DNA-enhanced CuAAC ligand enables live-cell detection of intracellular biomolecules

Background of the preprint

The copper-catalysed azide-alkyne cycloaddition (CuAAC) is a common strategy to combine an azide-bearing and an alkyne-bearing component. Importantly, this powerful method has a rapid rate of reaction, tolerates aqueous conditions, and is usually free of by-products—making it a bioorthogonal “click” strategy. Despite these advantages, CuAAC has not often been used for intracellular labelling. This is primarily due to copper toxicity, which is responsible for catalysing the generation of reactive oxygen species that are detrimental to the cells’ viability. In this preprint, Nian and colleagues develop a DNA oligomer-conjugated CuAAC accelerating ligand (BTT_(1,2)-DNA) and describe how it overcomes several limitations (Figure 1).

Figure 1. Advantages of the authors’ BTT(1,2)-DNA over current methods.

 

Key findings of this preprint

Nian and colleagues first synthesised the BTT_(1,2)‑DNA ligand, a mixture of DNA-bearing triazole products derived from precursors bearing either one or two azides. Notably, these products were complexed with Cu such that the ratio of Cu:BTT_(1,2)‑DNA ligand was 7.5:1 even after dialysis, which is supposed to remove Cu from the ligand. Subsequently, the authors compared the kinetics of the BTT_(1,2)‑DNA ligand in CuAAC to that of the non‑DNA bearing BTTAA (complexed with Cu so that the ratio of Cu:BTTAA was 1:2). They showed that BTT_(1,2)‑DNA ligand consistently outperformed BTTAA in accelerating the CuAAC reaction for various dyes. Impressively, the Cu-complexed BTT_(1,2)‑DNA ligand was able to catalyse the CuAAC at nanomolar ligand concentrations in the absence of free Cu(I).

Next, Nian and colleagues aimed to improve the reaction kinetics by increasing the local concentration of the reaction components. To do so, the authors used DNA splints of various lengths that were complementary to both the target 5’ alkyne DNA and the 3’ BTT_(1,2)‑DNA ligand to bring these reaction components into closer proximity to one another. They found that longer splints with more binding sites indeed accelerated the rate of the reaction more.

Finally, the authors demonstrated that their technique could be applied intracellularly. For these experiments, the authors used 5-ethynyl uridine, an alkyne-derivatised uridine analogue that can be metabolically incorporated into RNAs in live cells. In fixed cells, the BTT_(1,2)‑DNA ligand improved the fluorescent signal over 3-fold compared to BTTAA. In live HeLa cells, the BTT_(1,2)‑DNA ligand was effective in labelling nascent RNAs at concentrations as low as 5 μM. Apart from RNA, the authors also showed that the BTT_(1,2)‑DNA ligand was effective in labelling alkyne-bearing cell surface sialic acids. Importantly, because copper-induced cellular toxicity is an important downside in CuAAC, the authors also showed that the BTT_(1,2)‑DNA ligand exhibited reduced toxicity in live cells when compared to the commercial BTTAA ligand at the concentrations used in their experiments.

 

 

What I like about this preprint

This preprint by Nian and colleagues improves the traditional CuAAC reaction in several ways. First, the new ligand has lower copper-induced cellular toxicity, a common problem associated with CuAAC—this was likely made possible by eliminating the need for free Cu. Doing so has allowed the authors to track the incorporation of a target nucleotide in nascent RNA. Second, this strategy also exhibits improved properties, such as signal intensity and rate, compared to BTTAA when used on dyes.

 

Future directions

What I find most interesting is the authors’ strategy of using the DNA splint to enhance the CuAAC reaction. Potentially, this would be a powerful method to selectively target certain sequences over others. For instance, to target a particular sequence in cells, it may be possible to use a bespoke DNA splint to bring this sequence into closer proximity to the 3’ BTT_(1,2)‑DNA ligand than other sequences. Such a technique would be particularly useful for understanding how DNA is replicated and transcribed into RNA, benefitting fields like genetics and transcriptomics (and likely helping us to understand the epitranscriptome as well).

 

Questions for the authors

1. I find it interesting that the Cu was not dialysed out of the product mixture when purifying the BTT_(1,2)‑DNA ligand. Do you think this is representative of the binding strength of the Cu‑BTT_(1,2)‑DNA ligand complex? Given that this would also imply a reduced free Cu concentration in aqueous solution, might this also be the reason why your ligand is less toxic than BTTAA?
2. Interestingly, the BTT_(1,2)‑DNA ligand displays superior labelling properties (e.g. kinetics) over BTTAA, even in the absence of the DNA splint. Why might this be the case—which parts of the structure do you think have enhanced its properties over traditional smaller molecule ligands?

 

Acknowledgements

Images created using Microsoft Powerpoint and BioRender.

Tags: bioorthogonal chemistry, copper-catalysed azide-alkyne cycloaddition, dna functionalisation

doi: https://doi.org/10.1242/prelights.33434

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