Clustered CpG Sequences to Enhance Cytokine Secretion from Macrophages

Abstract

CG sequences (CpG ODNs) can activate innate immune and thus be potentially useful for immune stimulants in therapeutic use. By hybridizing a CpG ODN having a (dA)40 tail with poly(dT), we prepared a DNA duplex that multiple CpG ODNs are hanging from the duplex. IL-12 cytokine secretion reached the maximum at the composition that two to four CpG ODNs in one duplex. These phenomena can be rationalized by an allosteric or cluster effect, and the present clustered CpG system is the simplest and most elucidating tool to investigate the cluster effect of CpG ODNs.

Self-Assembled Multivalent DNA Nanostructures for Noninvasive Intracellular Delivery of Immunostimulatory CpG Oligonucleotides

“…Besides cellular uptake efficiency and stability, several spatial factors might also contribute to the stimulatory activities of CpG-bearing nanostructures, which include the accessibility of CpGs’ 5′-end, , the self-stabilized secondary structure, − and polyvalence of CpG motifs. ,, The tetra-CpG(IV) satisfies many of these requirements and shows excellent immunostimulatory activity. Compared with previously reported Y-shaped or dendrimer-shaped DNA structures, tetrahedral nanostructures not only are convenient to prepare with high yield but also provide a platform with small and compact structures that are easily cell-permeable. − Moreover, since DNA nanostructures are of uniform sizes and precise structures, it is possible to accurately place CpG motifs to any specific positions of the tetrahedron with predefined numbers and sequence design.…”

Self-Assembled DNA Dendrimer Nanoparticle for Efficient Delivery of Immunostimulatory CpG Motifs

“…Multiple CpG motifs on one unit have been reported to be effective in promoting the immunostimulatory activity of CpG ODN, and this can be rationalized by the cluster effect of CpG on the interaction with TLR9. 28 CpG loaded highly compact DNA nanostructures without open ends exhibited remarkable stability in serum, enhanced cellular uptake efficacy and strong TLR9-related immune stimulating activity. Further modifications can be implemented on these DNA dendrimer nanocarriers to allow precise control by loading amount of immune stimulatory molecules to realized adjustable immune responses.…”

“…Moreover, there were 48 CpG motifs in the DNA dendrimer with three neighboring CpG sequences in each loop structure. Multiple CpG motifs on one unit have been reported to be effective in promoting the immunostimulatory activity of CpG ODN, and this can be rationalized by the cluster effect of CpG on the interaction with TLR9 . CpG loaded highly compact DNA nanostructures without open ends exhibited remarkable stability in serum, enhanced cellular uptake efficacy and strong TLR9-related immune stimulating activity.…”

Enhanced Cytokine Secretion Owing to Multiple CpG Side Chains of DNA Duplex

“…Our latest letter showed that the enhanced cytokine secretion may be ascribed not only to the concentration effect but also to other structural factor of CpG ODN (Minari et al, 2008). The aim of the present paper is to explore this structural effect.…”

“…The sequences for CpG ODN and its negative control were 5′-TCC ATG ACG TTC CTG ATG-A 40 -3′ and 5′-TCC ATG AGC TTC CTG ATG-A 40 -3′ (containing GpC instead of CpG), and denoted by CpGA and non-CpGA, respectively (Mizu et al, 2004c;Anada et al, 2006;Ikeda et al, 2007;Shimada et al, 2007;Minari et al, 2008). Both sequences had poly(dA) 40 tail attached at the 3′ end in order to hybridize with poly(dT).…”