Highly Fluorescent 5‐(5,6‐Dimethoxybenzothiazol‐2‐yl)‐2′‐Deoxyuridine 5′‐Triphosphate as an Efficient Substrate for DNA Polymerases

Abstract

We herein describe the synthesis of fluorescent 5-(5,6-dimethoxybenzothiazol-2-yl)-2'-deoxyuridine 5'-triphosphate (d(bt)UTP) and primer extension reactions using d(bt)UTP. We also carried out primer extension reactions using the (bt)U template. B family DNA polymerases, such as KOD, Deep Vent (exo-), and 9°N(m) DNA polymerases, were effective for elongation with d(bt)UTP. Deep Vent (exo-) and KOD DNA polymerases have excellent fidelity for incorporating d(bt)UTP only at the site opposite the adenine template and only dATP when using the (bt)U template. Therefore, d(bt)UTP is an excellent fluorescent nucleotide that can be incorporated into DNA by DNA polymerases.

Inhibitory Effect of 8‐Halogenated 7‐Deaza‐2′‐deoxyguanosine Triphosphates on Human 8‐Oxo‐2′‐deoxyguanosine Triphosphatase, hMTH1, Activities

“…Next, detritylation with 3 % trichloroacetic acid in dichloromethane gave 5 – 8 in good yield. Finally, the corresponding triphosphates ( 9 – 12 ) were obtained by conventional methods (yields: 27 to 43 %) …”

Synthesis of 2′-O-Propargyl Nucleoside Triphosphates for Enzymatic Oligonucleotide Preparation and “Click” Modification of DNA with Nile Red as Fluorescent Probe

“…The fast, efficient, and reliable preparation of fluorescently labeled DNA and RNA is a topic of continuously important interest for both chemical bioanalytics and fluorescent cell imaging. − In particular, the enzymatic synthesis of fluorescent DNA pieces represents a growing research area. − The application of bioorthogonal and especially “click”-type ligations has been established over the past decade not only to introduce single labels, but also for sequential labeling of oligonucleotides. , This has been achieved primarily by making synthetically available the corresponding phosphoramidites as building blocks for automated DNA synthesis. Alternatively, providing modified nucleoside triphosphates has the advantage that the subsequent DNA polymerase-assisted preparation of oligonucleotides works under very mild conditions and allows in principle the synthesis of longer and highly modified sequences by PCR. , Corresponding 2′-deoxynucleoside triphosphates (dNTPs) that are modified at the base moiety have been tested successfully in primer extension (PEX) and PCR experiments. − Sugar-modified NTPs, especially those carrying the reactive group at the 2′-position, often fail to be accepted as substrates for DNA polymerases since they are recognized as RNA monomers. , We recently published “click”-type modifications at 2′- O -propargylated uridines that were incorporated synthetically into oligonucleotides using the corresponding phosphoramidite as a building block. − The advantage of 2′-labeling is that the thereby modified uridine in DNA is still able to recognize adenine in the counterstrand .…”