1989 — 1993 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
System For Large-Scale Automated Dna Sequencing @ University of Wisconsin Madison
We propose to develop efficient high-speed automated methods for the analysis of DNA sequence. Such methods are essential to the success of the Human Genome Initiative, in which the sequence of the human genome will be determined and used as a powerful tool for biological and medical research. Two aspects of sequence analysis will be addressed, the procedures in which DNA for sequencing is produced and fragmented, and the methods employed for separation and analysis. In the first area we will use the Polymerase Chain Reaction method in conjunction with support chemistry to produce pure sets of single-stranded DNA fragments for separation and detection. This chemistry will be automated using a combination of temperature ramping, filtration, and pipetting. We will also investigate the potential of phosphorothioate-based sequencing chemistry in automated sequencing. In the second area capillary electrophoretic separation of the DNA fragments will be investigated for its potential to dramatically increase speed and resolution in automated DNA sequencing.
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1 |
1989 — 1995 |
Smith, Lloyd [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Presidential Young Investigator Award @ University of Wisconsin-Madison
This award provides funds for a PYI award to an outstanding young scientist interested in the development of new bioanalytic techniques. His planned activities include further development of automated DNA and protein sequencing. Dr. Smith's previous work was central to the development of the first generation of automated DNA sequencers. The development of new instruments and associated techniques intended specifically for the chemical and physical analysis of molecules of biological interest has played a key role in the remarkable success of modern genetics and biochemistry. Continued progress in these development efforts, with the aim of further automation and increased sensitivity should have a significant effect on our understanding of the control of gene expression, of the adaptation and evolution of living organisms, and of the biochemistry that is basic to the biotechnology industry.
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0.915 |
1990 |
Smith, Lloyd M |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Mapping &Sequencing the Human Genome Symposium @ American Chemical Society
The Analytical Division of the American Chemical Society has asked me to organize the symposium for the August 1990 national meeting to be held in Washington, DC. The title will be "Mapping and Sequencing the Human Genome: New Challenges in Analytical Chemistry". The idea is to expose and educate the community of analytical chemists to some of the problems, challenges, and opportunities presented by the Human Genome Initiative (HGI). This appears to be a particularly propitious time to hold such a symposium, for several reasons. First, the field of analytical chemistry is close to unique, in that it has historically been concerned with the development of new technology as a goal within itself. This is in contrast to the situation in most of the biological and chemical sciences, where technology development is generally not considered to be a high priority. Second, the field of analytical to problems in analysis, separation, and purification. The problems presented by HGI are exactly the sort which such a group of researchers would find stimulating and challenging. Third, the HGI, at this time, is primarily a technology development program: that is, we need to develop powerful and effective tools for dissecting and analyzing genomes, from the megabase to the single nucleotide level. Once these tools have been developed, it will be possible to proceed rapidly in applying the new technology to genomic analysis. However, the groups which are most interested in using this map and sequence information, i.e., the medical and biological research communities, do not in general have tremendous expertise (as mentioned above) in the development of such tools. Fourth, the analytical community is in need of new problems to tackle. The problem which played an important role in the development of analytical chemistry as a field were often problems in structural analysis and trace element determination which were effectively addressed in large part decades ago. Although many new methods, particularly in the area of magnetic resonance and optical instrumentation, have been developed, the vast range of interesting new problems presented by the extremely rapid growth in the fields of biotechnology and genetic engineering would be of great interest. And finally, the resources available to scientists under the aegis of HGI, would be of great help to scientists who currently find the funding levels in their classical fields to be severely constrained. Thus, the idea behind the symposium is to help educate the analytical community about HGI, with the idea that this will stimulate their interest in taking on some of its challenges, and thereby help to achieve rapid progress in attaining the goals of HGI.
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0.904 |
1994 — 1996 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A System For Large-Scale Automated Dna Sequencing @ University of Wisconsin Madison
The major goal of this proposal is the development of an automated system for DNA sequence analysis. The system, based upon the shotgun strategy, will be developed in three phases, in which successively greater fractions of the sequencing process are automated. In Phase I the steps of sequencing reaction performance, sequencing reaction purification, gel loading, electrophoresis, and data analysis will be automated in an integrated format. In Phase II the system will be extended to include plaque picking, bacterial cell culture and template purification. In Phase III a limiting dilution front-end strategy for clonal isolation will be incorporated. Thus by the end of Phase III the system will automate all aspects of shotgun sequencing subsequent to the transformation of bacterial cells with recombinant DNA molecules (library construction). The projected operating cost at a throughput of 300,000 bases raw data (20 kb finished data in the worst-case scenario) per day is about $0.15/base finished data. This system will require two persons for operation.
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1 |
1997 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
System For Largescale Automated Dna Sequencing @ University of Wisconsin Madison
The major goal of this proposal is the development of an automated system for DNA sequence analysis. The system, based upon the shotgun strategy, will be developed in three phases, in which successively greater fractions of the sequencing process are automated. In Phase I the steps of sequencing reaction performance, sequencing reaction purification, gel loading, electrophoresis, and data analysis will be automated in an integrated format. In Phase II the system will be extended to include plaque picking, bacterial cell culture and template purification. In Phase III a limiting dilution front-end strategy for clonal isolation will be incorporated. Thus by the end of Phase III the system will automate all aspects of shotgun sequencing subsequent to the transformation of bacterial cells with recombinant DNA molecules (library construction). The projected operating cost at a throughput of 300,000 bases raw data (20 kb finished data in the worst-case scenario) per day is about $0.15/base finished data. This system will require two persons for operation.
