Bayne, Pauline Shaw. &lt;i&gt;A Guide to Library Research in Music&lt;/i&gt;. Reviewed by Lisa A. Lazar

Lazar, Lisa

doi:10.5860/0700405

Cited by 5 publications

(5 citation statements)

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“…Gels were imaged with a UV transilluminator GelDoc (BioRad) and gel densitometry was done using GelAnalyser. [35] Purification from excess staples was performed using PEG purification [23] , with resuspension into 1× TE buffer with varying concentrations of MgCl 2 (5 mM unless otherwise specified).…”

Section: Methodsmentioning

confidence: 99%

Passivating blunt-ended helices to control monodispersity and multi-subunit assembly of DNA origami structures

Berengut,

Berg,

Rizzuto

et al. 2023

Preprint

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DNA origami facilitates the synthesis of bespoke nanoscale structures suitable for a wide range of applications. Effective design requires prevention of uncontrolled aggregation, while still permitting directed assembly of multi-subunit superstructures. Uncontrolled aggregation can be caused by base-stacking interactions between arrays of blunt-ended helices on different structures, which are routinely passivated by incorporating disordered regions as either scaffold loops or poly-nucleotide brushes (usually poly-thymine) at the end of DNA helices. Such disordered regions are ubiquitous in DNA origami structures yet their exact design requirements in different chemical environments are ill defined. In this study, we systematically examine the use of scaffold loops and poly-nucleotide brushes for passivation and for controlling multi-subunit assembly. We assess the dependence of length and sequence for preventing aggregation amidst a titration of MgCl2concentrations and the suitability of each strategy for enabling controlled multi-subunit assembly. We then introduce a novel strategy where double-stranded DNA helices run orthogonal to arrays of blunt-ended DNA helices forming a steric shield that prevents base stacking. The results define the limitations of each method and important design considerations for achieving monodispersity. For example, poly-thymine brushes are most effective for achieving monodispersity in the broadest conditions whereas scaffold loops can facilitate directed multi-subunit assembly. Finally, orthogonal DNA helices remove the need for disordered regions altogether, prevent aggregation in a broad range of MgCl2concentrations and facilitate directed multi-subunit assembly. This study expands the design tools available and enables a more informed approach for achieving control of monodispersity and multi-subunit assembly in DNA origami structures.

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Section: Methodsmentioning

confidence: 99%

Passivating blunt-ended helices to control monodispersity and multi-subunit assembly of DNA origami structures

Berengut,

Berg,

Rizzuto

et al. 2023

Preprint

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“…The proteins of interest were detected using an enhanced chemiluminescence detection system (ECL, Amersham) on the Uvitec photodocumentation system (Alliance). The bands detected in the Western blot were quantified using GelAnalyzer 19.1 software [67]. The bands detected for anti-eIF5A and anti-hypusine were normalized in relation to the loading control bands (anti-eEF2), and the hypusination rate was calculated by dividing the values obtained for hypusinated eIF5A by the values quantified for total eIF5A.…”

Section: Methodsmentioning

confidence: 99%

An experimental target-based platform in yeast for screeningPlasmodium vivaxdeoxyhypusine synthase inhibitors

Silva,

Klippel,

Sigurdardóttir

et al. 2023

Preprint

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The enzyme deoxyhypusine synthase (DHS) catalyzes the first step in the post-translational modification of the eukaryotic translation factor 5A (eIF5A). This is the only protein known to contain the amino acid hypusine, which results from this modification. Both eIF5A and DHS are essential for cell viability in eukaryotes, and inhibiting DHS can be a promising strategy for the development of new therapeutic alternatives. The human and parasitic orthologous proteins are different enough to render selective targeting against infectious diseases; however, no DHS inhibitor selective for the parasite ortholog has previously been reported. Here, we established a yeast surrogate genetics platform to identify inhibitors of DHS fromPlasmodium vivax,one of the major causative agents of malaria. We constructed genetically modifiedSaccharomyces cerevisiaestrains expressing DHS genes fromHomo sapiens(HsDHS) orP. vivax(PvDHS) in place of the endogenous DHS gene fromS. cerevisiae. This new strain background was ∼60-fold more sensitive to an inhibitor of human DHS than the one previously used. Initially, a virtual screen using datasets from the ChEMBL-NTD database was performed. Candidate ligands were tested in growth assays using the newly generated yeast strains expressing heterologous DHS genes. Among these, two showed promise by preferentially reducing the growth of the PvDHS-expressing strain. Further, in a robotized assay, we screened 400 compounds from the Pathogen Box library using the sameS. cerevisiaestrains, and one compound preferentially reduced the growth of the PvDHS-expressing yeast strain. Western blot revealed that these compounds significantly reduced eIF5A hypusination in yeast. Our study demonstrates that this yeast-based platform is suitable for identifying and verifying candidate small molecule DHS inhibitors, selective for the parasite over the human ortholog.

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“…Unless otherwise stated, chemicals were purchased from Sigma‐Aldrich or Invitrogen. SDS PAGE densitometry analysis used GelAnalyzer and amyloid fibril dimensions were measured using ImageJ V1.51j8…”

Section: Methodsmentioning

confidence: 99%

Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold

Raynes

Domigan

Pearce

et al. 2018

Biotechnology Progress

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Tobacco etch virus (TEV) protease is widely used for the removal of poly-histidine affinity tags from proteins. In solution, it is a one-time use enzyme for tag cleavage that has low stability, and is therefore a good candidate for immobilization. Amyloid fibrils can act as a versatile nanoscaffold by providing a large surface area for biomolecule immobilization. Immobilization of TEV protease to amyloid fibrils grown from the surface of a small glass bead, using physisorption, successfully immobilized active TEV protease. The bead retained activity over several uses and successfully cleaved a poly-histidine tag from several his-tagged proteins. This is first time that TEV protease has been immobilized to insulin amyloid fibrils, or any protein based support. Such functionalized surface assembled amyloid fibrils show promise as a novel nanosupport for the creation of functional bionanomaterials, for example, active surface coatings for the production of fine chemicals, chemical detoxification, or biosensing. Insulin amyloid fibrils provide a new nanosupport for the immobilization of TEV protease, which could allow for the reuse of the enzyme, saving on production costs for recombinantly expressed poly-histidine tagged proteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.

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Bayne, Pauline Shaw. <i>A Guide to Library Research in Music</i>. Reviewed by Lisa A. Lazar

Cited by 5 publications

References 0 publications

Passivating blunt-ended helices to control monodispersity and multi-subunit assembly of DNA origami structures

Passivating blunt-ended helices to control monodispersity and multi-subunit assembly of DNA origami structures

An experimental target-based platform in yeast for screeningPlasmodium vivaxdeoxyhypusine synthase inhibitors

Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold

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