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