journal club

Multiplex single-molecule interaction profiling of DNA-barcoded proteins
Nature 2014, Church lab, Gu et al

presented by Pallav


  • current approaches = protein vs. library
  • rather do an everyobdy to everybody screen. One pot analysis not well-based screen


  • single-molecular interaction sequencing (SMI-seq)
  • barcode proteins with DNA.
  • on the mRNA add a barcode and a stalling tag downstream of a polypeptide linker. The ribosome now combines the mRNA and the protein together. (during an in vitro translation reaction following an in vitro transcription reaction).
  • can now mix proteins
  • barcoded RNA annealed to RNA with little tags.
  • create dilute array of polonies in acrylomide gel. Can now sequence.
  • have two different pools with two different primers. Only the ones with one of each primer can be decoded.
  • challenges: very different yields of different complexes. Can count total spots to get estimate of concentrations and thus get estimates of binding efficiency ?

ligand binding screening

  • small molecule binding to GPCR (g-protein coupled receptor) measure affinity based on GPCR’s change in affinity for binding protein arrestin. different ligand in each well.

library to library screening

  • library 1 antibody domains (a bunch of mutated variable forms)
  • library 2 human proteins
  • test 200 x 60. With Illumina reading propose could test 10,000 x 10,000
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