Abstract
Modified nucleoside triphosphates (dA(Hs)TP, dU(POH)TP, and dC(Val)TP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible with in vitro selection methods and can be used to develop artificial peptidases based on DNA.
Highlights
The selective scission of the amide bonds of proteins and peptides is of crucial importance for numerous biochemical and biotechnological applications[1] and plays a decisive role in physiological processes.[2,3] Considering the inertness of the peptide bond (t1/2 nonenz ∼ 500 years),[4,5] proficient proteolytic enzymes with high catalytic efficiencies need to be used in order to degrade proteins or peptides into smaller fragments or for promoting site-specific cleavage
The combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts
In order to assess the fidelity of the polymerase chain reaction (PCR) amplification with the modified dN*TPs 1–3, a sequencing experiment based on the dideoxynucleoside triphosphate method developed by Sanger et al was carried out (Fig. S6 and S7, respectively, Electronic supplementary information (ESI)†).[52,89]
Summary
The selective scission of the amide bonds of proteins and peptides is of crucial importance for numerous biochemical and biotechnological applications[1] and plays a decisive role in physiological processes.[2,3] Considering the inertness of the peptide bond (t1/2 nonenz ∼ 500 years),[4,5] proficient proteolytic enzymes with high catalytic efficiencies (kcat/KM) need to be used in order to degrade proteins or peptides into smaller fragments or for promoting site-specific cleavage. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions.
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