Enzymatic synthesis of 8-vinyl- and 8-styryl-2'-deoxyguanosine modified DNA--novel fluorescent molecular probes.

Fluorescent analogs of the natural nucleobases are widely used as molecular probes for investigating DNA hybridization and topology. In this study the guanosine analogs 8-vinyl- and 8-styryl-2'-deoxyguanosine were synthesized and converted into the corresponding 5'-triphosphates. These C8 modified nucleotides were processed by various DNA polymerases to create fluorescent DNA. Whereas the 8-styryl modified nucleotide somewhat hampers DNA synthesis 8-vinyl-2'-deoxyguanosine is processed by DNA polymerases emphasizing the broad applicability as a molecular probe for fluorescence spectroscopy.

Replacing 32 proline residues by a noncanonical amino acid results in a highly active DNA polymerase.

Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high molecular weight without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.

Enzymatic synthesis of perfluoroalkylated DNA.

Thymidine analogues 5-trifluoromethyl-, 5-pentafluoroethyl- and 5-(heptafluoro-n-propyl)-2'-deoxyuridines were synthesised and converted into the corresponding 5'-triphosphates 1a-c. Performing DNA polymerase-catalyzed primer extension reactions these modified nucleotides were incorporated into DNA to create perfluoroalkylated nucleic acids. Although single modified nucleotides were enzymatically incorporated and further elongated quite similar to the natural TTP, the enzymatic synthesis of multi-modified nucleic acids was initial only feasible with modifications at every fourth base. Nevertheless, as the effects of the modified dUTPs on DNA polymerases varied significantly with the used enzyme, Therminator DNA polymerase was proficient in incorporating 11 adjacent 5-trifluoromethyl-2'-deoxyuridine moieties.