Figure 1.

Peripheral elements regulate thermostability of the MALAT1 ENE triple helix. (A) Secondary structure of the full-length MALAT1 ENE triple helix (M1^TH) including Watson–Crick interactions (—), Hoogsteen interactions (), A-minor interactions (−−), and a wobble base pair (•). A 5′ GTP (grey) is added to the sequence to facilitate transcription. The A-rich tail is colored blue and structural regions are indicated in brackets. (B) The crystal structure of the MALAT1 triple helix core (PDB: 4PLX) (33). The three strands are colored orange, pink, and blue. The two short loops (gray) were engineered to facilitate crystallization. (C) Secondary structure of trimolecular M1^ABsT. M1^Bs is colored orange, M1^A is colored pink, and M1^Tail is colored blue, as in B. (D) Secondary structure of bimolecular M1^ET, which eliminates the basal linker. The ENE motif is colored cyan and the M1^Tail is colored as in C. (E) Secondary structure of bimolecular M1^AB, wherein the basal linker is included and the apical P2 helix is truncated. M1^A is colored as in C and M1^B is colored green. In C–E, a 5′ GTP (gray) is added to the sequence to facilitate transcription. (F) Normalized UV melt profiles for M1^TH (gray), M1^ABsT (orange), M1^ET (blue), M1^AB (green) from 20 to 85°C in 20 mM HEPES•KOH, pH 7.4, 1 mM MgCl₂, 25 mM NaCl and 25 mM KCl. Triplex formation for all constructs is explicitly demonstrated in Supplementary Figure S2. Triplex and duplex melting occur in the same transition for the bimolecular M1^AB. All experiments were performed in duplicate. A single representative melting profile is plotted. (G) Triplex melting temperatures (T_m,1) for the unimolecular, bimolecular, and trimolecular constructs, colored as in F. Large changes in T_m,1 for the bi- and tri-molecular RNA relative to the full-length RNA demonstrate the contributions of peripheral elements to triplex stability.