Genetically Encodable Design of Ligand “Bundling” on the Surface of Nanoparticles

Lu, Yao; Wang, Jianpeng; Wang, Jianhao; Wang, Lin; Au, Shannon Wing-Ngor; Xia, Junfeng

doi:10.1021/la302902m

Cited by 25 publications

(35 citation statements)

References 29 publications

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“…His‐tagged peptide has been applied to compete the His‐tagged peptide on QDs surface effectively . However, His‐tagged protein was hard to displace the surface bound His‐tagged peptide due to its large molecule . Using CE‐FL, we examined the changes of FRET signals in response to His‐PSP‐mediated cleavage of ATTO‐LEVH6‐QD substrate.…”

Section: Resultsmentioning

confidence: 99%

Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots‐peptide assembly

et al. 2017

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Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases.

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Section: Resultsmentioning

confidence: 99%

Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots‐peptide assembly

et al. 2017

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“…37 Moreover, their robust nature has been repeatedly confirmed in numerous cellular and enzymatic assays. 8,42,[44][45][46][47][48][49][50] To facilitate this, the luciferase is expressed with a terminal hexahistidine (His 6 ) as are the peptide sequences. Peptides were terminally labeled with Alexa Fluor 647 (AF647), Cy5.5 and Alexa Fluor 700 (AF700) and prepared as described.…”

Section: Resultsmentioning

confidence: 99%

An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots

et al. 2015

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The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of the energy transfer process(es) along with potential applications of such devices are finally discussed.

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“…Im can compete with histidine for binding sites, so high concentration of Im has been used to elute HAT‐tagged proteins from the nickel‐nitrilotriacetic acid (Ni‐NTA) resin. Displacing surface‐bound His‐tag by Im often provided a measure of QDs–ligand‐binding affinity .…”

Section: Resultsmentioning

confidence: 99%

In‐capillary probing QDs and HAT tag self‐assembly and displacement using Förster resonance energy transfer

Wang

Zhang

et al. 2015

Electrophoresis

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In-capillary probing QDs and HAT tag self-assembly and displacement using Förster resonance energy transfer HAT tag, a natural His affinity tag, is one of the most popular fusion tags. However, HAT tag containing three positive charges limited its self-assembly with quantum dots (QDs). Herein, ATTO 590-labeled HAT peptide was synthesized to self-assemble with QDs inside the capillary. QDs and ATTO 590-labeled HAT peptide were sequentially injected into the capillary and probed by fluorescence-coupled CE (CE-FL), showing an obvious Förster resonance energy transfer signal. Online self-assembly, separation, and detection were achieved within 10 min. CE-FL further revealed that imidazole and H 6 G 6 peptide could partially outcompete with HAT tag on the QDs surface inside the capillary. The displacement intermediates were separated clearly using CE-FL. Our study demonstrates the power of online CE-FL in analyzing the binding interaction between ligands containing positive charges and QDs.

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Genetically Encodable Design of Ligand “Bundling” on the Surface of Nanoparticles

Cited by 25 publications

References 29 publications

Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots‐peptide assembly

Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots‐peptide assembly

An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots

In‐capillary probing QDs and HAT tag self‐assembly and displacement using Förster resonance energy transfer

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