Polymerase-catalyzed synthesis of DNA from phosphoramidate conjugates of deoxynucleotides and amino acids

Some selected amino acids, in particular l-aspartic acid (l-Asp) and l-histidine (l-His), can function as leaving group during polymerase-catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. Although l-Asp-dAMP and l-His-dAMP bind, most probably, in a different way in the active site of the enzyme, aspartic acid and histidine can be considered as mimics of the pyrophosphate moiety of deoxyadenosine triphosphate. l-Aspartic acid is more efficient than d-aspartic acid as leaving group. Such P-N conjugates of amino acids and deoxynucleotides provide a novel experimental ground for diversifying nucleic acid metabolism in the field of synthetic biology

Iminodiacetic-phosphoramidates as metabolic prototypes for diversifying nucleic acid polymerization in vivo

Previous studies in our laboratory proved that certain functional groups are able to mimic the pyrophosphate moiety and act as leaving groups in the enzymatic polymerization of deoxyribonucleic acids by HIV-1 reverse transcriptase. When the potential leaving group possesses two carboxylic acid moieties linked to the nucleoside via a phosphoramidate bond, it is efficiently recognized by this error-prone enzyme, resulting in nucleotide incorporation into DNA. Here, we present a new efficient alternative leaving group, iminodiacetic acid, which displays enhanced kinetics and an enhanced elongation capacity compared to previous results obtained with amino acid deoxyadenosine phosphoramidates. Iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) is processed by HIV-1 RT as a substrate for single nucleotide incorporation and displays a typical Michaelis–Menten kinetic profile. This novel substrate also proved to be successful in primer strand elongation of a seven-base template overhang. Modelling of this new substrate in the active site of the enzyme revealed that the interactions formed between the triphosphate moiety, magnesium ions and enzyme's residues could be different from those of the natural triphosphate substrate and is likely to involve additional amino acid residues. Preliminary testing for a potential metabolic accessibility lets us to envision its possible use in an orthogonal system for nucleic acid synthesis that would not influence or be influenced by genetic information from the outside

Oxadiazole carboxamide nucleosides as probes for DNA polymerases

Azole carboxamide nucleosides represent a unique class of analogs that possess distinct electronic properties and structural versatility through the rotation of glycosidic and carboxamide bonds. Therefore, these nucleoside analogs can serve to probe various interactions within an active site of a DNA polymerase in order to understand the mechanics of DNA polymerase replication and fidelity. Structure-activity relationship suggested that contacts between the enzyme and the minor groove of a DNA duplex can be essential for incorporation and extension by a DNA polymerase. Electronic effects of azole nucleobases in DNA polymerase recognition were further explored with a series of oxadiazole carboxamide nucleosides. These C-nucleoside mimics have oxygen and nitrogen atoms at different positions of the heterocycle providing more tunable electrostatic contacts for DNA polymerase recognition and specificity. Conversion of oxadiazole nucleosides to triphosphates was successfully accomplished through enzymatic phosphorylation of oxadiazole diphosphates with nucleoside diphosphate kinase (NDPK). Our observations indicated that oxadiazole carboxamide dNTPs are incorporated by Taq DNA polymerase across from G and T nucleobases whereas Therminator DNA polymerase efficiently inserts these triphosphates opposite to all four natural bases. It was found that both Taq and Therminator DNA polymerases support the primer strand extension and DNA synthesis beyond the oxadiazole nucleobase. Due to incompatibility of oxadiazole carboxamide nucleosides with phosphoramidite synthesis, a method that utilized a solid support for the DNA polymerase mediated extension of an oligodeoxyribonucleotide strand with a modified nucleoside triphosphate was developed. This strategy involved grafting of derivatized latex beads with oligonucleotides and a non-covalent assembly of a primer/template duplex. The extension of the immobilized DNA duplex oxadiazole carboxamide nucleotides was successfully accomplished with several DNA polymerases