Project Overview

G-Quadruplexes are 4-stranded secondary structure made up of Guanine rich oligonucleotides (ssDNA and RNA). These structures are highly stable at physiological conditions with potential implications in Cancer and neurological disorders. I used the Heme:G-Quadruplex intrisic DNAzyme activity to map G-quadruplexes in living cells. Here is the description:

• High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ–heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn’t extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.