Compartmentalized Self‐Tagging for In Vitro‐Directed Evolution of XNA Polymerases

Pinheiro, Vitor B.; Arangundy-Franklin, Sebastian; Holliger, Philipp

doi:10.1002/0471142700.nc0909s57

Cited by 22 publications

(23 citation statements)

References 16 publications

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“…These libraries were subjected to selection by compartmentalized self‐tagging (CST) for the incorporation of dxT. Two modifications of this CST protocol were made: primers were tagged by biotinylated dC incorporation, given that short and destabilizing extension was expected, and no Mn 2+ was added.…”

Section: Resultsmentioning

confidence: 99%

“…Compartmentalized self‐tagging (CST) : Selection was performed by use of CST as described by Pinheiro et al., but in the absence of misincorporation‐promoting Mn 2+ ions, and labelling by incorporation of biotinylated dC during extension. An oil phase [mineral oil with Span 80 (4.5 %), Tween 80 (0.4 %) and Triton X‐100 (0.05 %)] and aqueous phase [Thermopol buffer (New England Biolabs, 1 ×), MgSO 4 (1 m m ), biotinylated dCTP (20 μ m ), equimolar dATP/dGTP mix (200 μ m ), dxTTP (200 μ m ), primer (0.38 μ m ), bovine serum albumin (BSA, 1 mg mL −1 ), dithiothreitol (DTT, 1 m m ), glycerol (10 % v / v )] were emulsified in a 4:1 ratio by adding the aqueous phase dropwise to the oil phase while stirring with a small magnet.…”

Section: Methodsmentioning

confidence: 91%

“…9° N ‐7 DNA polymerase with mutation A485L, and made exonuclease‐deficient with mutations D141A and E143A . With the aid of compartmentalized self‐tagging (CST), targeted libraries of this thermostable, archaeal family B polymerase were selected for incorporation of one or two 2′‐deoxyxylothymidine nucleotides (dxTs), followed by a number of natural nucleotides (dNTs). We report here on the characterization of selected mutants that allowed us to pinpoint a single amino acid substitution that provided improved dxNT incorporation and translocation.…”

Section: Introductionmentioning

confidence: 99%

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A Single Amino Acid Substitution in Therminator DNA Polymerase Increases Incorporation Efficiency of Deoxyxylonucleotides

et al. 2018

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Deoxyxylonucleic acid (dxNA) is a synthetic polymer that might have potential for heredity and evolution. Because of dxNA's unusual backbone geometry, sequence information stored in it is presumed to be inaccessible to natural nucleic acids or proteins. Despite a large structural similarity with natural nucleotides, incorporation of 2'-deoxyxylonucleotides (dxNTs) through the action of polymerases is limited. We present the identification of a mutant of the DNA polymerase Therminator with increased tolerance to deoxyxylose-induced backbone distortions. Whereas the original polymerase stops after incorporation of two consecutive dxNTs, the mutant is able to catalyse the extension of incorporated dxNTs with 2'-deoxyribonucleotides (dNTs) and the incorporation of up to four dxNTs alternates with dNTs, thereby translocating a highly distorted double helix throughout the entire polymerase. A single His-to-Arg substitution very close to the catalytic site residues is held to be responsible for interaction with the primer phosphate groups and for stabilizing nucleotide sugar-induced distortions during incorporation and translocation.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

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A Single Amino Acid Substitution in Therminator DNA Polymerase Increases Incorporation Efficiency of Deoxyxylonucleotides

et al. 2018

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“…This fulfills all prerequisites for the selective enrichment of active polymerases out of a pool of non-or less active variants, and presents an alternative to polymerase selection based on compartmentalized self-replication [22] or self-tagging [23,24]. The same setup might as well be used for selection and molecular evolution of other nucleic acid-modifying enzymes, e.g.…”

Section: Discussionmentioning

confidence: 98%

A novel helper phage for HaloTag-mediated co-display of enzyme and substrate on phage

Delespaul

Peeters

Herdewijn

et al. 2015

Biochemical and Biophysical Research Communications

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Phage display is an established technique for the molecular evolution of peptides and proteins. For the selection of enzymes based on catalytic activity however, simultaneous coupling of an enzyme and its substrate to the phage surface is required. To facilitate this process of co-display, we developed a new helper phage displaying HaloTag, a modified haloalkane dehalogenase that binds specifically and covalently to functionalized haloalkane ligands. The display of functional HaloTag was demonstrated by capture on streptavidin-coated magnetic beads, after coupling a biotinylated haloalkane ligand, or after on-phage extension of a DNA oligonucleotide primer with a biotinylated nucleotide by phi29 DNA polymerase. We also achieved co-display of HaloTag and phi29 DNA polymerase, thereby opening perspectives for the molecular evolution of this enzyme (and others) towards new substrate specificities.

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“…A recent adaptation of CSR has been compartmentalized self-tagging (CST) (Pinheiro et al, 2012;Pinheiro, Arangundy-Franklin, & Holliger, 2014), that allows efficient selection of polymerases for improved polymerization of nucleic acids with alternative sugar backbones, or xenonucleic acids (XNA). In CST, the selection step is achieved by primer extension, and not a PCR step.…”

Section: Csr For Polymerase Engineeringmentioning

confidence: 99%

Compartmentalized Self‐Replication for Evolution of a DNA Polymerase

Abil

Ellington

2018

CP Chemical Biology

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Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. E. coli host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR. CSR is a powerful method for engineering of polymerases since it allows selection under a variety of conditions, including the use of non-standard substrates. In this unit, we provide a step-by-step procedure for the selection of polymerases, using as an example the selection of reverse transcriptase activity starting from a library of Thermococcus kodakaraensis (KOD) DNA polymerase variants. © 2018 by John Wiley & Sons, Inc.

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Compartmentalized Self‐Tagging for In Vitro‐Directed Evolution of XNA Polymerases

Cited by 22 publications

References 16 publications

A Single Amino Acid Substitution in Therminator DNA Polymerase Increases Incorporation Efficiency of Deoxyxylonucleotides

A Single Amino Acid Substitution in Therminator DNA Polymerase Increases Incorporation Efficiency of Deoxyxylonucleotides

A novel helper phage for HaloTag-mediated co-display of enzyme and substrate on phage

Compartmentalized Self‐Replication for Evolution of a DNA Polymerase

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