Efficient cosubstrate enzyme pairs for sequence-specific methyltransferase-directed photolabile caging of DNA

Heimes, Michael; Kolmar, Leonie; Brieke, Clara

doi:10.1039/c8cc05913f

Cited by 27 publications

(18 citation statements)

References 33 publications

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“…In a similar manner, integration of 2-nitrobenzyl-caged 5-hydroxymethyl(hm)-cytosine and 5-hm-uracil into a DNA template controlled cell-free transcription with light [ 39 ]. A photocage was also installed on a nucleobase of a DNA template by using a methyltransferase with a 2-nitrobenzyl-modified S-adenosyl methionine substrate, which was used to control cell-free expression [ 40 ]. Enzymatic insertion of photocleavable biotin-coumarin-modified nucleobases into mRNA also enabled controlled expression in mammalian cells [ 41 , 42 ].…”

Section: Light-controlled Gene Expression Using Nucleic Acidsmentioning

confidence: 99%

Controlling gene expression with light: a multidisciplinary endeavour

Hartmann

Smith

Mazzotti

et al. 2020

Biochemical Society Transactions

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The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

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Section: Light-controlled Gene Expression Using Nucleic Acidsmentioning

confidence: 99%

Controlling gene expression with light: a multidisciplinary endeavour

Hartmann

Smith

Mazzotti

et al. 2020

Biochemical Society Transactions

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“…Here we report the development of the first highly selective non-covalent binder for a popular photocaging group that can be biosynthetically incorporated into recombinant proteins in yeast ( Lemke et al., 2007 ; Rakauskaitė et al., 2015 ) and mammalian ( Nguyen et al., 2014 ; Peeler et al., 2020 ) cells or installed de novo by chemical or chemoenzymatic labeling ( Anhäuser et al., 2018 ; Heimes et al., 2018 ). This new tool effectively expands the functionality of the photocaging moiety into a useful orthogonal affinity handle.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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Summary 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.

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“…[11] ForRNA photocaging,the same concepts and limitations apply.Here,the introduction of photocaging groups has been largely limited to chemical synthesis whereas post-transcriptional enzymatic modification was dependent on tags. [3a,12] We and others recently introduced MTases as tools to photocage DNAs equence-specifically, [13] but enzymatic sequence-specific photocaging of RNAs equences-be it by protein-or nucleic acid-based enzymes-has not been reported to the best of our knowledge.…”

Section: Introductionmentioning

confidence: 99%

Tag‐Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases

2020

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The mRNA modification N6‐methyladenosine (m6A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3‐METTL14 and METTL16 act as “writers” for different target transcripts and sequence motifs. The modification is perceived by dedicated “reader” and “eraser” proteins, but not by polymerases. We report that METTL3‐14 shows remarkable cosubstrate promiscuity, enabling sequence‐specific internal labeling of RNA without additional guide RNAs. The transfer of ortho‐nitrobenzyl and 6‐nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase‐catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo‐enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo‐enzymatic modification of different sites in a single RNA.

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Efficient cosubstrate enzyme pairs for sequence-specific methyltransferase-directed photolabile caging of DNA

Abstract: Efficient and selective methyltransferase-catalyzed transfer of photolabile groups onto DNA enables photoregulation of gene expression and can be performed even in the presence of AdoMet.

Cited by 27 publications

References 33 publications

Controlling gene expression with light: a multidisciplinary endeavour

Controlling gene expression with light: a multidisciplinary endeavour

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

Tag‐Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases

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