Site-Specific Fluorescent Labeling of DNA Using Staudinger Ligation

Abstract

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.

Selective Staudinger Modification of Proteins Containing p‐Azidophenylalanine

“…[14,15,18] Synthesis: Compound 1 was synthesized according to published procedures. [11,20] Compound 2 was prepared by the coupling reaction of 2-(diphenylphosphino)phenol (74 mg, 0.27 mmol) [21] and 5(6)-carboxyfluorescein (100 mg, 0.27 mmol) in the presence of dicyclohexylcarbodiimide (62 mg, 0.3 mmol) in anhydrous DMF (1 mL) at ambient temperature for 12 h, and purified by using preparative TLC to give a red powder (3 mg, 2 %); HRMS (ESI-TOF) for C 39 Enrichment experiment: Anti-fluorescein antibody (20 mg mL À1 , 250 mL per well) was coated in aqueous Na 2 CO 3 (0.1 M, pH 9.6) in eight wells of an immunoplate (Fisher) at 37 8C for 4 h. Wells were washed (3 , 0.9 % NaCl, 0.05 % Tween 20), blocked with BSA (0.5 %) overnight at 4 8C again, and then incubated at room temperature for 5 h with phage (100 mL) either from a ligation reaction or a control solution. After being washed, the phage was eluted from the plate with BSA-FITC conjugate (0.05 %).…”

The Staudinger Ligation—A Gift to Chemical Biology

“…[22] An oligonucleotide 45 modified at its 5' end with an azido group underwent selective reaction with the fluorescein-modified phosphane (Fam) 44 under aqueous conditions to produce the Fam-labeled oligonucleotide 46 in approximately 90 % yield (Scheme 10). The fluorescent oligonucleotide 46 was then used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which were analyzed with a fluorescence electrophoresis DNA sequencer.…”

Click chemistry with DNA

“…8 This reaction has been used extensively in various biological systems, [71][72][73] and has been employed to label DNA. 74,75 Applications of the Staudinger reaction in its ''traceless'' form are particularly interesting. 76 …”