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Updated: Jun 21, 2026

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes
Published on: July 5, 2024
Byproduct with altered fluorescent properties is formed during standard deprotection step of hexachlorofluorescein
A N Chuvilin1, M V Serebryakova, I P Smirnov
1Research Institute of Physical-Chemical Medicine, Moscow, Russian Federation. achuvilin@yandex.ru
Abstract:
HEX-labeled oligonucleotides obtained via typical synthetic protocols may contain more than 15% of material with altered spectral characteristics. We discovered hexachlorofluorescein residue transformation unknown earlier for standard DNA ammonolysis step. HEX residue reacts with ammonium hydroxide yielding acridine derivative, which has differed UV-VIS and fluorescent properties compared to HEX. Therefore, for critical bioassays where sensitivity and/or fluorescent signal differentiation (e.g., in quantitative or multiplexed assays) are essential, the careful RP-HPLC purification step is required.
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