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

Abstract

Polypurine reverse-Hoogsteen (PPRH) oligonucleotides are non-modified DNA molecules composed of two mirror-symmetrical polypurine stretches linked by a five-thymidine loop. They can fold into reverse-Hoogsteen hairpins and bind to their polypyrimidine target sequence by Watson-Crick bonds forming a three-stranded structure. They have been successfully used to knockdown gene expression and to repair single-point mutations in cells. In this work, we provide an in vitro characterization (UV and fluorescence spectroscopy, gel electrophoresis and nuclease assays) of the structure and stability of two repair-PPRH oligonucleotides and of the complexes they form with their single-stranded targets. We show that one PPRH oligonucleotide forms a hairpin, while the other folds, in potassium, into a guanine-quadruplex (G4). However, the hairpin-prone oligonucleotide does not form a triplex with its single-stranded target, while the G4-prone oligonucleotide converts from a G4 into a reverse-Hoogsteen hairpin forming a triplex with its target sequence. Our work proves, in particular, that folding of a PPRH oligonucleotide into a G4 does not necessarily impair sequence-specific DNA recognition by triplex formation. It also illustrates an original example of DNA structural conversion of a G4 into a reverse-Hoogsteen hairpin driven by triplex formation; this kind of conversion might occur at particular loci of genomic DNA.

Evidence for Reverse Hoogsteen Hairpin Intermediates in the Photocrosslinking of Human Telomeric DNA Sequences

“…B). The melting temperature of the reverse Hoogsteen hairpin conformation of Tel26 is also expected to be low in K + solution based on some recent data with A and G containing sequences . The transition temperature (reported as the inflection point in a melting curve) of a 13 bp reverse Hoogsteen hairpin containing 69% G's and 31% A's and no mismatches was 33°C in Na + , but could not be measured in K + solution due to the preferential formation of a G‐quadruplex.…”

Parallel Clamps and Polypurine Hairpins (PPRH) for Gene Silencing and Triplex‐Affinity Capture: Design, Synthesis, and Use

“…This subject was studied in detail by Solé et al. (), demonstrating that PPRHs can form triplexes with their target sequences even under conditions where they fold into G‐quadruplexes.…”

“…They also represent a tool to validate genes in proliferation and cancer (Villalobos et al., ), and can be used to target genes related to resistance (de Almagro, Coma, Noé, & Ciudad, ), as chemosensitizers (de Almagro, Mencia, Noé, & Ciudad, ), and in immunotherapy (Bener et al., ; Medina Enríquez, Félix, Ciudad, & Noé, ). Several examples of PPRHs for the inhibition of gene expression have been described (Ciudad et al., ; Félix, Ciudad, & Noé, ; Rodriguez et al., ; Rodríguez et al., ; Solé et al., ), as well as for the capture of miRNA (Ribes et al., ) and double‐stranded DNA for the analysis of DNA methylation status (Huertas et al., ).…”

“…A concern of G-rich sequences is the possibility to have competing G-quadruplex structures that may lower the efficacy of triplex formation. This subject was studied in detail by Solé et al (2017), demonstrating that PPRHs can form triplexes with their target sequences even under conditions where they fold into Gquadruplexes.…”

“…PolyPurine Reverse Hoogsteen (PPRH) technology is often used for targeting single‐ and double‐stranded nucleic acid sequences (Villalobos et al., ). Several examples of PPRHs for the inhibition of gene expression (Ciudad, Rodríguez, Villalobos, Félix, & Noé, ; Coma, Noé, Eritja, & Ciudad, ; Solé, Delagoutte, Ciudad, Noé, & Alberti, ), as well as for capture of miRNA (Ribes et al., ) and double‐stranded DNA for the analysis of DNA methylation status (Huertas et al., ), have been previously described.…”