FRET-Seq: a High-Throughput FRET-Based Screening Platform to Improve FRET Biosensors in Mammalian Cells

Abstract Genetically-encoded biosensors based on FRET have been widely used to dynamically monitor the activity of protein tyrosine kinases (PTKs) in living cell with high spatiotemporal resolution. However, the limitation in sensitivity, specificity, and dynamic range of FRET biosensors have hindered their broader applications. Here, we introduced a systematic platform, FRET-Seq, which integrates high-throughput FRET sorting and next-generation sequencing, to identify FRET biosensors with better performance from large-scale libraries directly in mammalian cells.

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Next-Generation Sequencing: An Emerging Tool for Drug Designing

Current Pharmaceutical Design ◽

10.2174/1381612825666190911155508 ◽

2019 ◽

Vol 25 (31) ◽

pp. 3350-3357 ◽

Cited By ~ 1

Author(s):

Pooja Tripathi ◽

Jyotsna Singh ◽

Jonathan A. Lal ◽

Vijay Tripathi

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Large Scale ◽

Massively Parallel Sequencing ◽

Genomic Research ◽

Biological Research ◽

Next Generation ◽

Human Welfare ◽

Drug Designing ◽

Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

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Development and Standardization of a High-Throughput Multiplex Immunoassay for the Simultaneous Quantification of Specific Antibodies to Five Respiratory Syncytial Virus Proteins

mSphere ◽

10.1128/msphere.00236-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 7

Author(s):

Rutger M. Schepp ◽

Cornelis A. M. de Haan ◽

Deidre Wilkins ◽

Hans Layman ◽

Barney S. Graham ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

High Throughput ◽

Large Scale ◽

Dynamic Range ◽

Valuable Insight ◽

Conformational Structure ◽

Multiplex Immunoassay ◽

Rsv Infection ◽

Syncytial Virus ◽

Antibody Levels

ABSTRACT Human respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in (premature) newborns and causes respiratory illness in the elderly. Different monoclonal antibody (MAb) and vaccine candidates are in development worldwide and will hopefully become available within the near future. To implement such RSV vaccines, adequate decisions about immunization schedules and the different target group(s) need to be made, for which the assessment of antibody levels against RSV is essential. To survey RSV antigen-specific antibody levels, we developed a serological multiplex immunoassay (MIA) that determines and distinguishes antibodies against the five RSV glycoproteins postfusion F, prefusion F, Ga, Gb, and N simultaneously. The standardized RSV pentaplex MIA is sensitive, highly reproducible, and specific for the five RSV proteins. The preservation of the conformational structure of the immunodominant site Ø of prefusion F after conjugation to the beads has been confirmed. Importantly, good correlation is obtained between the microneutralization test and the MIA for all five proteins, resulting in an arbitrarily chosen cutoff value of prefusion F antibody levels for seropositivity in the microneutralization assay. The wide dynamic range requiring only two serum sample dilutions makes the RSV-MIA a high-throughput assay very suitable for (large-scale) serosurveillance and vaccine clinical studies. IMPORTANCE In view of vaccine and monoclonal development to reduce hospitalization and death due to lower respiratory tract infection caused by RSV, assessment of antibody levels against RSV is essential. This newly developed multiplex immunoassay is able to measure antibody levels against five RSV proteins simultaneously. This can provide valuable insight into the dynamics of (maternal) antibody levels and RSV infection in infants and toddlers during the first few years of life, when primary RSV infection occurs.

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D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle

The Journal of General Physiology ◽

10.1085/jgp.201010591 ◽

2011 ◽

Vol 138 (2) ◽

pp. 211-229 ◽

Cited By ~ 22

Author(s):

Monika Sztretye ◽

Jianxun Yi ◽

Lourdes Figueroa ◽

Jingsong Zhou ◽

Leandro Royer ◽

...

Keyword(s):

Skeletal Muscle ◽

Adult Mouse ◽

Dynamic Range ◽

Endogenous Ligands ◽

Model Calculations ◽

Viable Cells ◽

Fret Biosensors ◽

Sensitivity Specificity ◽

Local Variability ◽

T System

Current fluorescent monitors of free [Ca2+] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca2+. “D4cpv-Casq1,” expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective KD of 222 µM and a dynamic range [(Rmax − Rmin)/Rmin] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, Rmax, and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca2+]SR in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca2+]SR was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca2+ concentration.

