In Vitro Selection of Highly Efficient G-Quadruplex-Based DNAzymes

Because of their ability to greatly enhance the low natural peroxidase activity of hemin, G-quadruplex-based DNAzymes have been widely used as an alternative to peroxidases for many colorimetric, chemiluminescent, or visual detections of metal ions, small molecules, nucleic acids, proteins, and cancer cells. To obtain G-quadruplex-based DNAzymes with better peroxidase activity, we designed three 81-nt ssDNA libraries containing 25%, 35%, and 45% guanine bases, respectively, at the 45-nt random regions to evolve hemin-binding DNA aptamers using hemin-agarose beads by SELEX (systematic evolution of ligands by exponential enrichment). Some G-rich sequences were obtained after 6 rounds of selection and optimized for stronger binding affinity to hemin and higher peroxidase activity. Our results show that the truncated aptamer [B7]-3-0 folds into compact parallel G-quadruplex structure and exhibits the highest peroxidase activity and strong binding affinity to hemin with 29 +/- 4 nM of K-d. It was found that the core G-motifs sequences with 5'-flanking nucleotides exhibit higher peroxidase activity than those with 3'-flanking nucleotides. The numbers of S'-flanking nucleotides also influence peroxidase activity. In addition, 2'-O-methyl modification facilitates the self-assembly of parallel G-quadruplex [B7]-3-0 and significantly promotes peroxidase activity. This study identifies a G-quadruplex sequence with peroxidase-like activity higher than any other sequences reported so far, which could be potentially used to improve the analytical performance of a wide variety of peroxidase-based bioassays.