Anton Simeonov Ph.D. National Institute of Health

George Church and Colleagues Provide an Elegant Solution to the Problem

Being able to derive screening data from very large-scale experiments without the need to test every interacting pair separately (as is commonly done in small-molecule high-throughput screening) has remained largely an elusive goal. George Church and colleagues provide an elegant solution to the problem by preparing proteins that are barcoded with a DNA sequence attached to them through in vitro translation and ribosome display (see figure below).

Schematics of protein barcoding methods. (a) Collective barcoding via ribosome display. A short synthetic barcoding sequence is joined to the 5′ end of DNA templates via PCR. PRMC complexes are formed via ribosome stalling triggered by a carboxy-terminal Escherichia coli SecM peptide. Displayed proteins bearing a C-terminal Flag tag are separated from the ribosomes by an E. coli TolA spacer domain. RBS, ribosomal binding site. (b) Individual barcoding via a HaloTag-mediated conjugation of proteins to a 220-base-pair (bp) double-stranded barcoding DNA with a HaloTag ligand modification (black triangle). Modifications are introduced to barcoding DNAs by PCR with modified primers.

In turn, the mixture of barcoded and displayed proteins could be analyzed through immobilization in a thin layer of polyacrylamide in which the individual protein nanospots were identified and quantified by amplification of the DNA barcode and subsequent sequencing, essentially visualizing and counting single-molecule occurrences (see figure below).  Using this technique, the authors* performed large-scale “library-against-library” screens in which two distinct protein families were crossed with each other, and the outcome of selective binding and resultant co-localization of uniquely barcoded cognate partners was measured through the single-molecule barcode detection. The same experiment was also conducted in a hybrid format in which a small molecule library was screened against a family of related G-protein coupled receptors (GPCR), all barcoded and present together in each well of the microtiter plate, along with potential cognate β-arrestin partners that were also barcoded. The screen thus allowed the detection of specific small-molecule disrupters of the various GPCR/β-arrestin interactions without the need to test each protein–protein pair separately.

Amplification and quantification of barcoding DNAs. (a) Schematic of in situ polony amplification and sequencing. Barcoded proteins were immobilized in a polyacrylamide (PAA) gel matrix attached to a Bind-Silane-treated glass slide. The slide was assembled into a flow cell, where barcoding DNAs were amplified in situ into polonies for DNA sequencing. (b) Representative merged images of polonies hybridized with Cy5 (red), Cy3 (green) and fluorescein (blue)-labelled oligonucleotides (320 objective magnification). (c) Polony quantification of mixed protein binders and antigens. The Pearson correlation coefficient r was calculated for different coverages grouped by dotted lines.

*Abstract from Nature 2014, Vol. 515:554–557

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 protein detection using optical methods 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 display 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) 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.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Nature titled "Multiplex single-molecule interaction profiling of DNA-barcoded proteins." Authors of the paper are Gu L, Li C, Aach J, Hill DE, Vidal M, Church GM.

Anton Simeoniv works at the NIH.