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Disruption of G-quadruplex motifs INNOVATION PROJECTS
a novel method to enhance promoter activity IIT DELHI

Nandhini Saranathan and Vivekanandan Perumal, IIT Delhi

Mammalian cell lines are the expression systems of choice to ensure appropriate post-translational modifications and
proper protein folding essential for functionality of the recombinant protein. However, the amount of DNA that can
be transfected into cells is limited (this is usually in the order of few hundreds of nanograms) and hence low yield is
a common problem associated with these expression systems. Risk of cytotoxicity associated with high DNA load and
liposomal delivery warrants optimization of reagent to DNA ratio. Vectors with the human cytomegalovirus (CMV)
immediate early promoter are most widely used for mammalian protein expression owing to high transcriptional
efficacy in a broad range of mammalian cell lines. Here, we identified G-quadruplexes, which are DNA secondary
structures in the CMV promoter. Subsequently, we also disrupt a G-quadruplex motif in the CMV promoter and
demonstrate increased gene expression. These results provide the first proof-of-concept that disruption of
G-quaduplexes in expression vectors may be used to enhance gene expression.