scholarly journals Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array

2019 ◽  
Vol 5 (8) ◽  
pp. eaav8185 ◽  
Author(s):  
Yi Zhang ◽  
Yoshihiro Minagawa ◽  
Hiroto Kizoe ◽  
Kentaro Miyazaki ◽  
Ryota Iino ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
High Throughput ◽  
High Throughput Screening ◽  
General Strategy ◽  
Dna Molecules ◽  
Single Dna Molecules ◽  
Droplet Array ◽  
Uniform Droplets ◽  
Increased Activity ◽  
Digital Counting

We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm2 to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution–predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents.

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

10.3791/60945 ◽  
2020 ◽  
Author(s):  
Yi Zhang ◽  
Kanako Kurosawa ◽  
Daisuke Nishiura ◽  
Mika Tei ◽  
Mikiko Tsudome
Keyword(s):  
Protein Synthesis ◽  
Massively Parallel ◽  
Dna Molecules ◽  
Single Dna Molecules ◽  
Droplet Array

scholarly journals High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

2001 ◽  
Vol 6 (6) ◽  
pp. 429-440 ◽  
Author(s):  
Michael W. Pantoliano ◽  
Eugene C. Petrella ◽  
Joseph D. Kwasnoski ◽  
Victor S. Lobanov ◽  
James Myslik ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Ligand Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
General Strategy ◽  
General Applicability ◽  
High Throughput Drug Screening ◽  
Compound Libraries ◽  
Thermal Shift

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

scholarly journals Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes byEscherichia coli

2019 ◽  
Author(s):  
Srijan Jindal ◽  
Lei Yang ◽  
Philip J. Day ◽  
Douglas B. Kell
Keyword(s):  
Steady State ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Dyes ◽  
Gene Knockout ◽  
Sybr Green ◽  
Efflux Transporters ◽  
General Strategy ◽  
Individual Gene

AbstractWe used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar findings were made with SYBR Green (the range being some 69-fold), though despite it too containing a benzimidazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains. Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of possibly broad and presently unknown specificity. This casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

High-throughput confinement and detection of single DNA molecules in aqueous microdroplets

2009 ◽  
pp. 6548 ◽  
Author(s):  
Monpichar Srisa-Art ◽  
Andrew J. deMello ◽  
Joshua B. Edel
Keyword(s):  
High Throughput ◽  
Dna Molecules ◽  
Single Dna Molecules

scholarly journals High-Throughput Screening for Protein Synthesis Inhibitors Targeting Aminoacyl-tRNA Synthetases

2017 ◽  
Vol 23 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Jiwon Kong ◽  
Pengfei Fang ◽  
Franck Madoux ◽  
Timothy P. Spicer ◽  
Louis Scampavia ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Synthesis Inhibitors ◽  
Chemical Libraries ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Reticulocyte Lysate ◽  
The Individual

Aminoacylation has been implicated in a wide variety of cancers. Aminoacyl-tRNA synthetases (ARSs) exist in large excess in tumor cells due to their increased demand for translation, whereas most other protein-synthesis apparatuses are quantitatively limited. Among other components that constitute the translation machinery—namely, tRNA, amino acid, ATP, and ARS—ARS is the only target that can be blocked by small molecules. No constitutively active ARSs have been reported, and mutations of ARS can cause inaccurate substrate recognition and malformation of the multi-ARS complex (MSC). Hence, interference of the activity is expected to be independent of genotype without developing resistance. Here, we report a high-throughput screening (HTS) system to find mammalian ARS inhibitors. The rabbit–reticulocyte lysate we used closely resembles both the individual and complexed structures of human ARSs, and it may predispose active compounds that are readily applicable for humankind. This assay was further validated because it identified familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for our purpose. The assay demonstrated excellent quality control (QC) parameters and reproducibility, and is proven ready for further HTS campaigns with large chemical libraries.

A bilayer cell-free protein synthesis system for high-throughput screening of gene products

FEBS Letters ◽  
2002 ◽  
Vol 514 (1) ◽  
pp. 102-105 ◽  
Author(s):  
Tatsuya Sawasaki ◽  
Yoshinori Hasegawa ◽  
Masateru Tsuchimochi ◽  
Nami Kamura ◽  
Tomio Ogasawara ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gene Products ◽  
Synthesis System ◽  
Free Protein ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

Forming a Large-Scale Droplet Array in a Microcage Array Chip for High-Throughput Screening

2019 ◽  
Vol 91 (16) ◽  
pp. 10757-10763 ◽  
Author(s):  
Jin-Gang Xu ◽  
Meng-Shi Huang ◽  
Hui-Feng Wang ◽  
Qun Fang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Droplet Array
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