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1 |
1998 — 1999 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development and Use of An Automated System For Sequencing At Megabase Level @ University of Wisconsin Madison
nucleic acid sequence; computer assisted sequence analysis; computer system design /evaluation; biomedical automation; computer program /software; gel electrophoresis; robotics; data collection methodology /evaluation; animal genetic material tag; human genetic material tag;
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1 |
1998 — 2004 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dna Sequencing by Charge Reduction Esi-Ms @ University of Wisconsin Madison
DESCRIPTION: (Applicant's Abstract) In this project we propose to develop a novel mass spectrometric method, Charge Reduction (CR) Electrospray Ionization (ESI) Mass Spectrometry (MS), for the analysis of enzymatic DNA sequencing reactions. A nanospray Charge Reduction source will be constructed and interfaced with a commercial Electrospray Time-of-Flight (ESI-TOF) instrument. This source will utilize the emitting radioisotope 210Po to ionize the source bath gas, producing ions which in turn neutralize the charges present on the ions generated in the ESI process. This charge neutralization process will greatly reduce the complexity of the mass spectra obtained in ESI-MS from large biopolymers such as DNA, thereby enabling the mass spectrometric analysis of biopolymer mixtures which are otherwise too complex to be useful. It is anticipated that the ability to reduce the number of charge states produced in the electrospray method, coupled with the intrinsic speed and accuracy of mass spectrometric analysis, will provide a viable route to the rapid mass spectrometric analysis of Sanger sequencing reactions in seconds, a dramatic improvement over the capabilities of electrophoretic methods.
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1 |
1998 — 1999 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Modular System For Large Scale Dna Sequencing @ University of Wisconsin Madison
The major goal of this Program Project is the development of robust, exportable technology for high throughput DNA sequencing, developed and tested in the context of a medium-scale production sequencing environment. The project builds upon the technology developed in the Smith laboratory at the University of Wisconsin (UW) over the past several years for an integrated system for sequencing comprised of three major parts: a high throughput robotics system for sample preparation, a novel electrophoresis platform for fluorescence-based automated DNA sequencing, and a software system for data analysis. Over 2 million bases of raw data have been produced with this system to data, including the reactions, electrophoresis and fluorescence detection, and the sequence data processing and base-calling. All of the technology is robust, modular, exportable, and available to the community. Average read lengths are >1000 bases. The Program Project is structured as a close collaboration with the chromosome 19 mapping group at Lawrence Livermore National Laboratory, who are responsible for producing contiguous sequence-ready clones covering a region of 10 Mb of human DNA on chromosome 19 and 2 Mb of synthetic mouse DNA. Two major emphases of the technology development efforts are a) increasing throughput of the automated system to a level of 6 Mb/yr, and b) automated major aspects of the finishing process. The throughput of the integrated systems will be increased to a level of 6 Mb/yr by the end of the grant period, with staffing requirements of approximately 6 FTEs to operate at that level.
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1 |
2001 — 2003 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Surface Invader Assay For Snp Analysis On Dna Arrays @ University of Wisconsin Madison
DESCRIPTION (Applicant's Abstract): The goal of this proposal is to develop a single step method for the parallel scoring of single nucleotide polymorphisms (SNPs) in a surface array format. The approach is based upon a recently developed invasive cleavage assay for SNP scoring referred to as the 'Invader" assay (Third Wave Technologies, Inc.) As it is a signal amplification technology rather than a target amplification technology, this assay is not subject to the contamination/carryover problems characteristic of PCR. It is a very simple, robust, and isothermal assay well-suited for high throughput analysis. In the proposed surface-based version of the procedure (the "Surface Invader Assay"), addition to the surface of target human genomic DNA containing a given SNP allele will result in specific cleavage of a corresponding surface-immobilized probe oligonucleotide containing a fluorophore-quencher dye pair. This cleavage will occur between the fluorophore and the quencher, leaving the unquenched fluorophore attached to the surface. The fluorescence intensity of this unquenched fluorophore will be substantially greater than that of the quenched fluorophore, and thus the region of the DNA array containing that probe oligonucleotide will exhibit increased fluorescence intensity in a specific target-dependent fashion. By employing an array of such probe oligonucleotides, one for each SNP allele to be typed, a single addition of target human genomic DNA to the surface, along with the other needed assay reagents (buffer and enzyme), followed by incubation, washing, and fluorescence imaging steps, will yield the genotypes of the target DNA analyzed for each SNP represented in the array.
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1 |
2001 — 2003 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High Fidelity Surfaces and Dna Arrays @ University of Wisconsin Madison
DESCRIPTION (Investigator's Abstract): The principal goal of this proposal is the development of high quality surfaces and nucleic acid arrays for applications including the analysis of gene expression, of genetic variations, of protein: nuclei acid interactions, and optical mapping. The proposed work falls into two interrelated areas: the development of a variety of surfaces designed and optimized for particular applications; and underlying this, basic research to develop improved understanding and control of the chemistry of the surfaces themselves. The surfaces to be developed fall into three major categories: Uniform (unpatterned) surfaces, surfaces patterned in a square grid pattern, and nanostructured surfaces. The surfaces will be prepared with various chemical functionalities as determined by the application. These functionalities will include hydroxyl groups (substrates for light-directed DNA array fabrication); amino groups (substrates for optical mapping, noncovalent PCR fragment deposition, or covalent attachment of activated oligonucleotides); thiol groups (substrates for covalent attachment of activated oligonucleotides); and thio-reactive or amino-reactive groups (for covalent attachment of oligonucleotides or PCR amplicons). Both vapor-deposited gold films on glass slides, and atomically flat silicon wafers will be employed as substrates. DNA molecules will be attached either by direct chemical coupling or by electrostatic interactions onto vapor-deposited gold thin films that have been chemically modified with densely packed self-assembled monolayers of alkanethiols that are terminated with various functional groups. Attachment to atomically flat hydrogen-terminated silicon surfaces will be accomplished by the UV-mediated reaction of functionalized aliphatic alkenes to form Si-C bonds, also suitable for further modification or direct DNA attachment. The surfaces will be required to meet stringent performance criteria, including physical and chemical stability, thermal stability, amenability to hybridization and enzymatic modification, control of the density of surface-bound DNAs, and reproducibility of preparation.
