Development of benzoquinoquinoxaline derivatives as triplex-specific probes : Recognition of DNA structures at repeats sequences

• Repeat sequences are associated with several human diseases, such as Friedreich’s ataxia, polycystic kidney disease and cancer. These sequences can form non-B-DNA structures, including triplex (H-DNA) DNA, and are associated with genomic instability and altered gene expression. The occurrence of triplex structures in vivo and identification of their links to biological processes have been challenging. The lack of effective probes has restrained the study of triplex structures in living cells. Here, the triplex binding small molecule benzoquinoquinoxaline (BQQ) and its derivatives were developed as tools to study triplex formation at genomic repeat sequences. The triplex binding efficiency towards both purine and pyrimidine triplex motifs was determined for BQQ, the DNA cleaving BQQ-1,10-(ortho)-phenanthroline (BQQ-OP) and the fluorescent BQQ-Bodipy compounds. BQQ was shown to have the most stabilising effect on both triplex motifs. Moreover, H-DNA structure formation at a pkd1 derived sequence was demonstrated for the first time by BQQ-OP at physiologically relevant conditions. H-DNA formation was also shown at (GAA)n repeats associated with Friedreich’s ataxia and the structure was further analysed on one nucleotide resolution, confirming that (GAA)n repeats form a pyrimidine H-DNA. However, a mixture of different isomers formed at longer (GAA)n repeats. To this end, the interaction between the peptide nucleic acids (PNA) and BQQ was investigated. PNA is a DNA mimic that binds sequence-specifically to dsDNA and can form several PNA-DNA complexes. The results of PNA binding to frataxin (GAA)n expansion in plasmid were evaluated, and in the presence of GAA-PNA no triplex structure could be detected by BQQ-OP cleavage. When the structure formed in the presence of either GAA-PNA or CTT-PNA was further analysed, it was found that GAA-PNA formed a duplex invasion complex preventing H-DNA formation, whereas CTT-PNA formed a triplex invasion complex.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Triplex

H-DNA

BQQ

BQQ-OP

BQQ-Bodipy

triplet repeat

DNA

non-B-DNA

pkd1

frataxin

Friedreich's ataxia

Molecular biology

Molekylärbiologi

Molecular Biology

molekylärbiologi

Publikations- och innehållstyp

vet (ämneskategori)

dok (ämneskategori)