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

Abstract

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.