Project acronym AutoCAb
Project Automated computational design of site-targeted repertoires of camelid antibodies
Researcher (PI) Sarel-Jacob FLEISHMAN
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Consolidator Grant (CoG), LS9, ERC-2018-COG
Summary We propose to develop the first high-throughput strategy to design, synthesize, and screen repertoires comprising millions of single-domain camelid antibodies (VHH) that target desired protein surfaces. Each VHH will be individually designed for high stability and target-site affinity. We will leverage recent methods developed by our lab for designing stable, specific, and accurate backbones at interfaces, the advent of massive and affordable custom-DNA oligo synthesis, and machine learning methods to accomplish the following aims:
Aim 1: Establish a completely automated computational pipeline that uses Rosetta to design millions of VHHs targeting desired protein surfaces. The variable regions in each design will be encoded in DNA oligo pools, which will be assembled to generate the entire site-targeted repertoire. We will then use high-throughput binding screens followed by deep sequencing to characterize the designs’ target-site affinity and isolate high-affinity binders.
Aim 2: Develop an epitope-focusing strategy that designs several variants of a target antigen, each of which encodes dozens of radical surface mutations outside the target site to disrupt potential off-target site binding. The designs will be used to isolate site-targeting binders from repertoires of Aim 1.
Each high-throughput screen will provide unprecedented experimental data on target-site affinity in millions of individually designed VHHs.
Aim 3: Use machine learning methods to infer combinations of molecular features that distinguish high-affinity binders from non binders. These will be encoded in subsequent designed repertoires, leading to a continuous “learning loop” of methods for high-affinity, site-targeted binding.
AutoCAb’s interdisciplinary strategy will thus lead to deeper understanding of and new general methods for designing stable, high-affinity, site-targeted antibodies, potentially revolutionizing binder and inhibitor discovery in basic and applied biomedical research.
Summary
We propose to develop the first high-throughput strategy to design, synthesize, and screen repertoires comprising millions of single-domain camelid antibodies (VHH) that target desired protein surfaces. Each VHH will be individually designed for high stability and target-site affinity. We will leverage recent methods developed by our lab for designing stable, specific, and accurate backbones at interfaces, the advent of massive and affordable custom-DNA oligo synthesis, and machine learning methods to accomplish the following aims:
Aim 1: Establish a completely automated computational pipeline that uses Rosetta to design millions of VHHs targeting desired protein surfaces. The variable regions in each design will be encoded in DNA oligo pools, which will be assembled to generate the entire site-targeted repertoire. We will then use high-throughput binding screens followed by deep sequencing to characterize the designs’ target-site affinity and isolate high-affinity binders.
Aim 2: Develop an epitope-focusing strategy that designs several variants of a target antigen, each of which encodes dozens of radical surface mutations outside the target site to disrupt potential off-target site binding. The designs will be used to isolate site-targeting binders from repertoires of Aim 1.
Each high-throughput screen will provide unprecedented experimental data on target-site affinity in millions of individually designed VHHs.
Aim 3: Use machine learning methods to infer combinations of molecular features that distinguish high-affinity binders from non binders. These will be encoded in subsequent designed repertoires, leading to a continuous “learning loop” of methods for high-affinity, site-targeted binding.
AutoCAb’s interdisciplinary strategy will thus lead to deeper understanding of and new general methods for designing stable, high-affinity, site-targeted antibodies, potentially revolutionizing binder and inhibitor discovery in basic and applied biomedical research.
Max ERC Funding
2 337 500 €
Duration
Start date: 2019-01-01, End date: 2023-12-31
Project acronym MapCat
Project High spatial resolution mapping of catalytic reactions on single nanoparticles
Researcher (PI) Elad Gross
Host Institution (HI) THE HEBREW UNIVERSITY OF JERUSALEM
Call Details Starting Grant (StG), PE4, ERC-2018-STG
Summary Catalytic nanoparticles are heterogeneous in their nature - and even within the simplest particles structural and compositional differences exist and affect the overall performances of a catalyst. Thus non-disruptive, detailed chemical information at the nanoscale is essential for understanding how surface properties direct the reactivity of these particles. Infrared spectroscopy offers a low-energy route towards conducting in-situ, high spatial resolution mapping of catalytic reactions on the surface of single nanoparticles, yielding the influence of various physiochemical properties on the catalytic reactivity.