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1 |
2002 — 2006 |
Smith, Lloyd (co-PI) [⬀] Hamers, Robert [⬀] Van Der Weide, Daniel (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Nirt: Nanotubes and Nanowires as Biological Sensors and Actuators: Approaching the Single-Molecule Limit @ University of Wisconsin-Madison
This proposal was submitted in response to the solicitation "Nanoscale Science and Engineering" (NSF 01-157). The aim of this project is the development and use of biologically modified nanotubes and nanowires as electrical probes of biological activity. Researchers will develop a new type of nanoscale probe, the "nano-coax", that can serve as a molecular probe only nanometers in dimensions. This new probe involves attaching biomolecules specifically to the end of silicon nanowires and carbon nanotubes, providing a very highly localized sensing region. The electrical response of the nanoprobe will be measured over a wide frequency range from kilo-Hertz to Giga-Hertz. The researchers will also explore the use of the nanoprobe as a molecular-scale actuator, using an applied electrical control signal to induce a change in activity of biological molecules tethered to the end. The research involves an interdisciplinary team of chemists, molecular biologists, and electrical engineers. The outcome of the research will be the development of a new set of bioanalytical tools able to rapidly detect biological species with unprecedented selectivity and sensitivity approaching the single-molecule limit. Successful use of nanoprobes as biological actuators would permit the direct manipulation of biological processes at the nanometer scale. The research involves a large component of graduate and undergraduate education and training. Graduate students will work together with faculty and undergraduate students as part of an interdisciplinary team. Faculty researchers will train graduate students and undergraduate students in state-of-the-art methods of materials fabrication and biological analysis, providing a workforce well-trained for industrial and academic research.
This project is aimed at the development of a revolutionary kind of biological sensor. Recent advances have led to the development of tiny wires ("nanowires") only a few nanometers in diameter. In this research project, an interdisciplinary team of scientists will fabricate nanowires and then attach biological molecules, such as DNA and proteins, to them. The researchers will then investigate the electrical signals generated when these "nanoprobes" interact with other biological molecules. The research has the potential to lead to major advances in the development of highly sensitive detectors able to identify minute quantities (perhaps as little as a single molecule) of biological molecules. The researchers will also explore the use of nanometer-sized probes to induce changes in biological activity, with long-term potential for biomedical applications. This project is co-supported by the Division of Materials Research, the Chemistry Division, the Division of Bioengineering and Environmental Systems, and the Chemical and Transport Systems Division.
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0.915 |
2002 |
Smith, Lloyd M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Single Droplet Electrospray Ionization @ University of Wisconsin Madison
DESCRIPTION (provided by applicant): We propose to develop a new generation of mass spectrometric instruments with vastly increased sensitivity, approaching the single molecule level; greatly enhanced capability for the analysis of complex mixtures and multisubunit protein complexes. The research presented here focuses on development of a new time-of-flight mass spectrometer which utilizes single charged droplets as ion sources in combination with an aerodynamic lens to focus the ions onto the center axis of the time of flight region of the mass spectrometer. Single droplets will be generated by a piezoelectric dispenser and transmitted through an aerodynamic lens where they will be desolvated to produce analyte ions. The aerodynamic lens system will replace the standard nozzle-skimmer and collisional cooling systems commonly employed in ESI-TOF. The fact that the ions will travel along a single defined trajectory, rather than along a multitude of trajectories as in conventional electrospray, will permit the collection efficiency of the ions into the mass spectrometer to be greatly increased. Ions exiting the aerodynamic lens will be electrostatically accelerated and time-of-flight mass analyzed as in conventional matrix assisted laser desorption ionization (MALDI) mass spectrometry. Calculations of the increase in detection efficiency that in theory is attainable with this approach suggest that as much as a 10exp12 fold increase could be realized; although practical considerations may limit the achievable increase, it seams clear that a very substantial sensitivity gain is to be expected. Additionally, the proposed instrument greatly simplifies the spectrometer design which should make possible the construction of a simple, robust, and cost effective mass spectrometer.
