Guanine-rich nucleic acids readily adopt a quadruple helical secondary structure via formation of G-quartets stabilized by monovalent cations such as Na+ or K+. Mounting evidence from research of in vivo G-DNA structures are involved in DNA replication, repair, various genetic disorders, and cancer. When studying specific guanine-rich oligonucleotides assembled in vitro, the resulting G-DNAs aggregate into larger supramolecular polymers called G-wires. These aggregation patterns are important functional and structural information what varies for different sequences of G-DNA. In this experiment the guanine-rich oligonucleotides (GROs) sequences used were 5’-dTGGGGT-3’ (GRO 6), 5’-dTGGGGG-3’ (GRO8a), and 5’-dGGGGGT-3’ (GRO8b). The goal of this study was to test what factors can affect these patterns and the extent of structural differences in very similar GROs 8a and 8b which vary by the location of one nucleotide with GRO 6 being a reference control. Analysis of the structural G-DNA differences was performed by using polyacrylamide gel electrophoresis (PAGE). It was observed that changing the percentage of acrylamide does have an impact on mobility of the G-DNA but minimum effect on aggregation. A general trend that increasing acrylamide gel percentage increased the mobility difference between a monomer and dimer G-DNA structure. The varying gel percentages did not directly affect if the samples were monomers or dimers. In addition, a G-DNA binding dye methylene blue (MB) was tested at varying concentrations to see if it changed the aggregation patterns. It was also observed that the MB when added to the samples during assembly effects the aggregation patterns specifically to GRO 8b causing a split between monomers and dimers in the sample. The exact binding method of MB with G-DNA is still unknown but aggregation of parallel stranded GRO 8b is hypothesized to be 5’-Head to 5’-Head (HTH) stacking meaning MB could bind 5’ and compete for binding. Considering the variety of DNA functions G-DNA contributes to shows the importance of understanding the structural mechanisms and aggregation patterns.

Analysis of G-quadruplex stacking modes by gel electrophoresis

ACS Spring Meeting 2021

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