| Summary: | DNAzymes are single-stranded DNA molecules capable of catalysis, and they are the DNA counterparts of ribozymes and protein enzymes. M²⁺-independent RNA cleaving DNAzymes are intriguing due to their robust activity that is not compromised by low Mg²⁺ that is found in cells. Hence, they hold great promise for RNA regulation in vivo. To mimic the metal independent protein endonuclease RNase A, three chemically modified nucleotides dAimTP, dCaaTP, and dUgaTP, that are adorned with respective side-chain functionalities of histidine, lysine, and arginine, have been simultaneously introduced in in vitro selections by our group, and led to the development of families of M²⁺-independent DNAzymes targeting chimeric DNA/RNA substrates, attaining kobs as high as ~0.6 min-¹ at pH 7.4, 37°C. In order to further select such DNAzymes capable of highly efficient all-RNA cleavage and multiple-turnover, a novel unimolecular selection scheme containing an all-RNA substrate derived from the HIV-1 LTR-promoter allows a direct selection of all-RNA cleavers meanwhile fostering the subsequent conversion of the cis-cleaving species into a trans-acting catalyst was constructed. An optimized in vitro selection cycle combining selection, re-selection, and evolution that permitted greater sequence space sampling and pursuit of catalytically improved sequences through generation-specific mutagenesis was designed. The application of the novel construct in the optimized in vitro selection cycle gave rise to two families of desired DNAzyme candidates. Under simulated physiological conditions (pH 7.45, 150 mM K⁺, 0.5 mM Mg²⁺, 37°C), the best representative, Dz7-38-32t, attained kcat and KM values of ~0.24 min-¹ and 2.72 µM, respectively, corresponding to a catalytic efficiency of ~10⁵ M-¹min-¹. Dz7-38-32 can be spontaneously taken up by HeLa cells after 45 h incubation at 0.9 µM due to its similarity to cell penetrating peptides (CPPs) regarding the appended functionalities. To understand fundamental aspects by which these three modified bases function in in vitro selection, investigations of biophysical and enzymatic properties of them in the context of discretely modified oligonucleotides were performed. These studies identified certain shortcomings in the use of modified nucleosides while providing clear evidence of negligible mutagenicity in terms of both primer extension and enzymatic read-through. === Science, Faculty of === Chemistry, Department of === Graduate
|
|---|