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1 |
2005 — 2008 |
Skop, Ahna (co-PI) [⬀] Smith, Lloyd (co-PI) [⬀] Moss, Richard Stretton, Antony (co-PI) [⬀] Sussman, Michael [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of a Maldi-Toftof Mass Spectrometer For Wisconsin Researchers @ University of Wisconsin-Madison
At the University of Wisconsin-Madison and in other universities throughout the state, there are over a thousand faculty performing research within the life sciences. Fundamental to many of these studies is a mass spectrometric-assisted proteomic analysis of the cells, tissues and organs that make up the organisms. The UW -Madison Biotechnology Center Mass Spectrometry/Proteomic Facility was established in 1998 as a centralized facility for the purpose of acquiring mass spectrometers and making them available to the research community on a fee-for-service basis. During the six years since inception, it has proven successful in meeting the proteomic needs of nearly one hundred different academic laboratories in a cost effective, top quality manner. However, a major deficiency for investigators using this facility has been the inability to obtain protein sequence from very small samples of tissue, a common handicap of biological research. A second limitation is the ability to routinely perform quantitative differential proteomic experiments using isotope-assisted tandem mass spectrometry. The MALDI-based tandem mass spectrometer known commonly as a MALDI-TOFTOF is an instrument that is uniquely capable to address both of these problems. Surprisingly, there is not a single MALDI-TOFTOF available in the state. Wisconsin researchers currently must drive/fly to Chicago or Boston to obtain precious instrument time. Lack of ready access to this instrument locally has hindered our researchers in obtaining the critical information needed for converting genomic sequence 'data' into a real understanding of life processes. The purpose of this award is to acquire this instrument and make it available to academic researchers statewide. The instrument will be placed within the existing UWBC Core Mass Spectrometry/Proteomics Facility, ensuring that (i) it will be accessible to a large diverse group of researchers, (ii) that it will heavily used and (iii) that it will be well maintained for optimal sensitivity and overall reliability.
The broader impacts of this project are several-fold. First, the research users represent a large number of different academic disciplines, from Animal/Food Science to Engineering to Prebiotic Chemistry to the 'traditional' Molecular, Cellular and Organismal Biology areas. Six years of prior experience in operating expensive and sophisticated mass spectrometers ensures that the operation of this MALDI-TOFTOF will be well organized, accessible and affordable for the entire community. In addition, the university has strived to ensure that the UWBC core facilities in general, and the Mass Spectrometry/Proteomics in particular, are utilized for education at all levels, including undergraduates and high schools, both locally and in Wisconsin communities with underrepresented groups, such as economically impoverished schools in Milwaukee and Native American Indians in small communities of northern Wisconsin. This instrument will advance discovery and promote teaching and generally enhance the academic infrastructure, not only at UW-Madison, but also, all over the state.
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0.915 |
2005 — 2007 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Snp Analysis by Surface Invasive Cleavage On Dna Arrays @ University of Wisconsin Madison
DESCRIPTION (provided by applicant): In this competitive renewal proposal we seek to continue the development of "surface invader" DNA arrays for high throughput SNP analysis. During the prior grant period we developed the surface invader assay and demonstrated "proof-of-principle" for the parallel analysis of SNPs directly from unamplified human genomic DNA samples on spotted DNA arrays. Now we desire to take this technology to the next step, i.e. to implement this chemistry in a high-density DNA array format, where the DNA array is manufactured by photolithographic methods. There are three issues that will be addressed to enable fabrication of the needed DNA arrays. First, chemistry will be implemented for the photolithographic synthesis of DNA molecules in the 5'->3' direction. Second, chemistry will be developed and implemented to allow the photolithographic synthesis of two different sequences intermixed in a single array element; both are needed to form the / necessary ternary complex on the surface. Third, glassy carbon and/or diamond thin film substrates will be developed for use in photolithographic DNA synthesis. A secondary thrust of this proposal will be to investigate an approach to detection based upon the detection of single molecule cleavage events on the surface. The rolling circle amplification reaction can make DNA molecules hundreds of thousands of bases long from a single initiation site. As it is very easy to detect such long DNA molecules, this raises the possibility of directly detecting individual cleaved DNA molecules on the surface; in fact, our preliminary results have shown this capability. This is intriguing, as it opens the possibility of analyzing very low levels of nucleic acid targets, perhaps as little as a single molecule, with many applications in areas such as gene expression analysis, infectious disease diagnostics, biowarfare agent detection, and genotyping from minute samples such as buccal swabs or fingerprick blood samples.
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1 |
2005 — 2006 |
Smith, Lloyd M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Supercritical Fluid Extraction For Membrane Proteomics @ University of Wisconsin Madison
[unreadable] DESCRIPTION (provided by applicant): [unreadable] The goal of this proposal is to develop a new approach to the mass spectrometric analysis of membrane, proteins. The proposed research addresses the critical problem with current membrane proteomics studies: namely, the efficient extraction of membrane proteins from biological samples in a form suitable for chromatography and high-throughput mass-spectrometrie analysis. Membrane proteins are typically solubilized in detergents: however, detergent solutions are not amenable to mass spectrometric analysis. This has presented a tremendous obstacle to the mass spectrometric analysis of membrane proteins. We propose to address this problem by using supercritical carbon dioxide as a mass spec- compatible solvent for membrane proteins. This solvent is a fluid with excellent dissolving power and complete transparency to a mass spectrometer. The work will proceed in two phases: in the first phase the basic feasibility of the approach will be evaluated by conducting studies on synthetic peptides and commercially available membrane proteins. The goal will be to demonstrate that these hydrophobic molecules can be solubilized in supercritical CO2, and that once solubilized they can be mass analyzed. In the second phase of the work the solubilized samples will be separated by supercritical fluid chromatography (SFC), a well-established method capable of fast separation with high resolution for non-polar compounds. This separation capability is critical to the analysis of complex mixtures. The approach will then be extended to the analysis of more complex biological samples such as a purified PhotoSystem I complex containing approximately 13 proteins and Fibroblast Growth Factor (FGF) receptor expressed in animal cell lines. The successful development of this new technology for membrane proteomics will address a critical gap in current proteomics efforts. [unreadable] [unreadable]
