Alteration of the CpG ODN core sequence influences its biological functions

AIM:To investigate whether alteration of the core sequence of CpG ODN change its cellular interanalization and immunological activity.METHODS:6-FAM labeled CpG ODN was artificial synthesized and the CpG dinucleotide was altered by cytosine methylation(mCpG ODN)and/or CG sequence reversal(GpC ODN).RAW264.7 cells were cultured in vitro.After stimulated with different 6-FAM labeled ODN,the accumulation of ODN in the cells were measured by flow cytometry.RAW264.7 cells grown on glass coverslips were pretreated with each ODN and subsequently the localization of ODN in cells were observed by confocal scanning microscopy.Meanwhile,the level of TNF-α in supernatant was tested by ELISA.RESULTS:The alteration of the core sequence of CpG ODN didn't influence its accumulation in RAW264.7 cells.In vitro,6-FAM labeled ODN was internalized into RAW264.7 cells and a few green fluorescent dots were widely distributed over cytoplasm.Large amount of TNF-α were released from RAW264.7 cells stimulated by CpG ODN.However,after methylation of cytosine in CpG dinucleotide,the TNF-α release was significantly decreased(P0.01).At the same time,CG sequence reversal of CpG dinucleotide completely abolished the stimulated activity of CpG ODN.CONCLUSION:It's essential for the biological function of CpG ODN with certain CG sequence.The methylation of cytosine in CpG dinucleotide doesn't influence the internalization and localization of CpG ODN in RAW264.7 cells,but partially inhibit its immunological activity.