Structure‐Specific Recognition of Friedreich's Ataxia (GAA)_n Repeats by Benzoquinoquinoxaline Derivatives

Abstract

Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)n repeats form non-B-DNA structures, including triple helix H-DNA and higher-order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non-B-DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)n binding by triplex-stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex-DNA-cleaving BQQ-1,10-phenanthroline (BQQ-OP) compounds. We also examined the ability of these compounds to act as structural probes for H-DNA formation within higher-order structures at pathological frataxin sequences in plasmids. DNA-complex-formation analyses with a gel-mobility-shift assay and sequence-specific probing of H-DNA-forming (GAA)n sequences by single-strand oligonucleotides and triplex-directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)n repeats under physiologically relevant conditions.

Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

“…Nevertheless, triplex formation in the presence of GAA-PNA was not detected using the BQQ-OP assay. Interestingly, we did not observe a purine motif TFO-DNA triplex nor a purine motif H-DNA in earlier studies [56]. Based on these observations and that the triplex formed by CTT-PNA binding resulted in a clear BQQ-OP cleavage, we conclude that a purine motif triplex is not formed in the presence of GAA-PNA.…”

“…Binding occurs with moderate efficiency at physiological pH. Conversely, purine motif triplex formation was not detected at this site when using the corresponding homopurine TFO [56]. Here, we aimed to test whether PNA would behave differently when targeted to FRDA expanded GAA repeats.…”

“…We recently showed that expanded (GAA) 115 repeats form a pyrimidine motif triplex in supercoiled plasmids by using the BQQ-OP assay [56]. Also, binding of a single strand CTT oligonucleotide (identical in sequence and length to the currently used CTT-DNA, Table 1) to the H-DNA forming plasmid enhanced triplex-formation significantly, whereas an analogous GAA oligonucleotide (corresponding to GAA-DNA, Table 1) had no such effect.…”

“…Additionally, we have converted BQQ to a triplex-specific cleaving agent (BQQ-OP) by conjugation to a 1,10-phenanthroline ligand [52]. In the presence of Cu 2+ and a reducing agent BQQ-OP causes dsDNA cleavage specifically at the site of formation of a triplex, and we have previously demonstrated the ability of BQQ-OP to probe triplex formation of both H-DNA and TFO-directed triplex structures in plasmids in vitro [55,56]. …”

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

“…A Triplex Stabilizing Compound BQQ Inhibits ChlR1 Unwinding-BQQ is a highly specific triplex-stabilizing compound that is able to bind both pyrimidine and purine motif triple-helix structures (44,(51)(52)(53). To confirm the stabilizing ability of BQQ on our triplex DNA structures, we used flush and plasmid triplex DNA to verify whether BQQ can prevent these DNA substrates from unwinding by ChlR1.…”