CG dinucleotides

In eukaryotes, DNA methylation is important in regulation of gene function. The predominant prodnct of methylation in the DNA of vertebrates is 5-methylcytosine. This methylated base is found largely in CG dinucleotides in palindromic sequences. These may occur in control regions upstream of transcribed DNA sequences. There is considerable evidence to strongly suggest that DNA methylation in vertebrates turns off gene expression. 

Immunotoxicity of Oligonucleotide Therapeutics The pro-inflammatory activity of the ASOs produces a constellation of effects, which includes splenomegaly, lymphoid hyperplasia, and multi-organ lymphohistiocytic cell infiltrate driven by the production of pro-inflammatory cytokines [57,63,75]. Incorporation of immunostimulatory sequences like unmethylated CG dinucleotides mimic bacterial DNA [76], Unmethylated CG dinucleotides interact with receptors of the innate immune system such as TLR9 to produce a shift toward T-helper type 1 immunity [77,78], Oligonucleotides with these motifs modulate the immune system. We have recently reported on a direct evaluation for effects on immune function. 

Methylation of cytosine to form 5-methylcytosine occurs frequently about 70% of CpG dinucleotides in the human genome are methylated. Although not inherited, interest in this 5th base has increased recently as correlations with cancer have been reported. " CpG islands are about 1000 bases in length and are often found near the 5 -end of genes. These regions consist of clusters of CG dinucleotides that are usually not methylated in normal cells. However, CpG methylation correlates with condensed chromatin structure and promoter inactivation an important example occurs in tumor-suppressor genes. 

Homology to the A phage and E. coli sequence has been found close to the proposed recombination breakpoints [132]. An unusually high frequency of the CG dinucleotides has also been correlated with the occurrence of homologous recombination [133]. The significance of these observations remains unclear. 

HPLC analysis of the products respectively exhibits one peak for the GG dinucleotides, two peaks for the CG dinucleotides, three peaks for the GC dinucleotides and one peak for the CC dinucleotides. In each case these peaks represent at least 95% of the overall product. Sephadex and gel permeation chromatographies show that the complexes formed are monomeric. In the CG cases the two complexes equilibrate after HPLC separation, as do the two complexes corresponding to peaks 1 and 3 (elution order) in the GC cases. 

EXAMPLE 7.9 What is the explanation for the low frequency of CG dinucleotides in mammalian genomes ... 

EXAMPLE 7.10 What is notable about the CG dinucleotide in eukaryotes ... 

DNA isolates from bacteria ehcit antitumor activity [33, 34], augment NK cell activity [35, 36] and induce interferon-a, interferon-p, and interferon-y production [36]. DNA from different bacterial species, but not mammalian DNA, stimulates prohferation of murine lymphoc3des [37]. Further studies showed that synthetic oligonucleotides containing certain self-complementary paUndromic sequences with CG dinucleotides, similar to those present in bacterial DNA, increase the cytolytic function of NK cells and induce secretion of IFN-y [38, 39]. Synthetic oligonucleotides containing CpG motifs were also shown to induce immune responses [40]. 

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