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Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: SMALL BUSINESS PHASE II | Award Amount: 526.00K | Year: 2013

This Small Business Innovation Research (SBIR) Phase II project proposes to develop a high-throughput, multi-analyte chromatin immunoprecipitation (ChIP) assay. ChIP is a widely used technique among life science and biomedical researchers seeking to understand how the epigenetic mechanism of histone post-translational modifications impacts the varied biological functions that are regulated through chromatin-protein interactions. Analysis ranges from single gene (using PCR) through to genome-wide (next generation sequencing (NGS)). As envisioned, the proposed technology will transform the lengthy and cumbersome multi-day ChIP into a high-throughput compatible single-day experiment. An antibody is linked to oligonucleotides containing NGS platform-compatible tags and bar-code sequences for simultaneous analysis of multiple DNA-protein interactions. The insertion of the oligonucleotides into chromatin at sites flanking antibody-bound chromatin fragments will enable direct detection (by PCR or NGS) of DNA fragments associated with the protein(s) of interest. Phase II efforts will focus on developing robust and reproducible methods for both antibody conjugation and technology validation with a panel of antibodies specific for varying classes of chromatin associating proteins, including histone and non-histone targets. DNA libraries produced by the novel method and that of traditional ChIP will be compared first by quantitative PCR for gene-specific analysis and subsequently genome-wide, by NGS.

The broader impact/commercial potential of this project, if successful, will be the development of a high throughput multi-analyte ChIP assay that will have significant impact scientifically and commercially in the life sciences and biomedical research arenas. In less than five years post-launch, it is projected to replace traditional current ChIP methods, which represents 20-25% of the epigenetic research tools market estimated at $175-245M in 2010. The life sciences research tool market is currently estimated at $42 billion, with the epigenetic sector enjoying high growth fueled by a shift of researchers to purchase commercial epigenetic products rather than in-house made, and by advances in NGS, which has accelerated genome-wide epigenetic analyses. The development of this method will open epigenetic analysis to virtually all researchers by eliminating technical barriers and by significantly reducing sample size requirements associated with traditional ChIP, including a potential for single cell analysis. Development of this technology will spur the creation of additional novel technologies such as homogeneous ChIP for high-throughput screening, multi-analyte ChIP, and open the door for environmental, nutrition, and toxicology disciplines to study the epigenetic profiles of any eukaryotic organism a genome wide scale.


Patent
Active Motif, Inc. | Date: 2014-05-22

Disclosed herein are compositions, methods and kits useful for epigenetic analysis based on the use of transposons that are targeted to specific regions of chromatin based on DNA-DNA interactions, protein-protein interactions, RNA-RNA interactions, and nucleic acid-protein interactions.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.22K | Year: 2015

DESCRIPTION provided by applicant Long non coding RNAs lncRNAs have recently emerged as regulators of gene expression functioning via mechanisms that involve chromatin modification transcription and post transcriptional processing LncRNAs exceed the number protein coding genes are transcribed by the same cellular machinery and have similar structural features as messenger RNAs Recent studies have shown that correct orchestration of lncRNA expression is necessary for normal central nervous system development and their dysregulation has been implicated in the etiology of several human neurological diseases In contrast to advances in lncRNA expression profiling much less is known about their function Gaining insight into the mechanisms by which lncRNAs function requires the development of techniques that enable the identification of their genomic targets One such technique is RNA antisense purification RAP which uses a pool of overlapping antisense biotinylated probes to capture lncRNAs and the associated DNA is used to identify the genomic binding patterns Developed only recently RAP has been use to profile the genomic targets of a handful of lncRNAs in cultured cell lines This Phase I SBIR proposal intends to establish the robustness of RAP in neurobiological model systems and to assess its potential for the co incident identification of lncRNA associated proteins This would enable scientists to directly identify which lncRNAs and proteins are both present at a given genomic locus using the same technique Aim efforts will establish transfer of technical know how of using RAP to target three lncRNAs whose genomic distribution has been determined in specific cell lines Aim efforts will establish the feasibility of applying RAP to human and mouse neural cell lines and to perfused mouse brain The DNA targets of three lncRNAs known to be expressed in brain will be determined by next generation sequencing To establish whether RAP can be used to isolate and identify lncRNA associated proteins by mass spectrometry the lncRNA HOTAIR which is known to interact with the Polycomb Repressive Complex PRC will be used for proof of concept PRC is a chromatin modifying complex containing the EZH and SUZ proteins The goal of Aim is to adapt RAP to enable the successful identification of PRC constituent proteins EZH or SUZ peptides in HOTAIR containing complexes isolated by RAP Successful completion of these objectives will form the basis of future Phase II efforts where the potential of utilizing RAP in neurological systems will be further developed into a suite of enabling tools products and services that will accelerate the functional analysis of lncRNAs in the etiology of neurological behaviors and diseases The products envisioned include custom synthesis of lncRNA probe sets RAP assay kits containing a detail protocol and reagents and providing profiling of lncRNA associated proteins or targeted genomic regions as a service PUBLIC HEALTH RELEVANCE Long non coding RNAs lncRNAs represent a new class of genes involved in regulating gene expression despite of their apparent lack of protein coding capacity Dysregulation of lncRNA expression has been associated with the development of several neurological diseases including Alzheimerandapos s and Huntingtonandapos s but little is known about how lncRNAs function This Phase I proposal describes feasibility studies for applying a newly developed technique used to determine the genomic targets of lncRNAs to neurobiology model systems which if successful will further our understanding of how lncRNAs function in the central nervous system and how their dysregulation contributes to the development of neuropathologies