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Remote fingerstick blood collection for SARS-CoV-2 antibody testing

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2020-0713-sa ◽

2020 ◽

Author(s):

Wilfredo F. Garcia-Beltran ◽

Tyler E. Miller ◽

Grace Kirkpatrick ◽

Andrea Nixon ◽

Michael G. Astudillo ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Dynamic Range ◽

Rapid Development ◽

Venous Blood ◽

Blood Collection ◽

Antibody Assay ◽

Serum Samples ◽

Blood Draw ◽

Serological Tests

ABSTACT The rapid worldwide spread of severe acute respiratory system coronavirus 2 (SARSCoV-2) infection has propelled the rapid development of serological tests that can detect anti-SARS-CoV-2 antibodies. These have been used for studying the prevalence and spread of infection in different populations, helping establish a recent diagnosis of coronavirus disease 2019 (COVID-19), and will likely be used to confirm humoral immunity after infection or vaccination. However, nearly all lab-based high-throughput SARS-CoV-2 serological assays require a serum sample from venous blood draw, limiting their applications and scalability. Here, we present a method that enables large scale SARS-CoV-2 serological studies by combining self or office collection of fingerprick blood with a volumetric absorptive microsampling device (Mitra, Neoteryx, LLC) with a high-throughput electrochemiluminescence-based SARS-CoV-2 total antibody assay (Roche Elecsys, Roche Diagnostics, Inc.) that is emergency use authorization (EUA) approved for use on serum samples and widely used by clinical laboratories around the world. We found that the Roche Elecsys assay has a high dynamic range that allows for accurate detection of SARS-CoV-2 antibodies in serum samples diluted 1:20 as well as contrived dried blood extracts. Extracts of dried blood from Mitra devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies as compared to neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of Mitra dried blood collection device with the Roche Elecsys SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies.

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Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

10.1101/2021.03.26.437268 ◽

2021 ◽

Author(s):

Angela Mc Ardle ◽

Aleksandra Binek, ◽

Annie Moradian ◽

Blandine Chazarin Orgel ◽

Alejandro Rivas ◽

...

Keyword(s):

High Throughput ◽

Whole Blood ◽

Large Scale ◽

Dynamic Range ◽

Profile Analysis ◽

Protein Biomarkers ◽

Throughput Analysis ◽

Broad Dynamic Range ◽

Plasma Fractions ◽

Deep Profile

Background: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid–throughput analysis for three blood–based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood. Methods: Optimal conditions for sample preparation and DIA–MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high–throughput or deep profile analysis with mid–throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5—6–point dilution curve. Result: Using our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high–throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid–throughput workflows. Conclusion: The workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

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Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

Biosensors ◽

10.3390/bios8040099 ◽

2018 ◽

Vol 8 (4) ◽

pp. 99 ◽

Cited By ~ 12

Author(s):

Tory Schaaf ◽

Ang Li ◽

Benjamin Grant ◽

Kurt Peterson ◽

Samantha Yuen ◽

...

Keyword(s):

High Precision ◽

Small Molecule ◽

High Throughput ◽

Fluorescence Lifetime ◽

High Throughput Screening ◽

Structural Changes ◽

Dynamic Range ◽

Resonance Energy ◽

Fluorescence Measurements ◽

Fret Biosensors

We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

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Integration of FRET and sequencing to engineer kinase biosensors from mammalian cell libraries

Nature Communications ◽

10.1038/s41467-021-25323-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Longwei Liu ◽

Praopim Limsakul ◽

Xianhui Meng ◽

Yan Huang ◽

Reed E. S. Harrison ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

High Throughput ◽

Mammalian Cells ◽

Large Scale ◽

Cell Activation ◽

Resonance Energy ◽

Cell Receptor ◽

Dynamic Imaging ◽

Cancer Drug

AbstractThe limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.

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Colorimetric High-Throughput Screen for Detection of Heme Crystallization Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01466-08 ◽

2009 ◽

Vol 53 (6) ◽

pp. 2564-2568 ◽

Cited By ~ 26

Author(s):

Margaret A. Rush ◽

Mary Lynn Baniecki ◽

Ralph Mazitschek ◽

Joseph F. Cortese ◽

Roger Wiegand ◽

...

Keyword(s):

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Dynamic Range ◽

Colorimetric Assay ◽

Microtiter Plate ◽

Viability Assay ◽

Parasite Viability ◽

Screening Platform ◽

Intraerythrocytic Stage

ABSTRACT Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay's reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.

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High-throughput microfluidic cell sorting platform (MICS)

10.21203/rs.2.10282/v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

David Philpott ◽

Peter Aldridge ◽

Barbara Mair ◽

Randy Atwal ◽

Sanna Masud ◽

...

Keyword(s):

Protein Expression ◽

Cell Viability ◽

High Throughput ◽

Cell Sorting ◽

Mammalian Cells ◽

Large Scale ◽

Genetic Screens ◽

Large Numbers ◽

Conventional Cell ◽

Set Up

Abstract Genome-scale functional genetic screens can be used to interrogate determinants of protein expression modulation of a target of interest. Such phenotypic screening approaches typically require sorting of large numbers of cells (>108). In conventional cell sorting techniques (i.e. fluorescence-activated cell sorting), sorting time, associated with high instrument and operating costs and loss of cell viability, are limiting to the scalability and throughput of these screens. We recently established a rapid and scalable high-throughput microfluidic cell sorting platform (MICS) using immunomagnetic nanoparticles to sort cells in parallel capable of sorting more than 108 HAP1 cells in under one hour while maintaining high levels of cell viability (Ref. 1). This protocol outlines how to set-up MICS for large-scale phenotypic screens in mammalian cells. We anticipate this platform being used for genome-wide functional genetic screens as well as other applications requiring the sorting of large numbers of cells based on protein expression.

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