Polymerase-catalyzed Primer Extension Research Articles

Back Cover: Regulated Incorporation of Two Different Metal Ions into Programmed Sites in a Duplex by DNA Polymerase Catalyzed Primer Extension (Angew. Chem. Int. Ed. 26/2014)

Back Cover: Regulated Incorporation of Two Different Metal Ions into Programmed Sites in a Duplex by DNA Polymerase Catalyzed Primer Extension (Angew. Chem. Int. Ed. 26/2014)

GFP-like Fluorophores as DNA Labels for Studying DNA–Protein Interactions

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.

Synthesis of Hydrazone‐Modified Nucleotides and Their Polymerase Incorporation onto DNA for Redox Labeling

Abstract5‐(5‐Formylthiophen‐2‐yl)cytosine, 7‐(5‐formylthiophen‐2‐yl)‐7‐deazaadenosine 2′‐deoxyribonucleosides, and their 5′‐O‐triphosphates (dNTPs) were converted into the 2,4‐dinitrophenylhydrazone or nitrobenzofurazanyl. The hydrazone‐modified dNTPs were enzymatically incorporated into DNA by polymerase catalyzed primer extension (PEX). This direct incorporation of hydrazone‐linked dNTPs was compared to previously reported incorporation of aldehyde‐modified dNTPs followed by postsynthetic hydrazone formation on DNA to show that the direct incorporation can be used for incorporation of more hydrazone units, however, cleaner PEX products are formed by incorporation of aldehydes and subsequent reaction with hydrazines. Extensive study of electrochemical behavior of the nitroarylhydrazone‐linked nucleosides and DNA was performed confirming the potential utility of the hydrazone, nitroaryl, and benzofurazane groups for redox labeling of DNA.