Fig. 1: The MIPSA method. | Nature Biomedical Engineering

Fig. 1: The MIPSA method.

From: Unbiased discovery of autoantibodies associated with severe COVID-19 via genome-scale self-assembled DNA-barcoded protein libraries

Fig. 1

a, Schematic of the recombined pDEST–MIPSA vector with key components highlighted: UCI (blue), RBS (yellow), N-terminal HaloTag (purple), FLAG epitope (orange), ORF (green) and the I-SceI restriction endonuclease site (black) for vector linearization. b, Schematic showing IVT-RNA from the vector template shown in a. Isothermal base-balanced UCI sequence: (SW)18–AGGGA–(SW)18. c, Cell-free translation of the RNA–cDNA shown in b. HaloTag protein forms a covalent bond with the HaloLigand-conjugated UCI-containing cDNA in cis during translation. d, RT primer positions tested for impact on translation. e, α-FLAG western blot analysis of translation in presence of RT primers depicted in d (NC, negative control, no RT primer). f, Western blot analysis of TRIM21 protein translated from RNA carrying the UCI-cDNA primed from the −32 position, either conjugated (+) or not (−) with the HaloLigand, IPed with healthy control (HC) or Sjögren’s syndrome (SS) plasma. g, qPCR analysis of the immunoprecipitated TRIM21 UCI. Fold difference is by comparison with the HaloLigand (−) HC IP.

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