DNA
polymerases can process a wide variety of structurally diverse
nucleotide substrates, but the molecular basis by which the analogs
are processed is not completely understood. Here, we demonstrate the
utility of environment-sensitive heterocycle-modified fluorescent
nucleotide substrates in probing the incorporation mechanism of DNA
polymerases in real time and at the atomic level. The nucleotide analogs
containing a selenophene, benzofuran, or benzothiophene moiety at
the C5 position of 2′-deoxyuridine are incorporated into oligonucleotides
(ONs) with varying efficiency, which depends on the size of the heterocycle
modification and the DNA polymerase sequence family used. KlenTaq
(A family DNA polymerase) is sensitive to the size of the modification
as it incorporates only one heterobicycle-modified nucleotide into
the growing polymer, whereas it efficiently incorporates the selenophene-modified
nucleotide analog at multiple positions. Notably, in the single nucleotide
incorporation assay, irrespective of the heterocycle size, it exclusively
adds a single nucleotide at the 3′-end of a primer, which enabled
devising a simple two-step site-specific ON labeling technique. KOD
and Vent(exo-) DNA polymerases, belonging to the B family, tolerate
all the three modified nucleotides and produce ONs with multiple labels.
Importantly, the benzofuran-modified nucleotide (BFdUTP) serves as
an excellent reporter by providing real-time fluorescence readouts
to monitor enzyme activity and estimate the binding events in the
catalytic cycle. Further, a direct comparison of the incorporation
profiles, fluorescence data, and crystal structure of a ternary complex
of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides
a detailed understanding of the mechanism of incorporation of heterocycle-modified
nucleotides.
Synthetic
anion transmembrane transporters are adding new aspirations
for treating channelopathies by replacing defective ion channels.
The availability of such suitable candidates is still infrequent due
to the associated toxicity. Here, we report 3-(1H-1,2,3-triazol-1-yl)benzamides as transmembrane anion carriers, nontoxic
to cells. The selective and electrogenic chloride transport activity
was established by fluorescence and ion selective electrode-based
assays. MQAE assay confirmed the chloride uptake into the cells by
the nontoxic compounds.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.