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1 |
2008 — 2012 |
Smith, Lloyd M |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Proteomics Core @ University of Wisconsin Madison
Amino Acids; Applications Grants; Arg-Lys; Categories; Cells; Charge; Chromatography, High Performance Liquid; Chromatography, High Pressure Liquid; Chromatography, High Speed Liquid; Chromatography, Mass; Classification; Code; Coding System; Complement; Complement Proteins; Complex Mixtures; Development; Dimensions; Discipline; Dissociation; ESI; Electron Transport; Electrospray Ionization; Endopeptidases; Esteroproteases; Evolution; Figs; Figs - dietary; Gases; Genome; Goals; Grant Proposals; Grants, Applications; HPLC; High Pressure Liquid Chromatography; Histones; Individual; Informatics; Instrumentation, Other; Ions; MALD-MS; MALDI; MALDI-MS; Mass Chromatography; Mass Spectrum; Mass Spectrum Analysis; Metabolic Glycosylation; Methods; Modification; Numbers; Organism; P01 Mechanism; P01 Program; Peptidases; Peptide Hydrolases; Peptide Peptidohydrolases; Peptides; Phase; Phosphorylation; Photometry/Spectrum Analysis, Mass; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Pro-Arg; Process; Program Project Grant; Program Research Project Grants; Proteases; Protein Modification; Protein Modification, Post-Translational; Protein Phosphorylation; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteinases; Proteins; Proteolytic Enzyme; Proteolytic Enzymes; Proteomics; R01 Mechanism; R01 Program; RPG; Records; Research Grants; Research Program Projects; Research Project Grants; Research Projects; Research Projects, R-Series; Sampling; Shotguns; Solutions; Source; Spectrometry; Spectrometry, Mass; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Support of Research; Systematics; Technology; aminoacid; arginine-lysine; arginyllysine; base; electron transfer; experiment; experimental research; experimental study; falls; gene product; glycosylation; hESC; human ES cell; human ESC; human embryonic stem cell; innovate; innovation; innovative; instrumentation; living system; mass spectrometer; matrix assisted laser desorption ionization; new technology; prolylarginine; protease; proteinase; research study; success; tandem mass spectrometry; technology development
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1 |
2008 — 2011 |
Smith, Lloyd [⬀] Blackwell, Helen (co-PI) [⬀] Shortreed, Michael |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Stable Metal:Amorphous Carbon Substrates For Combined Spr and Ms Analysis of Bioaffinity Interactions @ University of Wisconsin-Madison
The Analytical and Surface Chemistry Program in the Division of Chemistry, with co-funding from the Biomolecular Systems Cluster in the Division of Molecular and Cellular Biosciences, is supporting Profs. Lloyd Smith and collaborators Helen Blackwell and Michael Shortreed at the University of Wisconsin to study the nature and significance of interactions between molecules in biological systems. Specifically, they are examining the "language" that bacteria use for communication involving diffusible small molecules (or "autoinducers") perceived by cognate protein receptors. Bacteria use this chemical language to assess local population densities in a process known as "quorum sensing" which enables regulation of critical processes both harmful and beneficial to their plant host. Methods to control bacterial quorum sensing in plant-associated bacteria would have a major impact on agricultural science, because >50% of crop disease worldwide is caused by quorum sensing-regulated behaviors in bacteria. Further, molecules that inhibit bacterial quorum sensing represent an entirely new class of anti-infectives that could have immediate impact on human health. The work aims to develop new technologies for the elucidation of this complex communication network - a fundamental scientific advance. The focus is to create a more general platform for the multiplex analysis of bioaffinity interactions and to develop and apply this platform in the context of bacterial quorum sensing. The platform combines three powerful capabilities: (1) label-free measurements of bioaffinity interactions; (2) a novel substrate, which permits versatile and stable biomolecule attachment; and (3) mass spectrometric (MS) analysis capability to identify unknown ligands binding to the surface. This combination of three powerful and synergistic capabilities in a single system will provide a tool of unprecedented utility for the analysis of bioaffinity interactions.
The research groups involved are interdisciplinary, training researchers at many different stages of their education. Current collaborations involve over 20 different groups on campuses worldwide, plus two companies, and span the biological and physical sciences. In conjunction with the research, the PIs engage in interdisciplinary undergraduate research training; graduate student professional development through the Delta program (the UW implementation of the NSF Center for the Integration of Research, Teaching and Learning program); professional development for junior and senior high-school instructors through regular lectures by UW faculty; and a day-long hands-on science experience for high-school AP-chemistry students.
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0.915 |
2011 — 2013 |
Smith, Lloyd M Soh, Hyongsok Tom [⬀] Stewart, Ron Thomson, James Alexander (co-PI) [⬀] |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Qpass: Quantitative Parallel Aptamer Selection System @ University of California Santa Barbara
Nucleic acid aptamers possess many useful features as affinity reagents, including facile chemical synthesis, reversible folding, thermal stability and low cost, making them a powerful alternative to antibodies and other protein-based reagents. However, over the past two decades, aptamers have suffered from the fact that 1) the conventional method of aptamer generation (SELEX) is lengthy, labor intensive and often does not yield aptamers with sufficient affinity (<1 nM) and specificity;2) there is no "standard protocol" that can be generally applied to most protein targets to generate aptamers;and 3) the characterization steps to measure the affinity and specificity of candidate aptamers are lengthy and resource-intensive, because each aptamer must be measured individually. We believe that these challenges arise from deficiencies in the conventional methodology of performing the selection, which has not changed significantly since its initial description 20 years ago. We also believe that these problems can be solved, by systematically taking fundamentally different approaches towards the three central stages of the process - selection, analysis and characterization of the aptamers. We propose here the development of such a system. We will combine three distinctly novel technologies -microfluidic selection, next-generation aptamer sequencing, and SPR Imaging - to develop the Quantitative Parallel Aptamer Selection System (QPASS) platform. The QPASS platform will generate specific aptamers with sub-nanomolar affinities (Kd) for a wide range of protein targets within 3 rounds of selection, identify a pool of the best candidates by next generation DNA sequencing and bioinformatic analysis, and home in on the optimal aptamer sequence by the parallel synthesis and measurement of the affinities of thousands of aptamer candidates. Individually, each component represents a significant technological advance. Combined this integrated approach offers an opportunity to revolutionize the process of aptamer generation.