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2016

Summary RNA plays a central role in numerous cellular processes Over RNA modifications have been characterized of which twelve have been identified in messenger RNA mRNA With the exception of the mRNA cap structure little is known about their function The discovery that the FTO gene initially linked to obesity and energy homeostasis is an oxidative demethylase eraser of methyl adeonsine m A in RNA has spurred interest in understanding the biological function of RNA modifications and how their dysregulation may impact disease The identification of hydroxymethyl adenine hm A and formyl adensosine f A as demethylation intermediates with the potential for independent biological function has resulted in the coining of the terms epitranscriptome and epi modifications drawing comparison to the dynamic regulation of methylcytosine m C and hydroxymethylcytosine hm C in DNA The epitranscriptome field is still in its discovery phase with an unmet need for highly sensitive and specific reagents for the visualization isolation and analysis of the biological function of RNA epi modifications This Phase I proposal intends to develop and validate an RNA epi modification multi epitope tag MET recombinant antibody toolbox wherein the antibody heavy chain is engineered to contain the sequences recognized by the protein ligase SortaseA and the biotin ligase BirA thereby enabling targeted end user customizable labeling XHis tag is also present enabling purification of the functionalized antibody under mild conditions SortaseA can be used to attach a large repertoire of payloads ranging from fluorescent dyes to bioactive peptides Targeted biotinylation enables leveraging of avidin streptavidin based technologies for maximizing detection sensitivity or isolation efficiency in enrichment based detection methods Aim efforts will develop a specificity validation pipeline using existing RNA modification antibodies and DNA modification antibodies whose targets are also found in RNA In Aim recombinant MET antibodies will be generated to two RNA epi modifications using the Aim specificity pipeline to identify highly specific antibodies Aim efforts will establish proof of concept for the targeted conjugation of MET antibodies to a fluorophore using Sortase A or to biotin using BirA Future Phase II efforts will refine these methodologies and will be expand to include development of kits and reagents for end user customized antibody conjugates and assay systems for the visualization isolation and analysis of RNA epi modifications in a variety of cell and tissue types These antibodies and kits will serve as enabling tools allowing the discovery of the fundamental mechanisms that regulate RNA epigenetics and could eventually be used in clinical or diagnostic applications Project Narrative While the biological significance of chemical modifications found on RNA molecules and their interacting proteins is still in its discovery phase already it is known that the dysfunction of these proteins are involved in obesity mental retardation and possibly substance abuse This grant proposes to develop a highly specific and sensitive tool kit of antibodies that will allow for the visualization isolation and study of these biomolecules which will further our understanding of their role in human diseases


