Effect of polyethylene glycol as a molecular crowding agent on reducing template consumption for preparation of molecularly imprinted polymers

Man, Jia; Sun, Guang-Yin; Zhao, Yong Xin; Liu, Zhao‐Sheng; Aisa, Haji Akber

doi:10.1039/c6ay00383d

Cited by 7 publications

(4 citation statements)

References 28 publications

(34 reference statements)

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“…Molecular crowding in the intracellular environment caused by the presence of soluble and insoluble macromolecules such as proteins, nucleic acids, ribosomes and carbohydrates has been shown to impact both structure and stability of proteins in cells as well as enzyme kinetics 78 – 83 including a decrease in the activity of hepatitis C virus NS3/4A protease 84 and an increase in the proteolytic activity of SARS-CoV M pro 82 . For this, we included 25% (weight/volume; w/v) of 8000 Da polyethylene glycol (PEG 8000), which is a non-toxic, hydrophilic polyether that serves as a crowding agent and has been extensively utilized to mimic molecular crowding in vitro 85 , 86 , in the assays and monitored cleavage of the M pro sensors with 500 nM M pro under varying concentrations of GC376. Inclusion of 25% PEG 8000 resulted in a substantial increase in the rate of proteolytic cleavage of the M pro sensors in the absence of GC376 (Fig.…”

Section: Resultsmentioning

confidence: 99%

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

et al. 2022

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The main protease, Mpro, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting Mpro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with Mpro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in Mpro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

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Section: Resultsmentioning

confidence: 99%

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

et al. 2022

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“…We took advantage of the slow rate of the M pro sensor cleavage under 500 nM M pro to determine the effect of molecular crowding on the proteolytic activity of the protein.Molecular crowding in the intracellular environment caused by the presence of soluble and insoluble macromolecules such as proteins, nucleic acids, ribosomes and carbohydrates has been shown to impact both structure and stability of proteins in cells as well as enzyme kinetics 99-104 including a decrease in the activity HCV NS3/4A protease 105 and an increase in the proteolytic activity of SARS-CoV M pro103 . For this, we included 25% (weight/volume; w/v) of 20000 Da (20K) polyethylene glycol (PEG), which is a non-toxic, hydrophilic polyether that serves as a crowding agent and has been extensively utilized to simulate the molecular crowding in vitro 106, 107 , in the assays and monitored cleavage of the M pro sensors under 500 nM M pro under varying concentrations of GC376. Inclusion of 25% PEG 20K resulted in a substantial increase in the rate of proteolytic cleavage of the M pro sensors in the absence of GC376 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For this, we included 25% (weight/volume; w/v) of 20000 Da (20K) polyethylene glycol (PEG), which is a non-toxic, hydrophilic polyether that serves as a crowding agent and has been extensively utilized to simulate the molecular crowding in vitro 106,107 , in the assays and monitored cleavage of the M pro sensors under 500 nM M pro under varying concentrations of GC376. Inclusion of 25% PEG 20K resulted in a substantial increase in the rate of proteolytic cleavage of the M pro sensors in the absence of GC376 (Fig.…”

Section: In Vitro Assays Reveal Molecular Crowding-mediated Increase ...mentioning

confidence: 99%

A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

Geethakumari

Ahmed

Rasool

et al. 2022

Preprint

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The SARS-CoV-2 main protease, Mpro, is critical for its replication and is an appealing target for designing anti-SARS-CoV-2 agents. In this regard, a number of assays have been developed based on its cleavage sequence preferences to monitor its activity. These include the usage of Fluorescence Resonance Energy Transfer (FRET)-based substrates in vitro and a FlipGFP reporter, one which fluoresces after Mpro-mediated cleavage, in live cells. Here, we have engineered a pair of genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based sensors for detecting SARS-CoV-2 Mpro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the Mpro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in Mpro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based Mpro sensor. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensor developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

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“…Ovando-Medina et al [15] researched the effects of monomer addition rate and surfactant concentration on the kinetics, particle size, copolymer molecular weight, and composition in the semicontinuous heterophase copolymerization of vinyl acetate and butyl acrylate and compared against the results of the seeded semicontinuous process. Jia et al [16] investigated the effect of polyethylene glycol (PEG) as a molecular crowding agent on reducing template consumption in the preparation of molecular imprinted polymers (MIPs). The polymerization was realized in situ, used acrylamide as a functional monomer and ethylene glycol dimethacrylate as the cross-linking monomer.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and characterization of butyl acrylate-co-poly (ethylene glycol) dimethacrylate obtained by microemulsion polymerization

Alvarado

Rabelero

Aguilar

et al. 2020

Designed Monomers and Polymers

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The synthesis and characterization of copolymers of n-Butyl Acrylate (BA) and Poly(ethylene glycol) dimethacrylate (PEGDMA) were realized by microemulsion. In this synthesis, the relation of PEGDMA 10, 20, 30, 40 and 50% wt with respect to BA was changed. The copolymers obtained were characterized by the determination of conversions (gravimetry), infrared spectroscopy: Fourier transform (FTIR), dynamic light scattering (DLS), thermogravimetry (TGA) and differential scanning calorimetry (DSC). The results confirmed the synthesis of BA-co -PEGDMA copolymers by the identification of characteristic FTIR bands and which determined the glass transition temperature of the copolymers. The conversions were found in the range of 85% to 90%. Within the stability of the produced latex, it was observed that at 10% and 30% wt. of PEGDMA the systems were stable, but when more PEGDMA was added up 40% to 50% wt., the system became unstable. The stability of produced latexes depends on the PEGDMA contents and this must be less than 30% wt.; meanwhile the PEGDMA content greater than 30% wt. leads to unstable latexes, forming clots. Copolymers showed single glass transition temperatures between −53.37°C and −16.58°C, depending on the composition of PEGDMA in the copolymers. Resulting in the different arrangements of units of PEGDMA along in the chain affected the thermal properties of the final copolymers.

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Effect of polyethylene glycol as a molecular crowding agent on reducing template consumption for preparation of molecularly imprinted polymers

Abstract: The efforts in the present work focus on the topic of reducing template consumption in the preparation of molecularly imprinted polymers (MIPs).

Cited by 7 publications

References 28 publications

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

A Genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor

Synthesis and characterization of butyl acrylate-co-poly (ethylene glycol) dimethacrylate obtained by microemulsion polymerization

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