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.

Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-modified DNA

“…When E. coli SS320 cells are transformed with plasmid pEVOL-pAzF, which encodes a mutant Methanocaldococcus jannaschii aminoacyl-tRNA synthetase, the stop codon directs the incorporation of the unnatural amino acid p -azidophenylalanine ( p AzF) when it is supplied to the growth media 23 . Superinfection of SS320/pEVOL-pAzF harbouring the pFAB-Sf phagemid with Y30 helper phage and growth in media supplemented with p AzF was optimized to produce phage particles containing phagemid DNA, and displaying zero to one copy of the encoded pIII-Sf fusion, with the remainder of the pIII proteins containing the N-terminal p AzF, which was used to attach a primer/template via strain-promoted click chemistry 24 (Fig. 1a and b, see also Supplementary Fig.…”

Photocontrolled micellar aggregation of amphiphilic DNA-azobenzene conjugates

“…To react an oligonucleotide with hydrophobic molecules, which typically involves organic solvents, optimization of the reaction conditions is required, including the choice of catalyst, solvent combination, and ligand-to-copper catalyst ratios, to achieve reliable and efficient reactions [15,41]. However, copper is cytotoxic, and even residual trace amounts of copper remaining in the reaction products may severely limit the practical use of Click chemistry in a biological context [42]. Therefore, application of copper-free Click chemistry is becoming increasingly more important, especially for the functionalization of biomolecules [33,[42][43][44].…”

“…However, copper is cytotoxic, and even residual trace amounts of copper remaining in the reaction products may severely limit the practical use of Click chemistry in a biological context [42]. Therefore, application of copper-free Click chemistry is becoming increasingly more important, especially for the functionalization of biomolecules [33,[42][43][44]. Similar to copper-catalyzed Click chemistry, the copper-free Click chemistry between azide and cyclooctyne groups retains the advantages of high speed, high yield, high selectivity, and mild reaction conditions.…”

Oligonucleotide Tagging for Copper-Free Click Conjugation

“…However, the use of copper for oligonucleotide conjugation may be compromised due to potential metal-catalyzed strand degradation and/or difficulties in final purification [13][14][15]. Although new ligands reduce the chance of undesired chain cleavage during copper-catalyzed click reaction [16][17][18], strain-promoted azide-alkyne cycloaddition (SPAAC) offers the possibility of oligonucleotide conjugation in the absence of copper [19][20][21][22][23] as demonstrated for oligonucleotides labeled with plain cyclooctyne (OCT) [15] or the more reactive dibenzofused cyclooctyne DIBO [24]. Most recently, Brown et al [25,26] further extended the latter approach by ON incorporation of aminoalkyl thymidine derivatives, followed by selective N-acylation with azide or cyclooctyne after cleavage from support.…”