Monomeric G-Quadruplex-Based CpG Oligodeoxynucleotides as Potent Toll-Like Receptor 9 Agonists

Abstract

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine–phosphate–guanine (CpG) motifs trigger the immune response by stimulating endosomal Toll-like receptor (TLR) 9. Natural linear ODNs are susceptible to nuclease degradation, thereby limiting their clinical applications. Here, we designed monomeric G-quadruplex-based CpG ODNs (G4 CpG ODNs) containing CpG motifs in the central loop region of the G4 structure. The monomeric G4 CpG ODNs were more stable in serum than the linear ODNs. The monomeric G4 CpG ODNs containing two or three CpG motifs induced the production of immunostimulatory cytokines interleukin (IL)-6, IL-12, and interferon (IFN)-β in mouse macrophage-like RAW264 cells. We also showed that the number of CpG motifs and the number of nucleotides between the CpG motif and G-tracts define the efficacy of the G4 CpG ODNs in activating TLR9. Incubating human peripheral blood mononuclear cells with G4 CpG ODNs promoted IL-6 and IFN-γ production, confirming their stimulatory effects on human immune cells. Mice given intraperitoneal injections of G4 CpG ODNs produced higher plasma IL-6 compared with injections of linear ODNs. These findings provide further understanding of the parameters governing the immunostimulatory activity of G4 CpG ODNs, thereby providing insights into the rational design of highly potent G4 CpG ODNs for vaccine adjuvants.

Enhancement of the Immunostimulatory Effect of Phosphodiester CpG Oligodeoxynucleotides by an Antiparallel Guanine-Quadruplex Structural Scaffold

“…To measure the CD spectrum, 2 µM CpG ODNs were dispensed into a 1-cm path length quartz cuvette (model T-18-ES-10, 1 cm path length, JASCO) and analyzed using a JASCO J-1500 spectropolarimeter at a scan speed of 50 nm/min, response time of 2.0 s, bandwidth of 1.0 nm, resolution of 0.2 nm, and sensitivity of 100 millidegrees (mdeg). CD spectra were recorded as the average of five replications over the spectral range of The CD melting experiments were performed from 5 • C to 90 • C with a temperature gradient of 1 • C/min based on the measurement of absorbance at 295 nm, as previously reported [9]. The Tm value was calculated by determining the upper and lower baseline values [18].…”

“…These results indicated that the insertion of the CpG motif in the first and third loops preserved the antiparallel topology, whereas CpG ODNs with additional CpG motifs in the second loop failed to maintain the antiparallel topology. Next, we evaluated the immunostimulatory activity of G4-based CpG ODNs by examining mRNA expression in mouse macrophage-like RAW264 cells, since the transcript levels of inflammatory cytokine and type I interferon induced by CpG ODNs show the same tendency as protein secretion levels of these factors [9,19,20]. As shown in Figure 3, the position of the CpG motifs influenced cytokine production.…”

“…Furthermore, G2.0.0 showed a dosedependent immunostimulatory effect on RAW264 cells (Figure S2). Next, we evaluated the immunostimulatory activity of G4-based CpG ODNs by examining mRNA expression in mouse macrophage-like RAW264 cells, since the transcript levels of inflammatory cytokine and type I interferon induced by CpG ODNs show the same tendency as protein secretion levels of these factors [9,19,20]. As shown in Figure 3, the position of the CpG motifs influenced cytokine production.…”

“…Non-canonical guanine-quadruplex (G4) structures can increase the nuclease resistance of CpG ODNs with a PD backbone [ 9 , 10 , 11 ]. The hybrid type G4 CpG ODNs with CpG motif in the central loop showed high immunostimulatory effect with acquired high nuclease resistance and cellular uptake [ 9 ].…”

Time-resolved NMR-spectroscopic studies of conformational dynamics in DNA G-quadruplexes

“…259,315 Besides the potential role of DNA G-quadruplexes in the telomeric repeats, their function is discussed in basically all contexts that are related to genomic DNA. 247,251,259,315 This includes in particular replication [316][317][318][319] , epigenetic processes such as CpG methylation 255,[320][321][322][323] or alterations to the chromatin structure 257,258,294 , genomic instability 96,253 and transcription 324,325 . Within the following chapters, the focus will be on DNA G4s as transcription regulatory elements.…”