Immunostimulatory CpG Oligonucleotides Form Defined Three‐Dimensional Structures: Results from an NMR Study

Abstract

The DNA eicosamer 5'-TCCATGACGTTCCTGATGCT-3' is known to stimulate the innate immune system of vertebrae. The immunostimulatory activity is based on the activation of Toll-like receptor 9 (TLR9). While it is known that the CG dinucleotide of the eicosamer has to be unmethylated, the structural basis of the recognition of the DNA through the receptor remains unclear. Oligodeoxynucleotides containing the sequence of the eicosamer, or a portion thereof, ranging in length from hexamer to pentaeicosamer were studied by (1)H NMR spectroscopy. Based on two-dimensional NMR spectra, a number of resonances could be unambiguously assigned. For all oligonucleotides, structural transitions were detected upon heating, as monitored by the line width and chemical shift of low-field resonances. This includes the TC dinucleotide of the 5'-terminal portion, which does not have any clear base-pairing partners. The melting transitions, together with the NOESY cross-peaks, demonstrate that structure formation occurs well beyond the core hexamer 5'-GACGTT-3', a fact that may be important for understanding the molecular recognition by the Toll-like receptors of the innate immune system.

Structural requirements for uptake and recognition of CpG oligonucleotides

“…However, the most efficient murine CpG-ODN contain the CG dinucleotide sequence within a self-complementary motif. Moreover, recent results using NMR spectroscopy indeed confirm partial duplex formation of minimal CpG-ODN (He et al, 2007).…”

“…Furthermore, duplex recognition is also favored due to thermodynamic reasons. This hypothesis of duplex recognition, which has been substantiated by us in different ways, has enabled us to design multiple new ODN with TLR9 activity (Narayanan et al, 2003;He et al, 2007).…”

Modifications in Small Interfering RNA That Separate Immunostimulation from RNA Interference

“…The major groove is the site most frequently involved in sequence-specific recognition of DNA by proteins (31). Duplex-forming potential is known to affect RNAi and the structural characteristics of certain immunostimulatory CpG oligonucleotides (32). Thymidine and 5-methylcytidine are sterically more demanding, and 7-deazaguanosine lacks N7 as a hydrogen bond acceptor (Fig.…”

“…The following can be stated without any doubts, however: 1) compound 1 does show signs of structure formation in its UV-melting curve, 2) the low temperature transition in this curve is significantly reduced for 1dT, and 3) uridine residues are involved in forming base pairs in all sequences evaluated with structure prediction algorithms thus far. Because defined three-dimensional structures are advantageous for selective molecular recognition (32), this finding may not be accidental.…”

Newly identified CpG ODNs, M5-30 and M6-395, stimulate mouse immune cells to secrete TNF-α and enhance Th1-mediated immunity

“…Several factors affecting of the ability of CpG ODNs to function as TLR9 ligands have been reported (Yi et al, 1998;Hartmann and Krieg, 2000;Hartmann et al, 2003). For example, early studies showed that a species-specific difference in optimal CpG motif sequences with GTCGTT being optimal for human (Bauer et al, 2001) and GACGTT being optimal for mice (He et al, 2007). The immunostimulatory efficiency of CpG motifs is enhanced by the number and spacing of CpG motifs.…”