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 500.00K | Year: 2013

This Small Business Innovation Research (SBIR) Phase II project proposes to develop a high-throughput, multi-analyte chromatin immunoprecipitation (ChIP) assay. ChIP is a widely used technique among life science and biomedical researchers seeking to understand how the epigenetic mechanism of histone post-translational modifications impacts the varied biological functions that are regulated through chromatin-protein interactions. Analysis ranges from single gene (using PCR) through to genome-wide (next generation sequencing (NGS)). As envisioned, the proposed technology will transform the lengthy and cumbersome multi-day ChIP into a high-throughput compatible single-day experiment. An antibody is linked to oligonucleotides containing NGS platform-compatible tags and "bar-code" sequences for simultaneous analysis of multiple DNA-protein interactions. The insertion of the oligonucleotides into chromatin at sites flanking antibody-bound chromatin fragments will enable direct detection (by PCR or NGS) of DNA fragments associated with the protein(s) of interest. Phase II efforts will focus on developing robust and reproducible methods for both antibody conjugation and technology validation with a panel of antibodies specific for varying classes of chromatin associating proteins, including histone and non-histone targets. DNA libraries produced by the novel method and that of traditional ChIP will be compared first by quantitative PCR for gene-specific analysis and subsequently genome-wide, by NGS. The broader impact/commercial potential of this project, if successful, will be the development of a high throughput multi-analyte ChIP assay that will have significant impact scientifically and commercially in the life sciences and biomedical research arenas. In less than five years post-launch, it is projected to replace traditional current ChIP methods, which represents 20-25% of the epigenetic research tools market estimated at $175-245M in 2010. The life sciences research tool market is currently estimated at $42 billion, with the epigenetic sector enjoying high growth fueled by a shift of researchers to purchase commercial epigenetic products rather than in-house made, and by advances in NGS, which has accelerated genome-wide epigenetic analyses. The development of this method will open epigenetic analysis to virtually all researchers by eliminating technical barriers and by significantly reducing sample size requirements associated with traditional ChIP, including a potential for single cell analysis. Development of this technology will spur the creation of additional novel technologies such as homogeneous ChIP for high-throughput screening, multi-analyte ChIP, and open the door for environmental, nutrition, and toxicology disciplines to study the epigenetic profiles of any eukaryotic organism a genome wide scale.


Patent
Active Motif, Inc. | Date: 2012-11-23

The invention provides novel methods and materials for genetic and genomic analysis using single or multiplex isolation of protein-associated nucleic acids, including transposase-assisted chromatin immunoprecipitation (TAM-ChIP) and antibody-oligonucleotide proximity ligation. These methods comprise tagging and isolating chromatin or other protein-associated nucleic acids and using antibody-oligonucleotide complexes that recognize the proteins associated with such nucleic acids.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2016

Project Summary The advent of the CRISPR Cas technology has revolutionized genome engineering resulting in a vast array of new applications based on the ability of a single guide RNA sgRNA to target a Cas protein in living cells Engineered DNA binding molecule mediated chromatin immunoprecipitation or enChIP was developed and patented by the Fujii lab at Osaka University and uses transient transfection of an sgRNA and an epitope tagged deactivated Cas dCas to target a specific genomic region This approach allows for the identification of DNA and proteins that interact with a specific genomic region without prior information as to what those interacting components are and thus provides a comprehensive understanding of locus specific genomic regulation The need for transfection limits enChIP to cells that can be grown and transfected in culture One of the challenges that plagues neuroscience research is the lack of cell culture models that accurately replicate neurological states To demonstrate enChIPandapos s usefulness for mental health research we will target binding sites for Tcf l part of the Wnt Catenin pathway in E mouse neurons and tissue The experiments described in this Phase I SBIR proposal will establish proof of concept that enChIP can be adopted for use in a cell free system which will enable its broad use for neuroscience research thereby increasing our understanding of genomic regulation in the nervous system In Aim we will establish conditions for in vitro assembly of the sgRNA dCas complex and specific targeting with genomic DNA Subsequent efforts will focus on determining optimal conditions for specific targeting of the sgRNA dCas RNP complex with fixed chromatin extracted from cancer cells and primary mouse neurons Aim and mouse brain tissues Aim Success of these Phase I efforts will result in the development of methods for the identification of proteins and DNA looping events at the targeted locus and will form the basis of future Phase II efforts which will expand to include identification of locus associated regulatory RNAs and the expansion of the technology to other cell and tissue types We envision that the commercial potential of cell free enChIP will be first be realized initially as a custom service and eventually as an assay kit consisting of reagents and a detailed protocol for the neuroscience research community and for the life sciences field in general Active Motif Project Narrative enChIP allows the unprecedented isolation of a single genomic locus along with any co bound proteins and nucleic acids but requires transfection of targeting guideRNA dCas constructs Here we propose the adaptation of enChIP to work on chromatin isolated from any cell type without the need for transfection and transient expression of the targeting constructs and expands the potential of this technology to neurobiological disease models Identifying novel proteins their isoforms or mutations and DNA looping events that are specific to a diseased state will offer unique therapeutic targets


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 150.00K | Year: 2012

This Small Business Innovation Research (SBIR) Phase I project proposes to develop a high-throughput, multi-analyte chromatin immunoprecipitation (ChIP) system. ChIP is a widely used technique among life science and biomedical researchers seeking map the specific DNA sequences that are bound by a DNA binding protein. Its use ranges from single gene analysis through to genome-wide applications that use next generation sequencing (NGS) technologies as readout. As envisioned, the proposed technology will transform the lengthy and cumbersome multi-day ChIP into a high throughput compatible single-day experiment. The technology will result in the insertion of oligonucleotides containing NGS platform-compatible tags and bar-code sequences into DNA fragments associated with the immunoprecipitating antibodies. Each antibody will be associated with a unique barcode that can also be used for direct DNA sequencing or PCR analysis. Feasibility of this approach will be achieved by systematic identification of optimal conditions for insertion of the bar-code containing oligonucleotides into chromatin, and then, with antibody-coupled oligonucleotides. Final validation will be performed at the genome scale using NGS to compare the genomic representation of the library produced by the novel method with that of traditional ChIP.