In the project my team will employ recently developed Infrared nanospectroscopy measurements to provide high spatial resolution mapping of catalytic reactions on the surface of metallic nanoparticles, while using chemically active N-heterocyclic carbene molecules as indicators for surface reactivity. With this setup I will address fundamental questions in catalysis research and identify, on a single particle basis and under reaction conditions, the ways by which the size, structure, composition and metal-support interactions direct the reactivity of metallic nanoparticles in hydrogenation, oxidation and functionalization reactions. My research group demonstrated recently the feasibility of this novel approach by which structure-reactivity correlations were identified within single nanoparticles. Knowledge gained in this project will provide in-depth understanding of the basic elements that control the reactivity of heterogeneous catalysts and enable the development of optimized catalysts based on rational design. Moreover, one can foresee wide application potential for this experimental approach in various other research fields like batteries and fuel cells, in which high spatial resolution analysis of reactive surfaces is essential for understanding structure-reactivity correlations.
Summary
Catalytic nanoparticles are heterogeneous in their nature - and even within the simplest particles structural and compositional differences exist and affect the overall performances of a catalyst. Thus non-disruptive, detailed chemical information at the nanoscale is essential for understanding how surface properties direct the reactivity of these particles. Infrared spectroscopy offers a low-energy route towards conducting in-situ, high spatial resolution mapping of catalytic reactions on the surface of single nanoparticles, yielding the influence of various physiochemical properties on the catalytic reactivity.
In the project my team will employ recently developed Infrared nanospectroscopy measurements to provide high spatial resolution mapping of catalytic reactions on the surface of metallic nanoparticles, while using chemically active N-heterocyclic carbene molecules as indicators for surface reactivity. With this setup I will address fundamental questions in catalysis research and identify, on a single particle basis and under reaction conditions, the ways by which the size, structure, composition and metal-support interactions direct the reactivity of metallic nanoparticles in hydrogenation, oxidation and functionalization reactions. My research group demonstrated recently the feasibility of this novel approach by which structure-reactivity correlations were identified within single nanoparticles. Knowledge gained in this project will provide in-depth understanding of the basic elements that control the reactivity of heterogeneous catalysts and enable the development of optimized catalysts based on rational design. Moreover, one can foresee wide application potential for this experimental approach in various other research fields like batteries and fuel cells, in which high spatial resolution analysis of reactive surfaces is essential for understanding structure-reactivity correlations.
Max ERC Funding
1 846 009 €
Duration
Start date: 2019-01-01, End date: 2023-12-31
Project acronym MIDNP
Project Metal Ions Dynamic Nuclear Polarization: Novel Route for Probing Functional Materials with Sensitivity and Selectivity
Researcher (PI) Michal LESKES
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), PE4, ERC-2018-STG
Summary Materials with specific electrical, optical or chemical properties often derive their special functions from small perturbations in their composition or structure. Thus, rational design of new functional materials demands sensitive and versatile determination of structural and compositional properties, a very difficult goal not presently available. The overarching goal of this ERC project is to develop a novel route for Magic-Angle Spinning Dynamic Nuclear Polarization (MAS-DNP) as an enabling methodology in materials science, introducing new opportunities for investigating and designing functional materials.
Solid State Nuclear Magnetic Resonance (ssNMR) spectroscopy is an excellent probe for local order/disorder, but unfortunately its sensitivity is limited. DNP, a process whereby the large electron spin polarization is transferred to the nuclear spins, had greatly expanded the range of materials systems and questions that can be probed by ssNMR. However, it commonly relies on the use of exogenous nitroxide radicals, thereby limiting its utilization in materials science to nonreactive surfaces.
