Cloneable inorganic nanoparticles

Hendricks, Alexander R.; Guilliams, Bradley F.; Cohen, Rachel S.; Tien, Tony; McEwen, Gavin A.; Borgognoni, Kanda M.; Ackerson, Christopher J.

doi:10.1039/d3cc01319g

Cited by 6 publications

(4 citation statements)

References 61 publications

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“…We developed a cloneable inorganic nanoparticle (cNP) paradigm (Figure 1, panel C). 10 This paradigm adapts in vitro reductive iNP synthesis 11,12 to the in vivo context. With in vitro synthesis, soluble metal cations are reduced (e.g., with BH4 -) to insoluble precipitates in the presence of ligands.…”

Section: A Cloneable Se Nanoparticle (Csenp) Enables Cloneable Contra...mentioning

confidence: 99%

Cloneable Selenium Nanoparticles As Multi-modal Bio-imaging Contrast Agents

Borgognoni,

Guilliams,

Cohen

et al. 2023

Preprint

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A cloneable NanoParticle (cNP) is an inorganic nanoparticle that is synthesized by a protein. The protein determines the elemental composition, size, morphology, and other properties of the nanoparticle. Here, we describe the use of a cloneable Selenium NanoParticle (cSeNP) as a cloneable imaging contrast agent in electron microscopy, fluorescence microscopy, and X-ray computed tomography. Combined, these three imaging modalities produce images of biological length scales spanning meters to angstroms but are difficult to correlate. The cSeNP is comprised of an NADPH dependent enzyme that reduces selenodiglutathione to zerovalent Se precipitates. SeNP binding peptides fused to the enzyme ligate the Se precipitates, retain them at the enzyme, and regulate SeNP size to ~5 nm inside cells. The cSeNP-protein can be genetically fused to any protein-of-interest, creating a chimeric protein-of-interest-cSeNP. The cSeNPs endow the protein-of-interest with distinguishable contrast in X-ray and EM images, due to the contrast of SeNPs in these imaging modalities relative to biological background. The cSeNPs can react spontaneously with transition metals such as Znn+ or Cun+, forming fluorescent metal-selenides, imageable in florescence microscopy. The cSeNP, therefore, represents a cloneable imaging contrast agent that facilitates location and correlation of proteins-of-interest across all biological length scales.

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Section: A Cloneable Se Nanoparticle (Csenp) Enables Cloneable Contra...mentioning

confidence: 99%

Cloneable Selenium Nanoparticles As Multi-modal Bio-imaging Contrast Agents

Borgognoni,

Guilliams,

Cohen

et al. 2023

Preprint

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“…Our motivation for altering metal(loid) reductase substrate specificity is driven by our interest in producing cloneable nanoparticles of distinct size/shape/elemental composition. 43 A cloneable inorganic nanoparticle is synthesized in physiological conditions and has a well-defined elemental composition, size, shape, and crystal structure. Because the protein determines these nanoparticle properties, and proteins are encoded in DNA, the properties of the nanoparticle are encoded in DNA.…”

Section: ■ Introductionmentioning

confidence: 99%

Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size

Hendricks,

Cohen,

McEwen

et al. 2024

ACS Chem. Biol.

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Glutathione reductase-like metalloid reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error-prone polymerase chain reaction was used to create a library of ∼10,000 GRLMR variants. The library was expressed in BL21Escherichia coli in liquid culture with 50 mM of SeO 3 2− present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with two mutations. One of the mutations (D−E) lacks an obvious functional role, whereas the other mutation is L− H within 5 Å of the enzyme active site. This mutation places a second H residue within 5 Å of an active site dicysteine. This GRLMR variant was characterized for NADPH-dependent reduction of GS-Se-SG, GSSG, SeO 3 2− , SeO 4 2− , GS-Te-SG, and TeO 3 2− . The evolved enzyme demonstrated enhanced reduction of SeO 3 2− and gained the ability to reduce SeO 4 2−. This variant is named selenium reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory-directed evolution methods.

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Section: Introductionmentioning

confidence: 99%

Laboratory Evolution of Metalloid Reductase Substrate Specificity and Product Size

Hendricks,

Cohen,

McEwen

et al. 2023

Preprint

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: Glutathione Reductase-Like Metalloid Reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error prone polymerase chain reaction was used to create a library of ~10,000 GRLMR variants. The library was expressed in BL21 Escherichia coli in liquid culture with 50 mM of SeO32- present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with 2 mutations. One of the mutations (D to E) lacks an obvious functional role, whereas the other mutation is L to H within 5 Å of the enzyme active site. This mutation places a second H residue within 5Å of an active site dicysteine. This GRLMR variant was characterized for NADPH dependent reduction of GS-Se-SG, GSSG, SeO32-, SeO42-, GS-Te-SG and TeO32-. The evolved enzyme demonstrated enhanced reduction of SeO32- and gained ability to reduce SeO42-. This variant is named Selenium Reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory directed evolution methods.

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Cloneable inorganic nanoparticles

Abstract: When a defined protein/peptide (or combinations thereof) control and define the synthesis of an inorganic nanoparticle, the result is a cloneable NanoParticle (cNP). This is because the protein sequence/structure/function is...

Cited by 6 publications

References 61 publications

Cloneable Selenium Nanoparticles As Multi-modal Bio-imaging Contrast Agents

Cloneable Selenium Nanoparticles As Multi-modal Bio-imaging Contrast Agents

Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size

Laboratory Evolution of Metalloid Reductase Substrate Specificity and Product Size

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