Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Abstract

Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.

Polypurine reverse-Hoogsteen (PPRH) oligonucleotides can form triplexes with their target sequences even under conditions where they fold into G-quadruplexes

“…We next investigated whether HpE6 and HpE2 motifs, alone and with their rep extensions, formed triplexes with their single-stranded polypyrimidine target sequences under salt conditions mimicking the intracellular environment, where HpE6rep6 and HpE2rep2 oligonucleotides were addressed in previous gene correction experiments2324, i . e .…”

“…Recently, we were able to correct single-point mutations in mammalian cell lines using two PPRH oligonucleotides, each bearing a sequence identical (except for the corrected single-point mutation) to the genomic sequence to be repaired2324. In this work, we investigated the structure and the stability of these two repair-PPRH oligonucleotides and of the complexes they form with their single-stranded target sequences.…”

“…Recently, our group succeeded in repairing single point mutations in cells using synthetic oligonucleotides that we named Polypurine reverse-Hoogsteen (PPRH) oligonucleotides222324. PPRH oligonucleotides are non-modified DNA molecules composed of two mirror-symmetrical polypurine stretches separated by five thymines.…”

“…PPRH oligonucleotides knock down the expression of target genes with several advantages, such as stability, low immunogenicity and cost, compared to other approaches3132. PPRH oligonucleotides have been used for gene silencing29303334 and, recently, for gene targeting to repair single-point mutations2324. Repair-PPRH oligonucleotides are formed by a PPRH motif bearing an extended DNA sequence homologous to the sequence to be repaired but containing the correct nucleotide (Fig.…”

“…In a previous work, we designed and used two repair-PPRH oligonucleotides, named HpE6rep6 and HpE2rep2 , to correct single-point mutations in the dihydrofolate reductase ( dhfr ) gene in mammalian cell lines (in exons 6 and 2, respectively)23. Each of the two repair-PPRH oligonucleotides, HpE6rep6 and HpE2rep2 , is composed of a PPRH motif ( HpE6 and HpE2 , respectively) designed to target a polypyrimidine/polypurine tract next to the dhfr gene sequence to be repaired, and of a 25 nucleotide single-stranded extension ( rep6 and rep2 , respectively) homologous to the sequence to be repaired (Table 1).…”