We propose to develop Metal Ions DNP (MIDNP) utilizing paramagnetic dopants as endogenous polarization agents in the bulk. To effectively harness the electron spin polarization of the dopants for higher sensitivity, we will (a) address challenges such as the effect of bonding, spin interactions and relaxation on DNP via a mechanistic study of carefully selected dopants in energy materials; (b) Develop new techniques for NMR spectral assignment and explore alternative DNP mechanisms for paramagnetic solids; (c) Expand the approach for sensitizing the detection of surfaces and interfaces and elucidate the critical role of surface chemistry in the efficacy of energy storage materials.
MIDNP will provide a novel, sensitive alternative for probing the structure and composition of new materials and will transform the utilization of ssNMR in the study of functional materials.
Summary
Materials with specific electrical, optical or chemical properties often derive their special functions from small perturbations in their composition or structure. Thus, rational design of new functional materials demands sensitive and versatile determination of structural and compositional properties, a very difficult goal not presently available. The overarching goal of this ERC project is to develop a novel route for Magic-Angle Spinning Dynamic Nuclear Polarization (MAS-DNP) as an enabling methodology in materials science, introducing new opportunities for investigating and designing functional materials.
Solid State Nuclear Magnetic Resonance (ssNMR) spectroscopy is an excellent probe for local order/disorder, but unfortunately its sensitivity is limited. DNP, a process whereby the large electron spin polarization is transferred to the nuclear spins, had greatly expanded the range of materials systems and questions that can be probed by ssNMR. However, it commonly relies on the use of exogenous nitroxide radicals, thereby limiting its utilization in materials science to nonreactive surfaces.
We propose to develop Metal Ions DNP (MIDNP) utilizing paramagnetic dopants as endogenous polarization agents in the bulk. To effectively harness the electron spin polarization of the dopants for higher sensitivity, we will (a) address challenges such as the effect of bonding, spin interactions and relaxation on DNP via a mechanistic study of carefully selected dopants in energy materials; (b) Develop new techniques for NMR spectral assignment and explore alternative DNP mechanisms for paramagnetic solids; (c) Expand the approach for sensitizing the detection of surfaces and interfaces and elucidate the critical role of surface chemistry in the efficacy of energy storage materials.
MIDNP will provide a novel, sensitive alternative for probing the structure and composition of new materials and will transform the utilization of ssNMR in the study of functional materials.
Max ERC Funding
1 700 000 €
Duration
Start date: 2019-01-01, End date: 2023-12-31
Project acronym MultiplexGenomics
Project Exploring the Epigenome by Multiplexed Physical Mapping of Individual Chromosomes
Researcher (PI) Yuval EBENSTEIN
Host Institution (HI) TEL AVIV UNIVERSITY
Call Details Consolidator Grant (CoG), PE4, ERC-2018-COG
Summary The genome is composed of the genetic code and a rich repertoire of epigenetic chemical DNA modifications, the Epigenome, with distinct signatures in health and disease. Unmasking the interplay between different genomic features is critical for understanding the operating system of life. Specifically, revealing long-range epigenetic regulation may uncover predisposition to cancer. Nevertheless, due to the short read-length of single-cell next-generation sequencing, there is no method today that can integrate multiple genomic observables, on the same genome and at the same time. The missing picture constitutes a major genomic “blind spot”, obscuring epigenetic regulation of gene expression. This project aims to provide a multiplexed view of the genome never before accessible. I will utilize single-molecule physical and chemical mapping of individual chromosomes to discover long-range epigenetic correlations, focusing on markers for predisposition to breast cancer. I will approach multiplexing by applying optical and electrical sensing concepts to detect chemical tags attached to long genomic DNA molecules. Equipped with a toolbox of biochemical DNA labeling reactions, I will develop a unique spectral imager for simultaneous acquisition of high-content genomic information from DNA stretched in nanochannel arrays. DNA tagging will also be used to enhance electrical contrast for nanopore epigenetic sequencing. Finally, by combining electric sensing inside nanochannels I will develop new integrated devices for electro-optical genomic analysis. Together, these developments cover the full range of genomic length scales and resolution. MultiplexGenomics will establish a groundbreaking experimental framework for genetic/epigenetic profiling of native chromosomal DNA. A successful completion of this project will make possible the discovery of novel control networks and hidden long-range regulation, opening new horizons for basic genomic research and personalized medicine.
