Synthesis and enzymatic incorporation of norbornene-modified nucleoside triphosphates for Diels–Alder bioconjugation

Schoch, Juliane; Jäschke, Andres

doi:10.1039/c3ra40373d

Cited by 14 publications

(9 citation statements)

References 26 publications

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“…Examples are the strain-promoted azide-alkyne cycloaddition (SPAAC) ( 6 ), also dubbed ‘copper-free click chemistry’, and Staudinger ligation ( 7 ), as well as the Diels-Alder reaction with inverse electron demand (DAR inv ) ( 8 ). All of the abovementioned chemistries have been exploited by us or others, to label RNA ( 9 – 21 ) and DNA ( 3 , 11 , 22 – 30 ). DNA labeling is required, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…to generate fluorescent, biotinylated, spin-labeled or nanoparticle-labeled probes for detection and purification ( 31 – 35 ), labeled functional DNAs such as DNAzymes ( 36 , 37 ) and is of great use in nanotechnology ( 38 ). For click-labeling of DNA, the reactive moieties are generally introduced co-synthetically either chemically or by template-dependent polymerases ( 11 , 22 , 25 – 28 ). The only available fully post-synthetic ‘click’-labeling methods for DNA use methyltransferases to decorate DNA with ‘clickable’ adenosine analogs ( 30 ).…”

Section: Introductionmentioning

confidence: 99%

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Nucleotidyl transferase assisted DNA labeling with different click chemistries

et al. 2015

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Here, we present a simple, modular and efficient strategy that allows the 3′-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3′-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nucleotidyl transferase assisted DNA labeling with different click chemistries

et al. 2015

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“…However, the KOD-XL DNA polymerase, an engineered DNA polymerase from the archaeon Thermococcus kodakarensis, successfully amplified a 429 nt long dsDNA containing around 600 norbornene moieties. 124 In this study, no fluorescent reporter was attached, although the click reaction with tetrazine-bearing fluorophores should be straightforward (see Introduction: Click chemistry).…”

Section: Direct Chemo-enzymatic Labelingmentioning

confidence: 99%

Covalent labeling of nucleic acids

2020

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“…Indeed, the rather poor functional arsenal of DNA could be diversified by the incorporation of a myriad of modifications including bile acids 9 , amino acids [10][11][12] , norbornene residues 13 , electrochemical tags 14 , organocatalytic side chains 15 , and even oligonucleotides 16 . The only restriction to this methodology is the substrate acceptance of the modified dNTPs by DNA polymerases.…”

Section: Introductionmentioning

confidence: 99%