Triptycene-based small molecules modulate (CAG)·(CTG) repeat junctions

Abstract

Nucleic acid three-way junctions (3WJs) play key roles in biological processes such as nucleic acid replication in addition to being implicated as dynamic transient intermediates in trinucleotide repeat sequences. Structural modulation of specific nucleic acid junctions could allow for control of biological processes and disease states at the nucleic acid level. Trinucleotide repeat expansions are associated with several neurodegenerative diseases where dynamic slippage is thought to occur during replication, forming transient 3WJ intermediates with the complementary strand. Here, we report triptycene-based molecules that bind to a d(CAG)·(CTG) repeat using a gel shift assay, fluorescence-quenching and circular dichroism.

Fig. 1. Slipped DNA junctions formed by (CAG)·(CTG) repeats.

Fig. 2. (a) Schematic of gel shift assay. (b) The folded TNR 3WJ was incubated with different concentrations of an inhibitor strand (I10) complementary to the 5′-end, resulting in formation of TNR–I10. Non-denaturing polyacrylamide gel ran in 1× TBE buffer at 4 °C.

Fig. 3. (a) Schematic of gel shift assay. The folded TNR 3WJ was incubated with an inhibitor strand complementary to the 5′-end, opening the junction structure (TNR–I10). Addition of triptycene results in reformation of the junction (TNR–Trip). (b) Structures of triptycene derivatives (Trip 1–4). (c) Gel shift assay where TNR–I10 was incubated with triptycene derivatives at a constant concentration. (d) A plot of the difference in band intensities of TNR and TNR–I10. Bars below zero in the plot indicated an increased amount of complex relative to 3WJ. (e) Gel shift assay in the presence and absence of Trip 3 and Trip 4. Samples contained 0.5 μM TNR alone (where minus sign is indicated) or 0.5 μM TNR and 1.5 μM I10 (where a plus sign is indicated). Increasing concentrations of Trip 3 were added (lane 3, 0 μM; lane 4, 0.01 μM; lane 5, 0.10 μM; lane 6, 0.50 μM; lane 7, 1.0 μM; lane 8, 5.0 μM) and Trip 4 (lane 11, 0 μM; lane 12, 0.01 μM; lane 13, 0.10 μM; lane 14, 0.50 μM; lane 15, 1.0 μM; lane 16, 5.0 μM; lane 17, 10.0 μM). Lanes 1 and 9 are loaded with a 25 base pair DNA ladder in which the band present corresponds to 25 bases. Free TNR junction and TNR–I10 complex are indicated. Non-denaturing polyacrylamide gel ran in 1× TBE buffer at 4 °C.

Fig. 4. Fluorescence-quenching assay and circular dichroism (CD). (a) TNR 3WJ labeled with a fluorophore and quencher. When folded, low fluorescence is observed. Addition of inhibitor I10 opens the junction, resulting in an increase in fluorescence (TNR*–I10). Addition of triptycene reforms the junction, resulting in quenching of fluorescence. (b) Titration of I10 to the folded junction results in an increase in fluorescence. Fluorescence assay was conducted in 50 mM sodium phosphate buffer at pH 7.2. (c) Titration of Trip 3 and Trip 4 to TNR*–I10 results in a decrease in fluorescence. (d) Temperature-dependent circular dichroism of the TNR junction. Temperature-dependent CD in the presence of Trip 3 (e) and Trip 4 (f). Circular dichroism measurements were conducted in 50 mM sodium phosphate buffer at pH 7.2.