Summary
The genome is composed of the genetic code and a rich repertoire of epigenetic chemical DNA modifications, the Epigenome, with distinct signatures in health and disease. Unmasking the interplay between different genomic features is critical for understanding the operating system of life. Specifically, revealing long-range epigenetic regulation may uncover predisposition to cancer. Nevertheless, due to the short read-length of single-cell next-generation sequencing, there is no method today that can integrate multiple genomic observables, on the same genome and at the same time. The missing picture constitutes a major genomic “blind spot”, obscuring epigenetic regulation of gene expression. This project aims to provide a multiplexed view of the genome never before accessible. I will utilize single-molecule physical and chemical mapping of individual chromosomes to discover long-range epigenetic correlations, focusing on markers for predisposition to breast cancer. I will approach multiplexing by applying optical and electrical sensing concepts to detect chemical tags attached to long genomic DNA molecules. Equipped with a toolbox of biochemical DNA labeling reactions, I will develop a unique spectral imager for simultaneous acquisition of high-content genomic information from DNA stretched in nanochannel arrays. DNA tagging will also be used to enhance electrical contrast for nanopore epigenetic sequencing. Finally, by combining electric sensing inside nanochannels I will develop new integrated devices for electro-optical genomic analysis. Together, these developments cover the full range of genomic length scales and resolution. MultiplexGenomics will establish a groundbreaking experimental framework for genetic/epigenetic profiling of native chromosomal DNA. A successful completion of this project will make possible the discovery of novel control networks and hidden long-range regulation, opening new horizons for basic genomic research and personalized medicine.
Max ERC Funding
2 750 000 €
Duration
Start date: 2019-10-01, End date: 2024-09-30
Project acronym NanoProt-ID
Project Proteome profiling using plasmonic nanopore sensors
Researcher (PI) Amit MELLER
Host Institution (HI) TECHNION - ISRAEL INSTITUTE OF TECHNOLOGY
Call Details Advanced Grant (AdG), PE4, ERC-2018-ADG
Summary To date, antibody-free protein identification methods have not reached single-molecule precision. Instead, they rely on averaging from many cells, obscuring the details of important biological processes. The ability to identify each individual protein from within a single cell would transform proteomics research and biomedicine. However, single protein identification (ID) presents a major challenge, necessitating a breakthrough in single-molecule sensing technologies.
We propose to develop a method for proteome-level analysis, with single protein resolution. Bioinformatics studies show that >99% of human proteins can be uniquely identified by the order in which only three amino-acids, Lysine, Cysteine, and Methionine (K, C and M, respectively), appear along the proteins’ chain. By specifically labelling K, C and M residues with three distinct fluorophores, and threading them, one by one, through solid-state nanopores equipped with custom plasmonic amplifiers, we hypothesize that we can obtain multi-color fluorescence time-trace fingerprints uniquely representing most proteins in the human proteome. The feasibility of our method will be established by attaining 4 main aims: i) in vitro K,C,M protein labelling, ii) development of a machine learning classifier to uniquely ID proteins based on their optical fingerprints, iii) fabrication of state-of-the-art plasmonic nanopores for high-resolution optical sensing of proteins, and iv) devising methods for regulating the translocation speed to enhance the signal to noise ratio. Next, we will scale up our platform to enable the analysis of thousands of different proteins in minutes, and apply it to sense blood-secreted proteins, as well as whole proteomes in pre- and post-metastatic cancer cells. NanoProt-ID constitutes the first and most challenging step towards the proteomic analysis of individual cells, opening vast research directions and applications in biomedicine and systems biology.
Summary
To date, antibody-free protein identification methods have not reached single-molecule precision. Instead, they rely on averaging from many cells, obscuring the details of important biological processes. The ability to identify each individual protein from within a single cell would transform proteomics research and biomedicine. However, single protein identification (ID) presents a major challenge, necessitating a breakthrough in single-molecule sensing technologies.
