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Strain-promoted "click" chemistry for terminal labeling of DNA.

ABSTRACT: 1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.

SUBMITTER: Marks IS

PROVIDER: S-EPMC3622500 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Strain-promoted "click" chemistry for terminal labeling of DNA.

Marks Isaac S IS Kang Jun Sung JS Jones Brady T BT Landmark Kevin J KJ Cleland Andrew J AJ Taton T Andrew TA

Bioconjugate chemistry 20110602 7

1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the ...[more]

PMID: 21539391

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Project description:In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with 18F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-containing residualizing moiety, and an 18F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb 2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and guanidine functionalities. The thus derivatized sdAbs were radiolabeled with 18F using an 18F-labeled aza-dibenzocyclooctyne derivative ([18F]F-ADIBO) via SPAAC, generating the desired conjugate ([18F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [18F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [18F]RL-II-2Rs15d and [125I]SGMIB-2Rs15d, and microPET/CT imaging of [18F]RL-II-2Rs15d and the [18F]RL-II-R3B23 control sdAb were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of 23.9?±?6.9% (n?=?8) was achieved for the SPAAC reaction between [18F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (Kd) and IRF for the binding of [18F]RL-II-2Rs15d to HER2 were 5.6?±?1.3?nM and 73.1?±?22.5% (n?=?3), respectively. The specific uptake of [18F]RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14-17% of the input dose at 1, 2, and 4?h, slightly higher than seen for co-incubated [125I]SGMIB-2Rs15d. The uptake of [18F]RL-II-2Rs15d in SKOV-3 xenografts at 1?h and 2?h p.i. were 5.54?±?0.77% ID/g and 6.42?±?1.70% ID/g, respectively, slightly higher than those for co-administered [125I]SGMIB-2Rs15d (4.80?±?0.78% ID/g and 4.78?±?1.39% ID/g). MicroPET/CT imaging with [18F]RL-II-2Rs15d at 1-3?h p.i. clearly delineated SKOV-3 tumors while no significant accumulation of activity in tumor was seen for [18F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [18F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is the first time SPAAC method has been used to label an sdAb with 18F, especially with residualizing functionality.

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Strain-promoted "click" chemistry for terminal labeling of DNA.

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Strain-promoted "click" chemistry for terminal labeling of DNA.

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