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0.942 |
2012 — 2014 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Mechanical Nanomembrane Detector For Time-of-Flight Mass Spectrometry @ University of Wisconsin-Madison
DESCRIPTION (provided by applicant): Existing detectors for time-of-flight (TOF) mass spectrometry of intact proteins in low charge states, such as microchannel plates and electron multipliers, rely upon the emission of secondary electrons for ion detection. Unfortunately, the efficiency of this secondary electron generation falls-off severely with increasing mass of the incident ions, dramatically reducing detection sensitivity and limiting the ability of TOF mass spectrometers to provide useful mass information on large biomolecules. This problem in ion detection is one of the major reasons that biological mass spectrometry as currently practiced is predominantly directed towards the analysis of small peptides rather than towards whole intact proteins, a critical limitation in the technology. We have developed a new type of ion detector to address this problem, based upon the mechanical deformation and vibration of a nanomembrane. An incoming ion packet initiates oscillations of the nanomembrane, which are then detected by corresponding oscillations in field emission electron current from the membrane. We propose here to develop our initial prototype detector into a powerful, robust, and well- characterized device for the mass spectrometry of intact proteins up to a megadalton in size. This new detector technology will open many new opportunities in biological mass spectrometry, in areas such as biomarker discovery and monitoring, the elucidation of protein variation, and the imaging of tissue by MALDI mass spectrometry. PUBLIC HEALTH RELEVANCE: This project seeks to develop a novel detector technology to dramatically improve the mass spectrometric analysis of intact high molecular weight proteins present at low abundance and/or in complex biological samples. Research in areas such as protein variation, biomarker discovery and mass spectrometric imaging will greatly benefit from this technology, opening new possibilities in the diagnosis and treatment of disease.
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2014 — 2017 |
Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development and Applications of High Density Rna Arrays @ University of Wisconsin-Madison
DESCRIPTION: The overall goals of this project are to refine and optimize a powerful new approach that we developed for the fabrication of high density RNA arrays and to demonstrate its utility in three important and interesting applications. These arrays are RNA analogs to the high density DNA arrays that have proven over the past two decades to be tremendously powerful tools for biomolecular analysis, particularly in the areas of global transcriptomics and genome-wide genetic variation analysis. In our approach, a high density DNA array is fabricated by standard photolithographic methods using photoprotected nucleoside phosphoramidites, the surface-bound DNA molecules are enzymatically copied into their RNA complements (also tethered to the surface), and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. Our strategy begins with technology development and optimization. The utility of RNA arrays depends strongly upon the array quality. Two key interrelated quality parameters are the RNA strand sequence fidelity and the RNA lengths that can be obtained. Other parameters of interest are the surface density of the RNA strands, the nature of the substrates employed for array fabrication, and the ability to employ modified nucleosides of various types. Tools needed to measure these parameters will be developed and used to guide optimization (maximize sequence fidelity and length) of the chemistry and enzymology employed for array fabrication. Other key facets of optimization include strand density, substrate types, and inclusion of various modified nucleosides. Technology development and optimization will be performed in the context of three separate applications that open new possibilities in biomolecular analysis: 1.) Identification and characterization of fluorescent RNA mimics of GFP; 2.) Determination of microRNA binding sites in 3'- and 5'- UTR mRNA sequences; and 3.) Determining the binding specificity of RNA-binding proteins. Taken together, the above program of targeted optimization and application of RNA array technology will yield a powerful and versatile new tool for biomolecular analysis that will open many new frontiers for research in the study of normal and disease biology.
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2015 — 2017 |
Jarrard, David F. (co-PI) [⬀] Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Sequence-Specific Capture For Discovering Protein-Incrna Interactions in Prostate Cancer @ University of Wisconsin-Madison
? DESCRIPTION (provided by applicant): Protein-nucleic acid interactions play key roles in both transcriptional and translational regulation, not only in the direct interaction of proteins with DA and mRNA to regulate these processes, but also the interactions among proteins, lncRNAs, and DNAs. Understanding these interactions requires knowledge of the molecules that are involved. lncRNAs interact with proteins to achieve several consequences in gene regulation. First, lncRNAs can act as a molecular sink for proteins that may interact with DNA or RNA. Their interaction with the lncRNA thus diverts the protein from binding to its primary target. lncRNAs can also act as guides for proteins, leading them to their DNA targets to either repress or activate transcription. Finally, lncRNAs can act as platforms upon which molecules can congregate to perform a function, such as histone modification, as a team at a specific location and time. Each of these functions requires the interaction of lncRNAs with a diverse set of proteins. Knowledge of the proteins bound to a specific lncRNA will allow for determination of the mechanisms by which that lncRNA affects gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of proteins. In this proposal we focus on the particular problem of identifying the proteins that are bound to specific lncRNA molecules, their relationship to the progression of prostate cancer and their potential as markers of the disease and its progression. We describe an RNA-centric approach to discovery of the lncRNA-binding proteins that is innovative compared with existing technologies in two major ways. First, crosslinking of proteins to lncRNAs is done in vivo under normal cellular conditions. Therefore, proteins and lncRNAs will be folded normally, will be present at their normal cellular concentrations, and will be in their normal cellular locations. Second, sequence-specific capture of the lncRNA is used to extract the RNA of interest along with its crosslinked proteins. This is a universal capture strategy that does not rely on the availability of any other capture reagent (e.g. antibody or aptamer). Following enrichment by sequence-specific capture, mass spectrometry will be utilized to identify and quantify the associated proteins.
