Protein interaction discovery using parallel analysis of translated ORFs (PLATO)

Zhu, Jian; Larman, H. Benjamin; Gao, Geng; Somwar, Romel; Zhang, Zijuan; Laserson, Uri; Ciccia, Alberto; Pavlova, Natalya N.; Church, George M.; Zhang, Wei; Kesari, Santosh; Elledge, Stephen J.

doi:10.1038/nbt.2539

Cited by 52 publications

(43 citation statements)

References 14 publications

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“…To that extent, we are focusing on developing methodologies for high-throughput capture of paired heavy and light chain sequences from single cells (22). Coupled with significant advances in DNA synthesis technology (23, 24), we should soon be able to assay a large immune repertoire against a large, synthetic library of antigens (e.g., autoantigens, allergens, infectious agents) (25)(26)(27)(28). Doing so will further the development of immune repertoire profiling and facilitate our progress toward the next generation of diagnostics, vaccines, and personalized therapeutic discovery.…”

Section: Discussionmentioning

confidence: 99%

High-resolution antibody dynamics of vaccine-induced immune responses

Laserson

Vigneault

Gadala-Maria

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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219

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The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.next-generation sequencing | influenza | immunology T he immune system is able to rapidly sense and respond to a vast array of invading organisms. Its arsenal contains systems that are immediately effective against commonly seen patterns (innate immunity) and systems that are capable of responding to novel invaders (adaptive immunity). Given the acute nature and diversity of infections, the immune system must be capable of rapid recognition of a pathogen, amplification of the response, and subsequent contraction of the response after the resolution of the infection. Adaptive immune responses rely on the continuous selection and amplification of specific clones from an enormous library of immune receptors (antibodies and T cell receptors). Specifically, stimulation of B-cell immunity results in the synthesis of antibodies that are secreted into the blood stream or into the mucosa as well as the programming of B memory cells that play a crucial role in the generation of rapid protective responses upon reinfection.Currently, many immunology studies depend on characterizing lymphocyte subsets (e.g., assaying cell-surface receptors) and the ability to correlate them to encoded genetic information (1). Recent advances in next-generation sequencing (NGS) (2) have enabled any DNA-encodable assay to produce massive amounts of data. Indeed, NGS has enabled unprecedented views into the immune repertoire, as its immune receptor diversity is genetically encoded within a complex collection of lymphocytes (3-8).The present study set out to dissect the rapid dynamics of the complete human peripheral antibody response against a controlled immune challenge (vaccination), without the a priori notion of cell state markers or functions...

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Section: Discussionmentioning

confidence: 99%

High-resolution antibody dynamics of vaccine-induced immune responses

Laserson

Vigneault

Gadala-Maria

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

183

219

View full text Add to dashboard Cite

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“…The PLATO assays were performed as previously described (3). Briefly, the plasmid DNA was PCR-amplified using the T7B (5′-ATACGAAATTAATACGA-CTCACTATAGGGA GACCACAACGG-3′) and TolAK (5′-CCGCACACCAGTAA-GGTGTGCGGTTTCAGTTGC CGCTTTCTTTCT-3′) primers, and then in vitrotranscribed using the RiboMAX Large-Scale RNA Production system-T7 kit (Promega).…”

Section: Methodsmentioning

confidence: 99%

“…Understanding and diagnosing autoimmune diseases benefit from precise knowledge of the targets of the immune system, which can serve both diagnostic and, potentially, therapeutic purposes. For these reasons, we and others have developed methods to identify targets of autoimmune disorders (1)(2)(3)(4).…”

mentioning

confidence: 99%

Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer

Shah

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Scleroderma is a chronic autoimmune rheumatic disease associated with widespread tissue fibrosis and vasculopathy. Approximately two-thirds of all patients with scleroderma present with three dominant autoantibody subsets. Here, we used a pair of complementary high-throughput methods for antibody epitope discovery to examine patients with scleroderma with or without known autoantibody specificities. We identified a specificity for the minor spliceosome complex containing RNA Binding Region (RNP1, RNA recognition motif) Containing 3 (RNPC3) that is found in patients with scleroderma without known specificities and is absent in unrelated autoimmune diseases. We found strong evidence for both intra- and intermolecular epitope spreading in patients with RNA polymerase III (POLR3) and the minor spliceosome specificities. Our results demonstrate the utility of these technologies in rapidly identifying antibodies that can serve as biomarkers of disease subsets in the evolving precision medicine era.

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“…Unlike other methods that only detect affinity-enriched proteins (e.g., PLATO 16 ), our approach simultaneously counts polonies of both unbound and bound proteins in a single solution. Thus, we sought to determine if it can provide a measure of protein binding affinities.…”

mentioning

confidence: 99%

Multiplex single-molecule interaction profiling of DNA-barcoded proteins

Aach

et al. 2014

Nature

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In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 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 display6 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)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over 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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Protein interaction discovery using parallel analysis of translated ORFs (PLATO)

Cited by 52 publications

References 14 publications

High-resolution antibody dynamics of vaccine-induced immune responses

High-resolution antibody dynamics of vaccine-induced immune responses

Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer

Multiplex single-molecule interaction profiling of DNA-barcoded proteins

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