Systematic and Quantitative Analysis of G-Quadruplex DNA Folding

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G-quadruplex (GQ) is a noncanonical secondary structure that forms in G-rich sequences of DNA or RNA. GQ has been implicated in gene regulation and human diseases, yet its biological role is still unclear. We sought to investigate the folding propensity of potential GQ forming sequences and measure the GQ folding strength in a systematic and quantitative manner. We prepared over 400 GQ forming sequences by modulating the loop length and sequence composition within the context of [G3 N G3 N G3 N G3] where “N” represents the loop. First, we used the induced fluorescence of N-methyl mesoporphrin IX (NMM) (1) to quantify the parallel conformation of GQ folding in both single (ss) and double stranded (ds) DNA. Second, we established DNA polymerization (DNAP) assay in which tight GQ folding inhibits DNA extension. The folding stability measured by DNAP showed a high correlation to the NMM fluorescence observed in ssDNA, suggesting that parallel conformation is likely responsible for the GQ folding strength. Third, we employed in vitro transcription assay in which mRNA synthesis is detected in real time. Taken together, we provide quantitative biochemical and biophysical platforms to profile GQ folding sequences in DNA and RNA.1. Kreig, A., Calvert, J., Sanoica, J., Cullum, E., Tipanna, R. and Myong, S. (2015) G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence. Nucleic Acids Res.