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2015 — 2018 |
Gould, Michael N Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Intact Proteoform Identification and Quantification @ University of Wisconsin-Madison
? DESCRIPTION (provided by applicant): The critical protein actors in biological systems are the intact proteoforms, namely the different forms of proteins produced from the genome in a variety of splice forms and adorned with a myriad of post-translational modifications that dramatically affect their function. Lamentably, today's bottom-up proteomic technologies, which identify and quantify peptides derived from proteins, rather than the proteins themselves, fail to deliver crucial protein-level information to biologists. We address this problem here, by developing a new paradigm for proteomic analyses, which uses intact mass and lysine count to identify and quantify the proteoforms present in a biological sample. Our strategy integrates state-of-the-art proteomic and genomic technologies. We couple a new generation of high resolution mass spectrometers and a novel isotopic tagging strategy to yield for each proteoform its accurate mass, its number of lysines, and its relative abundance. Parallel RNA-Seq analyses on the same sample will provide transcriptomic data to construct a tightly focused sample-specific proteoform database. Comparison of the mass spectrometric data with the proteoform database will reveal the proteoform identities present in the sample, and the isotopic tagging strategy will provide relative abundance. This information will comprise a revolutionary new tool for biology.
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2018 — 2020 |
Smith, Lloyd M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Novel Neucode Tagging Reagents For Identification and Quantification of Intact Proteoforms in Cancer Tissues @ University of Wisconsin-Madison
Project Summary/Abstract In this proposal we address a very substantial limitation in the ability of existing technologies to provide critical proteomic information from normal and cancer tissues. In standard proteomic analyses, proteins in complex protein mixtures such as total cell lysate are digested with proteases such as trypsin to produce peptides. These peptides are then analyzed by liquid chromatography and mass spectrometry (LC-MS), and the proteins present are identified by the presence of peptides contained within them. Relative or absolute quantification may be obtained if desired by a variety of different isotopic tagging strategies. While this strategy, termed ?bottom-up? proteomics, works well for identifying large numbers of proteins present in complex samples, it suffers from the loss of contextual information about the particular form of the proteins from which the peptides are derived, the ?proteoforms?. In order to understand the processes and pathways that are operative in cancer, it is essential to know the identities and abundances of these ?proteoforms? present in the tissues. Recently, a new approach has been developed for the identification and quantification of proteoforms in complex mixtures. In this approach two pieces of information are obtained for each proteoform in the sample: a highly accurate measurement of the proteoform intact mass, and the number of lysine residues it contains. This information allows identification and quantification of thousands of proteoforms. However, at present it can only be performed on cells grown in culture containing isotopically labeled lysine amino acids. This limitation makes it impossible to use the strategy for identification of proteoforms in cancer tissue samples. It is proposed here to develop an alternative means of introducing the isotopic tags needed for proteoform quantification and for the determination of the number of a targeted amino acid residue in each proteoform. Cysteine amino acids have been chosen in place of lysine amino acids because of their amenability to highly efficient tagging reactions, and suitable isotopic labels have been designed and will be synthesized and tested. This new chemistry will provide the power of the isotopic tagging strategy for proteomic analyses of cancer tissue samples. Such proteoform level knowledge of changes that occur in cancer will reveal a new universe of possible cancer biomarkers, as well as opening a currently closed avenue to understanding the proteomic changes that occur in cancer.
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2018 — 2021 |
Smith, Lloyd M |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Novel Technologies For Protein Analysis @ University of Wisconsin-Madison
Abstract The primary focus of my laboratory is the development of novel technologies for protein analysis, specifically centered around the concept of the proteoform. Proteoforms, each of which comprises a unique combination of amino acid sequence and post-translational modifications (PTMs), are the primary molecular effectors of cell function. Subtle sequence and PTM differences between proteoforms can completely alter their function and activity. We see comprehensive proteoform-level analysis of biological systems as absolutely essential to understanding their function, for both individual pathways and networks operative within cells, and more globally, to decipher the systems-biology-level dynamics and interactions that control cellular response. For example, one of our projects is to elucidate the interactome between specific nucleic acid sequences and the proteoforms bound to those DNA or RNA molecules. However, today's technology for global proteoform analysis in complex systems is in its infancy, offering both a great challenge and a great opportunity. We seek to develop novel strategies for comprehensive proteoform identification and quantification in complex systems. We envision combining information from multiple data streams, such as transcriptomic data (to reveal splice forms and genetic variation), bottom-up proteomics data (to reveal and localize PTMs), top-down proteomics data (to provide sequence tags for proteoform identifications), and intact mass measurements (to identify and quantify proteoforms, using information from all of the other data streams). Specific projects will develop the following: (1) robust tools for the construction of sample-specific proteoform databases; (2) new strategies for the discovery and localization of PTMs; (3) improved sample preparation, separation, and mass spectrometry methods for intact proteins; (4) synergistic approaches that utilize both intact mass measurements and selected top-down fragmentations to maximize proteoform identifications; and (5) visualization tools for proteoform families that show connections and changes between related proteoforms. We will integrate these methods and data streams together with powerful open-source software and accompanying protocols to make these capabilities widely available, enabling researchers everywhere to gain a deeper understanding of the functioning of their biological systems. We will apply our innovative tools to many cutting-edge projects with numerous collaborators, both because technology development is most meaningful in the context of relevant biological studies and because it will increase the adoption of proteoform analysis among scientists in the broader biomedical community.
