Synthesis and Evaluations of Cleavable Triazene‐Modified Nucleotide for DNA Sequencing

Abstract

A fluorescence‐labeled nucleotide with a cleavable triazene linker was designed and synthesized as a potential reversible terminator for DNA sequencing by synthesis (SBS). The key intermediate, a triazene‐modified nucleotide, was successfully synthesized through the triazenylation of an amino‐modified nucleotide, followed by fluorescence labeling to yield the desired product. The synthesized triazene‐modified nucleotide can effectively serve as a substrate for Klenow Fragment (exo‐) DNA polymerase and be incorporated into DNA strands. Once the first modified nucleotide is incorporated, no further extension is possible, even with an unblocked 3'‐OH group. After the fluorescent label is completely removed under acidic conditions, the triazene reversible terminator can be incorporated into DNA strands again, thereby enabling the DNA sequencing cycles.