Byproduct with Altered Fluorescent Properties Is Formed during Standard Deprotection Step of Hexachlorofluorescein Labeled Oligonucleotides

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.

A Solution to the Common Problem of the Synthesis and Applications of Hexachlorofluorescein Labeled Oligonucleotides

“…The amounts of amplicone were equal in every analysis, judjing by electrophoresis data as so as mentioned in [21]. …”

“…We compared the optical characteristics of HEX and ACR fluorophores (Table 1) from our earlier study [21] with HEX-T 10 (HT10), its ACR-analog (AT10), and their mixtures at different molar ratios.…”

“…We were surprised that prior to the completion of our study [21], the transformation of hexachlorofluorescein label with aqueous ammonia had not been described; as such, there were no recommendations for its prevention. In one study, wherein a new peak had been observed in an HPLC profile after ammonolysis of HEX-Dabsyl-ODN, the idea arose that this peak may have appeared due to degradation of the dye [23], without much further discussion or investigation ensuing.…”

“…We discovered that during standard DNA deprotection, the HEX-residue (Fig 1, I) reacts with aqueous ammonia to give a hexachloroarylacridine (ACR) derivative (Fig 1, II), which has differed UV-VIS and fluorescence properties [21]. ACR impurities in HEX-ODNs may significantly distort optical and physicochemical characteristics of the probe and may lead to imprecise, inaccurate, or false results.…”

Nucleotides and nucleic acids; oligo- and polynucleotides

“…179,180,568 Various fluorescein derivatives have been used, including a novel fluorescein phosphoramidite that has been described, 569 and a novel dC-modified derivative 139 as well as some modifications that are formed during oligonucleotide synthesis with the fluorescein HEX. 570 Other derivatives have been used to investigate DNA hybridisation under microwave conditions, 571 to monitor i-motif structures, 572 to measure endogenous RNA in living cells, 573 and in a new method for SNP typing. 574 Other fluorophores include the fluorescent guanine analogues 3MI or 6MI, 575 Cy-dyes to monitor antisense targeting in vivo, 576 the naturally fluorescent 8-aza-dG in hybridisation studies, 256 SYBR green to monitor silver ions in oligonucleotide structures, 577 a chemiluminescent analogue derived from acridine 578 and a ruthenium complex as part of an aptamer probe.…”