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2021 |
Jarrard, David F. (co-PI) [⬀] Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Sequence-Specific Hybridization Capture For Discovery of Proteoform?Lncrna Interactions in Prostate Cancer @ University of Wisconsin-Madison
Project Summary/Abstract Long noncoding RNAs (lncRNAs) are vital components of gene expression programs controlling cellular differentiation and function. lncRNAs act as scaffolds to recruit or sequester effector proteins. They may act in cis and trans at multiple genomic sites. lncRNAs modulate transcription, chromatin organization, RNA processing, and translation, and many questions remain unanswered regarding how they influence gene expression. Recent reviews have detailed several lncRNAs that play key roles in prostate cancer including regulation of processes in cancer cells such as proliferative signaling, replicative immortality, invasion and metastasis, evasion of growth suppressors, induction of angiogenesis and resistance to apoptosis. While much remains to be learned regarding the mechanisms of action involved in lncRNA function, it is abundantly clear that lncRNAs act in concert with associated proteins to carry out their roles. Distinguishing between aggressive and indolent prostate cancer is a major conundrum in cancer. In roughly one-third of the 140,000 intermediate grade cancers (Gleason 7) diagnosed annually, the disease follows a more aggressive course developing rapid progression of prostate specific antigen (PSA) after treatment and earlier metastasis and death. During the prior three-year grant period, we discovered several dozen lncRNAs that distinguish aggressive and indolent prostate cancers, of which four were validated using RT-qPCR. We also developed the HyPR-MS strategy to permit elucidation of specific RNA-protein interactomes. In this renewal proposal we seek to further develop and apply a suite of powerful new proteomics tools to study these and other prostate cancer-relevant lncRNAs and the proteins that associate with them, and use this capability to reveal important proteomic signatures to distinguish aggressive versus indolent prostate cancer. These studies will provide a novel and unprecedented view into the nature of the proteoform?lncRNA interactions that underlie lncRNA function. The delineation of differentially expressed lncRNAs and their associated proteins/proteoforms will provide insight into the biological mechanisms underlying disease progression and provide novel therapeutic targets for further development.
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2021 |
Bresnick, Emery H. (co-PI) [⬀] Smith, Lloyd M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
New Tools to Decipher the Role of Lncrnas and Their Protein Interactomes in Hematopoiesis @ University of Wisconsin-Madison
Project Summary/Abstract Bioanalytical tools to study long noncoding RNAs (lncRNAs) and their protein interactors are desperately needed. lncRNAs are critical elements in the transcriptional regulation of gene expression. They have been shown to function through several different mechanisms. They may serve directly as gene-regulatory factors, produced by transcription at the genomic site where they act. Alternatively, lncRNAs interact with proteins to control gene expression. For example, lncRNAs can act as a ?molecular sink? for proteins that interact with DNA or RNA; in this case, binding of the protein to the lncRNA competes with its interaction with its primary target. lncRNAs can also guide proteins to their DNA targets to either repress or activate transcription. They can act not only in cis, or near their site of transcription, but also in trans, at multiple genomic sites. Finally, lncRNAs can act as platforms upon which molecules can convene to perform a function as a team at a specific location and time (e.g. histone modification complexes). Each of these mechanisms requires the interaction of lncRNAs with a diverse protein cohort. Knowledge of the proteins bound to specific lncRNAs is thus essential information needed to understand mechanisms by which lncRNAs control gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of lncRNA- associated proteins. Despite the great importance of lncRNAs and the proteins that interact with them, there are significant limitations in the tools available to study them. We propose to develop and validate a suite of powerful new tools for the discovery, identification, and quantification of lncRNAs and for the comprehensive proteomic analysis of their protein interactomes. It is known that lncRNAs direct gene expression to modulate cell fate in the hematopoietic system, enabling diversification of gene programming during development. Disrupted lncRNA function can also contribute to malignant transformation in specific myeloid and lymphoid cancers. GATA factor-regulated lncRNAs, discovered in powerful genetic systems (wild type and Gata2 enhancer-mutant mice, GATA-1 genetic complementation system and GATA factor knockdowns in primary human erythroid precursor cells), control human erythroid precursor cell function and erythrocyte development. In initial profiling studies, we have identified 74 GATA factor-regulated lncRNAs. The performance of the new tools developed here will be thoroughly tested on a subset of these 74 lncRNAs to discover GATA factor-dependent regulatory circuits and networks that control hematopoiesis. The proposed research will drive state-of-the-art lncRNA identification and proteomic analysis of the lncRNA interactome, applied to one of the most important regulatory networks in hematopoiesis. We will bring a new and unprecedented level of visibility to the definition of the GATA factor-relevant lncRNA interactome, while developing powerful open-source software tools and detailed experimental protocols.
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