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Related Concept Videos

DNA Microarrays02:34

DNA Microarrays

Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...

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Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded

Michael Rappleye1,2, Sarah J Wait2,3, Justin Daho Lee2,3

  • 1Department of Bioengineering, University of Washington, 850 Republican Street, Seattle, Washington 98105, United States.

ACS Sensors
|November 13, 2023
PubMed
Summary
This summary is machine-generated.

Researchers developed a high-throughput platform, Opto-MASS, to rapidly engineer genetically encoded fluorescent indicators (GEFIs). This system accelerates the development of sensitive dopamine and opioid sensors for cellular monitoring.

Keywords:
GPCRdopaminefluorescent biosensorhigh throughput screeningopioidsprotein engineering

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Optogenetics

Background:

  • Genetically encoded fluorescent indicators (GEFIs) are crucial for monitoring cellular physiology but engineering them is slow.
  • Current methods involve laborious trial-and-error mutagenesis and in vitro testing of individual variants.
  • The vast protein mutational landscape hinders the development of sensitive and specific GEFIs.

Purpose of the Study:

  • To develop a high-throughput platform for rapid engineering of GEFIs.
  • To overcome the limitations of traditional GEFI optimization methods.
  • To accelerate the discovery of novel GEFI variants with improved performance.

Main Methods:

  • Developed the optogenetic microwell array screening system (Opto-MASS) for parallel screening of thousands of GEFI variants in mammalian cells.
  • Applied Opto-MASS to screen over 13,000 dopamine and 21,000 opioid sensor variants.
  • Utilized a multiplexed, high-throughput approach to functionally test GEFI variants.

Main Results:

  • Generated a novel dopamine sensor (dMASS^1) with a >6-fold signal increase in response to dopamine.
  • Developed a new opioid sensor (μMASS^1) exhibiting a ~4.6-fold signal increase to DAMGO.
  • Demonstrated Opto-MASS's capability to screen large variant libraries significantly faster than existing methods.

Conclusions:

  • Opto-MASS enables rapid engineering of novel GEFIs.
  • The platform significantly shortens optimization time for sensors with distinct biophysical properties.
  • This high-throughput system advances the development of optogenetic tools for cellular monitoring.