We propose to develop a method for proteome-level analysis, with single protein resolution. Bioinformatics studies show that >99% of human proteins can be uniquely identified by the order in which only three amino-acids, Lysine, Cysteine, and Methionine (K, C and M, respectively), appear along the proteins’ chain. By specifically labelling K, C and M residues with three distinct fluorophores, and threading them, one by one, through solid-state nanopores equipped with custom plasmonic amplifiers, we hypothesize that we can obtain multi-color fluorescence time-trace fingerprints uniquely representing most proteins in the human proteome. The feasibility of our method will be established by attaining 4 main aims: i) in vitro K,C,M protein labelling, ii) development of a machine learning classifier to uniquely ID proteins based on their optical fingerprints, iii) fabrication of state-of-the-art plasmonic nanopores for high-resolution optical sensing of proteins, and iv) devising methods for regulating the translocation speed to enhance the signal to noise ratio. Next, we will scale up our platform to enable the analysis of thousands of different proteins in minutes, and apply it to sense blood-secreted proteins, as well as whole proteomes in pre- and post-metastatic cancer cells. NanoProt-ID constitutes the first and most challenging step towards the proteomic analysis of individual cells, opening vast research directions and applications in biomedicine and systems biology.
Max ERC Funding
2 498 869 €
Duration
Start date: 2019-08-01, End date: 2024-07-31
Project acronym ProMiDis
Project A unified drug discovery platform for protein misfolding diseases
Researcher (PI) Georgios SKRETAS
Host Institution (HI) ETHNIKO IDRYMA EREVNON
Call Details Consolidator Grant (CoG), LS9, ERC-2018-COG
Summary It is now widely recognized that a variety of major diseases, such as Alzheimer’s disease, Huntington’s disease, systemic amyloidosis, cystic fibrosis, type 2 diabetes etc., are characterized by a common molecular origin: the misfolding of specific proteins. These disorders have been termed protein misfolding diseases (PMDs) and the vast majority of them remain incurable. Here, I propose the development of a unified approach for the discovery of potential therapeutics against PMDs. I will generate engineered bacterial cells that function as a broadly applicable discovery platform for compounds that rescue the misfolding of PMD-associated proteins (MisPs). These compounds will be selected from libraries of drug-like molecules biosynthesized in engineered bacteria using a technology that allows the facile production of billions of different test molecules. These libraries will then be screened in the same bacterial cells that produce them and the rare molecules that rescue MisP misfolding effectively will be selected using an ultrahigh-throughput genetic screen. The effect of the selected compounds on MisP folding will then be evaluated by biochemical and biophysical methods, while their ability to inhibit MisP-induced pathogenicity will be tested in appropriate mammalian cell assays and in established animal models of the associated PMD. The molecules that rescue the misfolding of the target MisPs and antagonize their associated pathogenicity both in vitro and in vivo, will become drug candidates against the corresponding diseases. This procedure will be applied for different MisPs to identify potential therapeutics for four major PMDs: Huntington’s disease, cardiotoxic light chain amyloidosis, dialysis-related amyloidosis and retinitis pigmentosa. Successful realization of ProMiDis will provide invaluable therapeutic leads against major diseases and a unified framework for anti-PMD drug discovery.
Summary
It is now widely recognized that a variety of major diseases, such as Alzheimer’s disease, Huntington’s disease, systemic amyloidosis, cystic fibrosis, type 2 diabetes etc., are characterized by a common molecular origin: the misfolding of specific proteins. These disorders have been termed protein misfolding diseases (PMDs) and the vast majority of them remain incurable. Here, I propose the development of a unified approach for the discovery of potential therapeutics against PMDs. I will generate engineered bacterial cells that function as a broadly applicable discovery platform for compounds that rescue the misfolding of PMD-associated proteins (MisPs). These compounds will be selected from libraries of drug-like molecules biosynthesized in engineered bacteria using a technology that allows the facile production of billions of different test molecules. These libraries will then be screened in the same bacterial cells that produce them and the rare molecules that rescue MisP misfolding effectively will be selected using an ultrahigh-throughput genetic screen. The effect of the selected compounds on MisP folding will then be evaluated by biochemical and biophysical methods, while their ability to inhibit MisP-induced pathogenicity will be tested in appropriate mammalian cell assays and in established animal models of the associated PMD. The molecules that rescue the misfolding of the target MisPs and antagonize their associated pathogenicity both in vitro and in vivo, will become drug candidates against the corresponding diseases. This procedure will be applied for different MisPs to identify potential therapeutics for four major PMDs: Huntington’s disease, cardiotoxic light chain amyloidosis, dialysis-related amyloidosis and retinitis pigmentosa. Successful realization of ProMiDis will provide invaluable therapeutic leads against major diseases and a unified framework for anti-PMD drug discovery.
