Directed In Vitro Evolution of Reporter Genes Based on Semi-Rational Design and High-Throughput Screening

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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Faculty Opinions recommendation of High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029996.351440 ◽

2005 ◽

Author(s):

Burckhard Seelig

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

In Vitro Evolution ◽

Double Emulsions ◽

Beta Galactosidase

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High-Throughput Screening of Enzyme Libraries: In Vitro Evolution of a β-Galactosidase by Fluorescence-Activated Sorting of Double Emulsions

Chemistry & Biology ◽

10.1016/j.chembiol.2005.09.016 ◽

2005 ◽

Vol 12 (12) ◽

pp. 1291-1300 ◽

Cited By ~ 147

Author(s):

Enrico Mastrobattista ◽

Valerie Taly ◽

Estelle Chanudet ◽

Patrick Treacy ◽

Bernard T. Kelly ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

In Vitro Evolution ◽

Double Emulsions

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Rational design of novel fluorescent enzyme biosensors for direct detection of strigolactones

10.1101/2020.03.10.986562 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rebecca J Chesterfield ◽

Jason H Whitfield ◽

Benjamin Pouvreau ◽

Da Cao ◽

Christine A Beveridge ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Rational Design ◽

Direct Detection ◽

Specific Inhibitor ◽

Striga Hermonthica ◽

Signalling Molecules ◽

Domain Insertion

AbstractStrigolactones are plant hormones and rhizosphere signalling molecules with key roles in plant development, mycorrhizal fungal symbioses, and plant parasitism. Currently, sensitive, specific, and high-throughput methods of detecting strigolactones are limited. Here, we developed genetically encoded fluorescent strigolactone biosensors based on the strigolactone receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica via domain insertion of circularly permuted GFP. The DAD2 biosensor exhibited loss of cpGFP fluorescence in vitro upon treatment with the strigolactones 5-deoxystrigol and orobanchol, or the strigolactone analogue GR24. The biosensor likewise responded to strigolactones in an in vivo protoplast system, and retained strigolactone hydrolysis activity. The ShHTL7 biosensor exhibited loss of cpGFP fluorescence upon GR24 treatment in vitro, and responded to a specific inhibitor of ShHTL7 but not DAD2, indicating that the biosensors retained the specificity of their parent receptors. These biosensors have applications in high-throughput screening, and may also have utility for studying strigolactone biology.

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A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

Nucleic Acids Research ◽

10.1093/nar/gkaa1213 ◽

2020 ◽

Author(s):

Yuru Wang ◽

Christopher D Katanski ◽

Christopher Watkins ◽

Jessica N Pan ◽

Qing Dai ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Targeted Mutagenesis ◽

Rna Repair ◽

Dna And Rna ◽

Modified Dna ◽

Dna Substrates ◽

Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

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Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18073332 ◽

2021 ◽

Vol 18 (7) ◽

pp. 3332

Author(s):

Olga V. Naidenko ◽

David Q. Andrews ◽

Alexis M. Temkin ◽

Tasha Stoiber ◽

Uloma Igara Uche ◽

...

Keyword(s):

Immune System ◽

High Throughput ◽

Environmental Protection Agency ◽

High Throughput Screening ◽

Molecular Mechanisms ◽

Specific Activity ◽

Laboratory Animals ◽

In Vitro Assays ◽

Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

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