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

Safitri, Fika Ayu; Tu, Anh Thi Tram; Hoshi, Kazuaki; Shobo, Miwako; Zhao, Dandan; Witarto, Arief Budi; Sumarsono, Sony Heru; Giri-Rachman, Ernawati Arifin; Tsukakoshi, Kaori; Yamazaki, Tomohiko

doi:10.3390/biom11111617

Cited by 5 publications

(6 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, we observed that apart from the hybrid, antiparallel G4 also exhibits high immunostimulatory activity, 27 indicating that G4 topology (the sequence of glycosidic bond angles within the quadruplex stem), 28 does not directly influence the TLR9 activation efficiency. The change in topology consequently results in a change in the folding manner of the loops connecting the G-quartets and charge distribution, which may be the factors directly influencing TLR9 activation.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides

Hoshi

et al. 2022

ACS Chem. Biol.

Self Cite

View full text Add to dashboard Cite

We previously reported that the formation of guanine-quadruplex (G4) structures provides phosphodiester oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine (CpG ODNs) with higher nuclease resistance and cellular uptake, thereby increasing their immunostimulation efficiency through TLR9 activation. CpG ODNs forming G4 structures (G4 CpG ODNs) are thus potential vaccine adjuvants against infectious diseases. However, the G4 structure changes topology depending on the surrounding environment. Recently, G4 ligands, which are small molecules that bind to G4 ODNs with high affinity, were reported to improve the stability of G4. In this study, we propose to increase the stability and function of G4 CpG ODNs using G4 ligands. We show the effects of two G4 ligands, named L2H2-6OTD (L2H2) and L2G2-2M2EG-6OTD (L2G2), on the topology, stability, and immunostimulatory properties of a monomeric hybrid-type G4 CpG ODN containing CpG motifs in the central loop, named GD3. We found that L2H2 helps maintain the hybrid G4 topology of GD3, whereas L2G2 induces parallel G4 formation. Both G4 ligands increase the thermodynamic and nuclease stability of GD3. However, only GD3 associated with L2H2 binds efficiently to TLR9 and evokes a higher immune response from mouse macrophage-like RAW264 cells. GD3 associated with L2G2 does not bind efficiently to TLR9 and elicits lower cytokine production. Our results demonstrate that the potential to enhance immunostimulatory properties depends on the ability of G4 ligands to maintain and stabilize the hybrid G4 of GD3. We anticipate that our findings will facilitate the development of more effective G4 CpG ODN-based vaccine adjuvants against infectious diseases.

show abstract

Section: ■ Results and Discussionmentioning

confidence: 99%

Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides

Hoshi

et al. 2022

ACS Chem. Biol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Although G4-CpG ODNs are unmodified ODNs, they acquire resistance to nucleases because of their compact structure. As a result, compared to linear-CpG ODNs, G4-CpG ODNs showed excellent immunostimulatory activity in immune cells and mice [ 23 , 24 , 25 , 26 ]. The electrostatic interaction between the positively charged amino group on ε-PLL and the negatively charged phosphate group on G4-CpG ODN caused ODN condensation into the complexes.…”

Section: Resultsmentioning

confidence: 99%

“…In our previous studies, we developed a structured CpG ODN named the guanine-quadruplex (G4)-based CpG ODN [ 23 , 24 , 25 , 26 ]. The presence of guanine-rich regions in G4-CpG ODNs allows linear CpG ODNs to easily form G4 structures in the presence of potassium and sodium ions, resulting in high nuclease resistance.…”

Section: Introductionmentioning

confidence: 99%

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-L-Lysine Induce Antibody Production as Vaccine Adjuvants

Zhao

Shobo

et al. 2022

Biomolecules

Self Cite

View full text Add to dashboard Cite

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-β. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.

show abstract

“…Previously, we reported that inserting CpG motifs into different loops of RE31, a sequence forming antiparallel G4 structure, leads to different immunostimulatory activities. Inserting the CpG motif into the first or third loop of RE31 preserves the anti-parallel structure of the wild-type, but the RE31 variant with CpG motifs in the third loop exhibits a significantly lower cytokine induction than the one with CpG motifs in the first loop 14 . The CpG motif located in the third loop of RE31 interacts with poly-guanine near the 3'-terminal and consequently forms a weaker interaction with TLR9 14 .…”

Section: Discussionmentioning

confidence: 97%

Influence of loop permutation on immunostimulatory activities of CpG oligodeoxynucleotides forming monomeric guanine-quadruplex structures

Hoshi

Yamazaki

2022

Biomed. Res. Ther.

View full text Add to dashboard Cite

Introduction: Existing research establishes the role of oligodeoxynucleotides (ODNs) containing cytosine-phosphate-guanine motifs (CpG ODNs) as adjuvants to vaccines against infectious diseases. However, the natural structure of ODN with phosphodiester (PD) linkages is prone to degradation by serum DNase, which limits the application of CpG ODNs. We recently engineered a monomeric guanine-quadruplex (G4)-structured CpG ODN (G4 CpG ODN) and determined that the monomeric G4 increased the DNase resistance, intracellular uptake, and cytokine induction of CpG ODN in vitro and in vivo. However, the effect of the primary sequence on G4 conformation and function of G4 CpG ODN is unclear. Methods: Therefore, in this study, we examine the role of loop permutation, a sequential order of three G4 loops, in G4 folding and immunostimulatory properties of G4 CpG ODNs. We explore the impact of loop permutation on the immunostimulatory activity of GD3_2L, a monomeric G4 CpG ODN that we reported previously. We exchanged the sequence of the second loop with the first and third loops of GD3_2L to obtain GD3_1L and GD3_3L, respectively. The structure of the obtained ODNs was determined by circular dichroism and polyacrylamide gel electrophoresis; the thermodynamic stability of ODN was evaluated through UV melting curve analysis. We assessed the immunostimulatory activity by examining the relative messenger RNA (mRNA) levels of cytokines produced in G4 CpG ODN-stimulated mouse macrophage-like RAW264 by reverse transcription-quantitative polymerase chain reaction method. Results: We found that changing the sequential order of CpG motifs from the second to the first loop resulted in a change in G4 conformation from hybrid to parallel and a decrease in the compactness and thermodynamic stability of GD3_2L. Moreover, swapping order between the second and third loop of GD3_2L led to a hybrid G4 structure that is as stable as GD3_2L but possesses a lower cytokine induction. Conclusion: In conclusion, the loop permutation does not always change the topology and structural compactness; instead, it changes the conformation of CpG motifs in the G4 structure, directly affecting the immunostimulatory properties. Our result demonstrates that the loop-transposition-mediated structural compactness decrease can, if exploited, increase the efficacy of immunostimulatory G4 CpG ODN.

show abstract

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

Cited by 5 publications

References 27 publications

Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides

Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-L-Lysine Induce Antibody Production as Vaccine Adjuvants

Influence of loop permutation on immunostimulatory activities of CpG oligodeoxynucleotides forming monomeric guanine-quadruplex structures

Contact Info

Product

Resources

About