Max ERC Funding
1 972 000 €
Duration
Start date: 2019-03-01, End date: 2024-02-29
Project acronym SM-Epigen
Project Revealing the Epigenetic Regulatory Network with Single-Molecule Precision
Researcher (PI) Efrat SHEMA-YAACOBY
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), LS9, ERC-2018-STG
Summary Genes and genomic elements are packaged by chromatin structures that regulate their activity. The fundamental unit of chromatin is the nucleosome, composed of an octamer of histones. The large numbers of histone modifications, chromatin remodelers and transcription factors (TFs) that interact with our genome has fuelled speculation that multiple elements act combinatorially to direct specific outcomes. However, the field lacks technologies for detection and analysis of such combinations, thus impeding our ability to test this hypothesis and shed light on human genome regulation.
Our recent proof-of-principle for a single-molecule system for mapping combinatorial chromatin modifications holds the technological solution. This powerful method can identify directly unique combinations of epigenetic marks and reveal regulatory modules that can only be ascertained by single-molecule studies.
The proposed project will scale-up and advance our technology to establish robust high-throughput systems for investigating combinatorial chromatin and TF interactions and identify their genomic locations, thus bridging the gap between single-molecule proteomics and genomics. We will apply it to address basic questions in epigenetic regulation during early development, and define the network of interactions between histone marks, DNA methylation and the core TFs in stem cells and differentiated cells. We will also harness our technology to reveal the tissue-of-origin of cell-free DNA circulating in our blood in the form of nucleosomes, and apply it to devise novel strategies for early detection of cancer and other diseases.
Successful implementation and dissemination of these novel systems will yield a transformative new technology for functional genomics that will unravel the chromatin language during early development. This work will open new research directions at the interface of genomics and proteomics, and pave the way for the development of therapeutic and diagnostic tools.
Summary
Genes and genomic elements are packaged by chromatin structures that regulate their activity. The fundamental unit of chromatin is the nucleosome, composed of an octamer of histones. The large numbers of histone modifications, chromatin remodelers and transcription factors (TFs) that interact with our genome has fuelled speculation that multiple elements act combinatorially to direct specific outcomes. However, the field lacks technologies for detection and analysis of such combinations, thus impeding our ability to test this hypothesis and shed light on human genome regulation.
Our recent proof-of-principle for a single-molecule system for mapping combinatorial chromatin modifications holds the technological solution. This powerful method can identify directly unique combinations of epigenetic marks and reveal regulatory modules that can only be ascertained by single-molecule studies.
The proposed project will scale-up and advance our technology to establish robust high-throughput systems for investigating combinatorial chromatin and TF interactions and identify their genomic locations, thus bridging the gap between single-molecule proteomics and genomics. We will apply it to address basic questions in epigenetic regulation during early development, and define the network of interactions between histone marks, DNA methylation and the core TFs in stem cells and differentiated cells. We will also harness our technology to reveal the tissue-of-origin of cell-free DNA circulating in our blood in the form of nucleosomes, and apply it to devise novel strategies for early detection of cancer and other diseases.
Successful implementation and dissemination of these novel systems will yield a transformative new technology for functional genomics that will unravel the chromatin language during early development. This work will open new research directions at the interface of genomics and proteomics, and pave the way for the development of therapeutic and diagnostic tools.
Max ERC Funding
1 500 000 €
Duration
Start date: 2018-11-01, End date: 2023-10-31
