Minimal Sequence Requirements for Oligodeoxyribonucleotides Activating Human TLR9

Pohar, Jelka; Krajnik, Alenka Kužnik; Jerala, Roman; Benčina, Mojca

doi:10.4049/jimmunol.1402755

Cited by 47 publications

(104 citation statements)

References 45 publications

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“…Our previous systematic analysis of the minimal sequence requirements for ODNs that activate hTLR9 (minH) confirmed that the 59TCG motif is essential for TLR9 activation but not sufficient (28), as indicated in the design of commonly used ODN2006 PTO (10,18,27,35,36). The ODNs with a 59TCG motif activate hTLR9 more effectively than mTLR9 as observed for the m109 and m127.…”

Section: Discussionmentioning

confidence: 92%

“…The single CG motif within the minM ODN is sufficient to effectively activate mTLR9 in comparison with the two CpGs required for hTLR9 (Fig. 5D) (28). The minimal sequence requirements for the ODN that activates hTLR9 are the stringent 59TCGT motif and an additional CG motif 6-10 nt distant from the 59CG motif.…”

Section: Discussionmentioning

confidence: 99%

“…The minimal ODNs that activate hTLR9 (e.g., minH-75) make up two stimulatory CG dinucleotides separated by 6-10 nt. The first CpG motif is preceded by the 59-thymidine (28). The sequence motifs of the minH-75 PD (h75) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PTO modifications lead to the increased unspecific binding to proteins (41,42), and an induction of the immune response independent of the CpG motif and TLR9 (43)(44)(45)(46), which obscures the motif of the physiologically relevant agonists. The minH (28) and minM ODN chimeras comprising a 39 PTO-linked poly-T tail to hinder the nucleases and PD-linked motifs (59TCGT and 59TCCT 3 CG for the minH and minM, respectively) to maintain the ODNs' species-specific specificity are as efficient stimulators of immune cells as ODNs with a PTO backbone.…”

Section: Discussionmentioning

confidence: 99%

“…Nucleotides adjacent to the CG dinucleotide are important for determining the agonists' potency and are an integral part of the CpG motif (10,28,39), probably reflecting the binding specificity of the site on the TLR9 ECD. Substitution of the thymidine adjacent to the CG with any other nucleotide reduces the ability of minM to activate TLR9.…”

Section: Discussionmentioning

confidence: 99%

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Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9

Pohar

Lainšček

Fukui

et al. 2015

The Journal of Immunology

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Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated CpG recapitulate the activation of TLR9 by microbial DNA. ODNs are potent stimulators of the immune response in cells expressing TLR9. Despite extensive use of mice as experimental animals in basic and applied immunological research, the key sequence determinants that govern the activation of mouse TLR9 by ODNs have not been well defined. We performed a systematic investigation of the sequence motif of B class phosphodiester ODNs to identify the sequence properties that govern mouse TLR9 activation. In contrast to ODNs activating human TLR9, where the minimal sequence motif for the receptor activation comprises a pair of closely positioned CpGs we found that the mouse TLR9 requires a single CpG positioned 4–6 nt from the 5′-end. Activation is augmented by a 5′TCC sequence one to three nucleotides from the CG. The distance of the CG dinucleotide of four to six nucleotides from the 5′-end and the ODN’s length fine-tunes activation of mouse macrophages. Length of the ODN <23 and >29 nt decreases activation of dendritic cells. The ODNs with minimal sequence induce Th1-type cytokine synthesis in dendritic cells and confirm the expression of cell surface markers in B cells. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9

Pohar

Lainšček

Fukui

et al. 2015

The Journal of Immunology

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Structural basis for species‐specific activation of mouse Toll‐like receptor 9

et al. 2018

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Single-stranded DNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs derived from microorganisms are recognized by Toll-like receptor (TLR) 9 and activate an innate immune response. TLR9 has two DNA-binding sites for CpG DNA and DNA containing cytosine at the second position from the 5'-end; both are required for efficient TLR9 activation in most vertebrate species. However, mouse TLR9 can be dimerized by CpG DNA only, although the underlying mechanism remains elusive. Here, we report the crystal structure of mouse TLR9 complexed with both DNAs. Although most TLR9-CpG DNA interactions are conserved among species, some are unique to mice and involved in species-specificity. These findings provide the structural basis for how mouse TLR9 dimerizes efficiently in response to CpG DNA to activate innate immunity.

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Tuning CpG motif position in nanostructured DNA for efficient immune stimulation

Tan,

Makiguchi,

Kusamori

et al. 2024

Biotechnology Journal

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It was previously demonstrated that polypod‐like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine–phosphate–guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single‐stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif‐containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2–5 CpG‐free ODNs, and investigated their immunostimulatory activity using Toll‐like receptor‐9 (TLR9)‐positive murine macrophage‐like RAW264.7 cells. Polypodnas with the CpG motif in the 5′‐overhang induced more tumor necrosis factor‐α release than those with the motif in the double‐stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single‐stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif‐containing nanostructured DNAs for immune stimulation.

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Minimal Sequence Requirements for Oligodeoxyribonucleotides Activating Human TLR9

Cited by 47 publications

References 45 publications

Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9

Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9

Structural basis for species‐specific activation of mouse Toll‐like receptor 9

Tuning CpG motif position in nanostructured DNA for efficient immune stimulation

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