triple helix was measured at neutral pH. All eleven modifications were found to either have no effect or to destabilize the R●D‐D triple helix, ranging from 2‐fold to complete disruption of binding. For each of the canonical R●D‐D base triples (U●A‐T and C●G‐C), the 2ʹ‐O‐methyl modifications were found to be the least destabilizing, whereas those that directly interfered with the Hoogsteen interactions, such as m3U, were the most destabilizing modifications. As the formation of R●D‐D triple helices in promoter regions of DNA leads to transcriptional enhancement and repression, this study reveals that some RNA modifications could potentially inhibit R●D‐D triplex formation as another level of transcriptional regulation.