Esterase-triggered fluorescence of fluorogenic oligonucleotides.
In the prooligonucleotide approach, a step of activation by cellular esterases is necessary for the removal of internucleoside phosphate masking groups and subsequent intracellular delivery of active antisense oligonucleotides. The efficacy of this approach implies that prooligonucleotides, once they are taken up by cells, are demasked by esterases during their course to their nucleic acid targets. In this regard, a method for labeling oligomers with esterase-activable fluorogenic tag was designed. The two phenolic functions of carboxyfluorescein were protected by pivaloyl groups, yielding a nonfluorescent lactone which was further activated as a N-hydroxysuccinimide ester. Two nuclease-resistant phosphorothioate 18-mer and methylphosphonate 19-mer oligodeoxynucleosides were attached to this biprotected fluorescein derivative via an amino linker at the 5'-end of the oligomers. The two conjugates were assayed for their carboxyesterase substrate ability in different biological media. In the presence of purified esterases or when incubated in serum or cell extracts, both oligonucleotide conjugates became fluorescent. In addition, the phosphorothioate oligoconjugate was microinjected into the cytoplasm of human fibroblasts, and a fast cytoplasmic release of fluorescence was observed with a rapid translocation of the fluorescent oligomer into the nucleus.
Laurent
A
A
Laboratoire de Chimie Bio-organique, UMR 5625 CNRS-UM II, Université Montpellier II, France.
Debart
F
F
Lamb
N
N
Rayner
B
B
D000641:Ammonia / Q000494:pharmacology; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D005347:Fibroblasts / Q000378:metabolism; D005452:Fluoresceins / Q000138:chemical synthesis / Q000378:metabolism* / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D008099:Liver / Q000201:enzymology; D008845:Microinjections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism* / Q000493:pharmacokinetics; D011355:Prodrugs / Q000138:chemical synthesis / Q000378:metabolism* / Q000493:pharmacokinetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D003470:Culture Media; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D011355:Prodrugs; C024098:6-carboxyfluorescein; D000641:Ammonia; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase