Synthesis of Fluorescence Turn‐On DNA Hybridization Probe Using the ^DEAtC 2′‐Deoxycytidine Analog

Abstract

DEAtC is a tricyclic 2’-deoxycytidine analogue that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with guanine base in a DNA–DNA duplex. The synthesis of DEAtC begins with 5-amino-2-methylbenzothiazole and provides the DEAtC nucleobase analogue over four synthetic steps. This nucleobase analogue is then silylated using BSA and conjugated to Hoffer’s chlorosugar to provide the protected DEAtC nucleoside in good yield. Following protective group removal and chromatographic isolation of the β-anomer, dimethoxytritylation and phosphoramidite synthesis offered the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEAtC amidite and a short deprotection time are used to maximize efficiency. By following the protocol described in this unit, the DEAtC fluorescent probe can be synthesized and incorporated into any desired synthetic DNA oligonucleotide.

Synthesis of 2′‐Deoxyuridine Modified with a 3,5‐Difluoro‐4‐Methoxybenzylidene Imidazolinone Derivative for Incorporation into Oligonucleotide Probes for Detection of HER2 Breast Cancer Marker

“…For automated DNA synthesis, controlled pore glass (CPG) support (200 nmol, pore size 500 Å, K&A Laborgeraete) and commercially available standard phosphoramidites (Eurogentec) should be used. The solid-phase synthesis and the purification of the modified oligonucleotides are general and are described in previously reported protocols (Kumar et al, 2017;Current Protocols articles: Sato &Matsuda, 2019 andTurner et al, 2018).…”