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We developed a new method for multiplexed probe immobilization on library beads for emulsion screening. This technique enables high-throughput screening of enzyme libraries for novel activities using fluorescent probes.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • Emulsions enable miniaturization and parallelization of high-throughput screening.
  • Localizing activity-based fluorescent probes within emulsion droplets is crucial but challenging.
  • Multiplexing probes in droplets is impractical, hindering the identification of specific library members.

Purpose of the Study:

  • To develop a robust method for multiplexed probe immobilization on library beads for emulsion screening.
  • To enable high-throughput screening of enzyme libraries for novel proteolytic specificity.
  • To overcome the limitations of probe localization and multiplexing in droplet-based assays.

Main Methods:

  • Quantified primers per bead using fluorescence in situ hybridization (FISH).
  • Leveraged unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates.
  • Programmed emulsion in vitro transcription/translation reactions with probe-hybridized bead libraries.
  • Analyzed results using fluorescence-activated cell sorting (FACS) for multiplexed activity-based screening.

Main Results:

  • Successfully immobilized multiple probes onto library beads.
  • Demonstrated multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries.
  • Identified novel proteolytic specificities within the enzyme mutant libraries.

Conclusions:

  • The developed approach enables multiplexed probe immobilization on library beads for emulsion screening.
  • This method facilitates high-throughput identification of enzyme variants with novel activities.
  • The modularity of the approach allows for extensibility to other enzyme classes and library types.