Design and synthesis of fluorescent-dye-labeled nucleotide with a new cleavable azo linker for DNA sequencing by synthesis.

Abstract

A new cleavable azo linker was synthesized and reacted with 5-iodo-2'-deoxyuridine, followed by triphosphorylation, and finally labeled with Cy3 to give the desired product dUTP-azo linker-Cy3 as a potential reversible terminator for DNA sequencing. The synthesized 3'-OH-unblocked nucleotide triphosphate can be faithfully incorporated into the DNA strands as catalyzed by DNA polymerase (Bst 3.0) with 100% yield. Meanwhile, further incorporation is terminated temporarily by the overall steric hindrance of the nucleotide triphosphate, ensuring that only one molecule can be incorporated into the DNA strand within one sequencing cycle even for a template containing multiple identical bases in a row. The next incorporation can proceed smoothly upon complete removal of the labeled dye with only aniline left on the elongated DNA strand. The synthesized nucleotide triphosphate is the first reversible terminator that can be considered crucial for DNA sequening. These preliminary evaluations indicate that the synthesized nucleotide triphosphate holds substantial potential value in DNA sequencing.