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Multiplex single-molecule interaction profiling of DNA-barcoded proteins


In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2,3,4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods5 is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables ‘library versus library’ screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

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Figure 1: Schematics of protein barcoding methods.
Figure 2: Amplification and quantification of barcoding DNAs.
Figure 3: Analyses of protein interactions via polony colocalization.
Figure 4: Parallel antibody binding profiling.

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This work was supported by a grant from the US Department of Energy (DE-FG02-02ER63445) to G.M.C. and a grant from the NIH NHGRI (HG001715) to M.V. and D.E.H. L.G. was supported by a postdoctoral fellowship from the Jane Coffin Childs Fund for Medical Research and a grant from Harvard Origins of Life Initiative. We thank W. Harper, L. Pontano Vaites, S. Elledge and J. Zhu for providing plasmids, F. Vigneault for comments on the manuscript, J. Lai and D. Breslau for assistance on imaging setup, and members of the Church and Vidal groups for constructive discussions.

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Authors and Affiliations



L.G. and G.M.C. conceived the technique; L.G. and C.L. performed experiments and analysed data; J.A. built the mathematical model and assisted the colocalization analyses; D.E.H. and M.V. assisted the production of barcoded proteins; L.G., J.A. and G.M.C. wrote the manuscript with help from the other authors.

Corresponding authors

Correspondence to Liangcai Gu or George M. Church.

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Competing interests

A provisional patent has been filed by the Harvard University Office of Technology Development.

Additional information

MATLAB scripts for imaging analyses, colocalization statistics and mathematical modelling can be found at

Extended data figures and tables

Extended Data Figure 1 Improved stability of PRMC complexes generated in a reconstituted E. coli IVT system.

a, Schematic of PRMC complex stability analysis by measuring the relative ratio of barcoding DNA to HaloTag-labelled protein. b, Comparison of PRMC complex stabilities in the E. coli recombinant-factor-reconstituted (PURE) and an E. coli crude extract (S30) IVT system. Nucleic acid degradation or ribosome dissociation can result in the loss of barcoding DNAs. IVT reactions were performed at 37 °C for 30 min and PRMC complexes were further incubated at room temperature for indicated periods of time before the affinity purification and the stability analysis. Means of three independent experiments ± standard deviations.

Extended Data Figure 2 HaloTag-based protein–DNA conjugation.

a, Schematic of the individual barcoding method adaptable to an automatic platform. Fusion proteins bearing an N- or C-terminal HaloTag and the affinity tags were purified and conjugated to a barcoding DNA bearing three different modifications. b, Agarose gel electrophoresis of the barcoding DNA and selected protein–DNA conjugates.

Extended Data Figure 3 Covalent immobilization of barcoding DNAs is required for in situ polony amplification.

a, b, Representative images of polonies amplified from barcoding DNA templates without (a) or with (b) 5′-acrydite modifications. Oversized polonies or polony clusters shown in a resulted from template-drifting-induced multiple seeding events during the amplification.

Extended Data Figure 4 Polony quantification of various barcoded proteins.

a, Plot showing the average number of polonies detected at a single imaging position against the average number of barcoding DNA templates predicted by real-time PCR quantification. Data represent mean values of 100 measurements; error bars, 95% CL. b, Log–log plot of total numbers of polonies detected against dilution factors. Data represent mean values of two technical replicates.

Extended Data Figure 5 Crosslinking efficiency of DsRed is improved by a lysine-rich TolA domain.

a, SDS–PAGE analysis of purified DsRed (Clontech Laboratories) and HaloTag–DsRed–TolA proteins before (lanes 1 and 3) and after (lanes 2 and 4) the crosslinking. 10 µM purified proteins were crosslinked by 1 mM BS(PEG)5 in 20 mM HEPES buffer, pH 8.0, 150 mM KOAc at 4 °C for 1 h. Proteins were stained with Coomassie blue. Only a minor band of the crosslinked dimer was observed for DsRed (lane 2); in contrast, HaloTag–DsRed–TolA was all crosslinked as a tetramer or a trimer (lane 4). Co-purified E. coli proteins (some protein bands below the major band in the lane 3), probably bound to TolA during the purification, and degradation products (due to the hydrolysis of an acylimine bond in the DsRed chromophore) were efficiently crosslinked to HaloTag–DsRed–TolA. b, Comparison of HaloTag-labelled DsRed and mCherry, a monomeric fluorescence protein, crosslinked at different conditions. Proteins labelled with Halo-TMR were analysed by fluorescent gel imaging. Only a minor fraction of HaloTag–mCherry–TolA, a control to show non-specific crosslinking, was crosslinked at increased protein concentrations. Intramolecularly crosslinked proteins show multiple bands or smears corresponding to different quaternary structures of the multidomain proteins stabilized by crosslinking. Bands of the crosslinked trimers show an increased intensity at higher BS(PEG)5 concentrations probably because the primary amine groups on surface are more quickly modified by BS(PEG)5, thus preventing the further crosslinking to form the tetramer.

Extended Data Table 1 Polony quantification of titrated binder proteins and antigens
Extended Data Table 2 ScFvs and human proteins used in the one-pot binding profiling
Extended Data Table 3 Comparison of protein interaction profiling technologies based on nucleic acid barcoding and high-throughput sequencing

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Notes, a Supplementary Discussion, Supplementary Figure 1 and Supplementary References. (PDF 678 kb)

Supplementary Table 1

The file contains the Ras and Raf-RBD binding assay. (XLSX 10 kb)

Supplementary Table 2

This file contains the GPCR screening assay. (XLSX 11 kb)

Supplementary Table 3

This file contains the ScFv binding profiling assay. (XLSX 142 kb)

Supplementary Table 4

This file contains the DNA constructs used in this study. (XLSX 31 kb)

Supplementary Table 5

The file contains the Sequences of expression vectors and synthetic oligos used in this study. (XLSX 25 kb)

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Gu, L., Li, C., Aach, J. et al. Multiplex single-molecule interaction profiling of DNA-barcoded proteins. Nature 515, 554–557 (2014).

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