Supplemental web site for

Multiplex single-molecule interaction profiling of DNA barcoded proteins


 Liangcai Gu1,*, Chao Li2, John Aach1, David E. Hill1,3, Marc Vidal1,3 & George M. Church1,2,*


1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.

3Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.


* To whom correspondence should be addressed: E-mail: (L.G.); (G.M.C.)

ABSTRACT: In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analyzed by DNA sequencing. This method allows precise quantification of various proteins with an array density theoretically higher than one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

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Last modified: 6/23/2014 4:48 PM