Short LNA-modified oligonucleotide probes as efficient disruptors of DNA G-quadruplexes

Abstract

G-quadruplexes (G4s) are well known non-canonical DNA secondary structures that can form in human cells. Most of the tools available to investigate G4-biology rely on small molecule ligands that stabilise these structures. However, the development of probes that disrupt G4s is equally important to study their biology. In this study, we investigated the disruption of G4s using Locked Nucleic Acids (LNA) as invader probes. We demonstrated that strategic positioning of LNA-modifications within short oligonucleotides (10 nts.) can significantly accelerate the rate of G4-disruption. Single-molecule experiments revealed that short LNA-probes can promote disruption of G4s with mechanical stability sufficient to stall polymerases. We corroborated this using a single-step extension assay, revealing that short LNA-probes can relieve replication dependent polymerase-stalling at G4 sites. We further demonstrated the potential of such LNA-based probes to study G4-biology in cells. By using a dual-luciferase assay, we found that short LNA probes can enhance the expression of c-KIT to levels similar to those observed when the c-KIT promoter is mutated to prevent the formation of the c-KIT1 G4. Collectively, our data suggest a potential use of rationally designed LNA-modified oligonucleotides as an accessible chemical-biology tool for disrupting individual G4s and interrogating their biological functions in cells.

Long-range DNA interactions: inter-molecular G-quadruplexes and their potential biological relevance

“…Consistently with our biophysical and biochemical data, we found that short LNA-modified probes significantly enhanced c-KIT1 geneexpression, whereas this effect was not observed when the isosequential unmodified DNA probe was used (Figure 6). 69 Taken together, our studies demonstrated that short LNA-modified oligonucleotides are viable chemical-biology tools that can individually target and disrupt specific G4s both in vitro and in cells. This opens-up new possibilities of the use of these sequence-specific probes to study the biological effect of disrupting individual G4s (rather than using unspecific small-molecules).…”

“…6). 69 Taken together, our studies demonstrated that short LNAmodified oligonucleotides are viable chemical-biology tools that can individually target and disrupt specific G4s both in vitro and in cells. This opens-up new possibilities of the use of these sequence-specific probes to study the biological effect of disrupting individual G4s (rather than using unspecific small-molecules).…”

“…6 ). 69 Intriguingly, we found that the G4-disrupting potential of the LNA-modified probes varied significantly depending on the placement of the LNA-modification in the complementary sequence. Specifically, probes with the LNAs placed complementary to the Gs (guanines) showed significantly lower G4-disrupting half-lives compared to probes where the LNA modification was in loops or randomly distributed.…”

“…Our studies revealed that the LNA-DNA complex induces an increase in gene expression without hampering biological processes such as transcription and replication, thus suggesting that this is a viable strategy to achieve G4-disruption in cells. 69…”

G4-DNA, Class Switch Recombination and Potential Additional Regulations in B-Lymphocytes

“…However, given the multiple roles of G4s in gene regulation their non-specific targeting throughout in the genome is clearly not satisfactory. The current development of new strategies able to target G4 structures within a given sequence [171,182] would certainly open new opportunities, by providing therapeutic means to target a single S region and then inhibit CSR to a single Ig gene with minimal side effects.…”