Abstract
Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selectively write, remove, and rewrite chemical functionality to a biomolecule, DNA in this case. Our method combines the precision and robustness of methyltransferase-directed labeling with the reversibility of acyl hydrazones and the efficiency of click chemistry. Underpinning the method is a new S-adenosyl-l-methionine derivative to site-selectively label DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide and can be removed (i.e., untagged) when needed at the acyl hydrazone via exchange with hydroxyl amine. The formed hydrazide-labeled DNA is a versatile intermediate that can be either rewritten to reset the original chemical handle or covalently reacted with a permanent tag. This ability to write, tag, untag, and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.
Highlights
Established and emerging approaches for studying biomolecules rely on their conjugation with chemical groups or functional tags (Figure 1A).[1−4] The introduced functionalities enable manipulations that go from simple extraction and purification from complex mixtures to advanced analytical studies
We have developed a new class of AdoMet derivatives, containing an acyl hydrazone or oxime linker and a terminal azide at the sulfonium center
To demonstrate that release was the result of the cleavage of the acyl hydrazone linker and not the result of nonspecific interactions of the excess of H2NOH·HCl, a sample of DNA was modified using M.MpeI and azide-containing AdoMet derivative 8
Summary
Established and emerging approaches for studying biomolecules rely on their conjugation with chemical groups or functional tags (Figure 1A).[1−4] The introduced functionalities enable manipulations that go from simple extraction and purification from complex mixtures to advanced analytical studies. To demonstrate that release was the result of the cleavage of the acyl hydrazone linker and not the result of nonspecific interactions of the excess of H2NOH·HCl, a sample of DNA was modified using M.MpeI and azide-containing AdoMet derivative 8 This cofactor lacks the required chemistry for reversible capture of the DNA (Figure 6B) but reacts in a similar fashion to 4a in the capture of MTase-labeled DNA (Figure 6A). These results demonstrate the potential of our approach, and of AdoMet derivative 4a to underpin the development of a mild and selective method to sort labeled DNA with high recovery efficiencies and functionality
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