The broader impact/commercial potential of this project is far reaching. The life sciences research tools market is currently estimated at $42 billion, with the epigenetic sector experiencing high growth fueled by researchers purchasing commercial epigenetic products rather than spending the time and effort to develop them in-house, and by advances in NGS, which has accelerated genome-wide epigenetic analyses. Successful development of the high-throughput, multi-analyte ChIP will have significant impact scientifically and commercially in the life sciences and biomedical research arenas. In less than five years post-launch, it is projected to replace traditional ChIP, which represents 20-25% of the epigenetic research tools market estimated at $175-245M in 2010. The development of this method will open epigenetic analysis to virtually all researchers by eliminating technical barriers, and by significantly reducing sample size requirements associated with traditional ChIP, including a potential for single cell analysis. Development of this technology will spur the creation of additional novel technologies such as homogeneous ChIP for high throughput screening, multi-analyte ChIP, and open the door for environmental, nutrition, and toxicology disciplines to study the epigenetic profiles of any eukaryotic organism on a genome wide scale.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2012

This Small Business Innovation Research (SBIR) Phase I project proposes to develop a high-throughput, multi-analyte chromatin immunoprecipitation (ChIP) system. ChIP is a widely used technique among life science and biomedical researchers seeking map the specific DNA sequences that are bound by a DNA binding protein. Its use ranges from single gene analysis through to genome-wide applications that use next generation sequencing (NGS) technologies as readout. As envisioned, the proposed technology will transform the lengthy and cumbersome multi-day ChIP into a high throughput compatible single-day experiment. The technology will result in the insertion of oligonucleotides containing NGS platform-compatible tags and "bar-code" sequences into DNA fragments associated with the immunoprecipitating antibodies. Each antibody will be associated with a unique barcode that can also be used for direct DNA sequencing or PCR analysis. Feasibility of this approach will be achieved by systematic identification of optimal conditions for insertion of the bar-code containing oligonucleotides into chromatin, and then, with antibody-coupled oligonucleotides. Final validation will be performed at the genome scale using NGS to compare the genomic representation of the library produced by the novel method with that of traditional ChIP. The broader impact/commercial potential of this project is far reaching. The life sciences research tools market is currently estimated at $42 billion, with the epigenetic sector experiencing high growth fueled by researchers purchasing commercial epigenetic products rather than spending the time and effort to develop them in-house, and by advances in NGS, which has accelerated genome-wide epigenetic analyses. Successful development of the high-throughput, multi-analyte ChIP will have significant impact scientifically and commercially in the life sciences and biomedical research arenas. In less than five years post-launch, it is projected to replace traditional ChIP, which represents 20-25% of the epigenetic research tools market estimated at $175-245M in 2010. The development of this method will open epigenetic analysis to virtually all researchers by eliminating technical barriers, and by significantly reducing sample size requirements associated with traditional ChIP, including a potential for single cell analysis. Development of this technology will spur the creation of additional novel technologies such as homogeneous ChIP for high throughput screening, multi-analyte ChIP, and open the door for environmental, nutrition, and toxicology disciplines to study the epigenetic profiles of any eukaryotic organism on a genome wide scale.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.83K | Year: 2014

DESCRIPTION (provided by applicant): Epigenetic mechanisms regulate gene expression potential and alterations in epigenetic marks, such as DNA methylation and histone modifications, have been associated with a variety of diseases. Tissue samples (both research and clinical) are often preserved by formalin fixation and paraffin embedment (FFPE), which allows for long term storage in minimally controlled environments. However there can be significant variability in the preparation of FFPE samples and the preservation conditions are harsh, making FFPE samples challenging for sophisticated downstream analyses. Clinical FFPE samples are often accompanied by valuable information including, histology, treatment course and patient outcome, thus they are an incredibly valuable source for linking basic and clinical research. The goal of this Phase I proposal is to provide the feasibility studies to use or newly developed transposase associate chromatin immunoprecipitation (TA-ChIP) to localize DNA methylation and

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