Selective immunocapture and light-controlled traceless release of transiently caged proteins.

The 4,5-dimethoxy-2-nitrobenzyl (DMNB) photocaging group introduced into small biomolecules, peptides, oligonucleotides, and proteins is commonly used for spatiotemporal control of chemical and biological processes. Here, we describe the use of a DMNB-selective monoclonal antibody for non-covalent capture of chemically or biosynthetically produced proteins containing surface-exposed DMNB caging groups followed by light-controlled traceless decaging and release of the bound proteins into solution for a variety of downstream applications. For complete details on the use and execution of this protocol, please refer to Rakauskaite et al. (2020).

Photocage-Selective Capture and Light-Controlled Release of Target Proteins.

Photochemical transformations enable exquisite spatiotemporal control over biochemical processes; however, methods for reliable manipulations of biomolecules tagged with biocompatible photo-sensitive reporters are lacking. Here we created a high-affinity binder specific to a photolytically removable caging group. We utilized chemical modification or genetically encoded incorporation of noncanonical amino acids to produce proteins with photocaged cysteine or selenocysteine residues, which were used for raising a high-affinity monoclonal antibody against a small photoremovable tag, 4,5-dimethoxy-2-nitrobenzyl (DMNB) group. Employing the produced photocage-selective binder, we demonstrate selective detection and immunoprecipitation of a variety of DMNB-caged target proteins in complex biological mixtures. This combined orthogonal strategy permits photocage-selective capture and light-controlled traceless release of target proteins for a myriad of applications in nanoscale assays.

Excision of the doubly methylated base N4,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins.

Cytosine (C) in DNA is often modified to 5-methylcytosine (m5C) to execute important cellular functions. Despite the significance of m5C for epigenetic regulation in mammals, damage to m5C has received little attention. For instance, almost no studies exist on erroneous methylation of m5C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m5C into N4,5-dimethylcytosine (m N4,5C) in DNA, m N4,5C is probably present in vivo We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m N4,5C residues in vitro The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m5C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m N4,5C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Biosynthetic selenoproteins with genetically-encoded photocaged selenocysteines.

Selenocysteine is a valuable component of both natural selenoproteins and designer biocatalysts; however the availability of such proteins is hampered by technical limitations. Here we report the first general strategy for the production of selenoproteins via genetically-encoded incorporation of a synthetic photocaged selenocysteine residue in yeast cells, and provide examples of light-controlled protein dimerization and targeted covalent labeling in vitro.

Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases.

S-Adenosylmethionine-dependent DNA methyltransferases (MTases) perform direct methylation of cytosine to yield 5-methylcytosine (5mC), which serves as part of the epigenetic regulation mechanism in vertebrates. Active demethylation of 5mC by TET oxygenases produces 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which were shown to be enzymatically excised and then replaced with an unmodified nucleotide. Here we find that both bacterial and mammalian C5-MTases can catalyze the direct decarboxylation of caC yielding unmodified cytosine in DNA in vitro but are inert toward fC. The observed atypical enzymatic C-C bond cleavage reaction provides a plausible precedent for a direct reversal of caC to the unmodified state in DNA and offers a unique approach for sequence-specific analysis of genomic caC.

DNA unmethylome profiling by covalent capture of CpG sites.

Dynamic patterns of cytosine-5 methylation and successive hydroxylation are part of epigenetic regulation in eukaryotes, including humans, which contributes to normal phenotypic variation and disease risk. Here we present an approach for the mapping of unmodified regions of the genome, which we call the unmethylome. Our technique is based on DNA methyltransferase-directed transfer of activated groups and covalent biotin tagging of unmodified CpG sites followed by affinity enrichment and interrogation on tiling microarrays or next generation sequencing. Control experiments and pilot studies of human genomic DNA from cultured cells and tissues demonstrate that, along with providing a unique cross-section through the chemical landscape of the epigenome, the methyltransferase-directed transfer of activated groups-based approach offers high precision and robustness as compared with existing affinity-based techniques.

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA-M.HhaI-AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA.