Philip J Santangelo
Georgia Institute Of Technology
Project start date: 2011-07-12
Project end date: 2013-06-30
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Philip J Santangelo
In Vivo Gene Expression Imaging For Cancer Analysis
Philip J Santangelo
Vivonetics, Inc. 311 Ferst Dr, L-1308 Atlanta, Ga 30332
Grant 1R41CA103103-01 from National Cancer Institute IRG: ZCA1
Abstract: We propose to develop a novel dual FRET molecular beacons technology for the early detection of cancer in living cells with high specificity, sensitivity and efficiency. Molecular beacons (MBs) are single-stranded oligonucleotides with a stem-loop hairpin structure and dual-labeled with a fluorophore at one end and a quencher at the other. Delivering MBs into cells will result in a fluorescence signal if the MBs hybridize to target mRNAs. Thus, when the target mRNAs corresponding to the molecular markers of a cancer are detected in cells, cancer cells (bright) can be distinguished from normal cells (dark). However, the conventional design of MBs would suffer from false positives in cancer cell detection due to degradation by cytoplasmic nucleases and nonspecific interactions. To overcome this difficulty, we have created the dual FRET MBs concept (i.e., to hybridize a pair of donor and acceptor MBs on the same target and detect the resulting FRET), and demonstrated its potential to significantly reduce or even eliminate the false positives. We have studied the energy transfer between MBs with different dye molecule pairs, developed lanthanide dyes and performed time-resolved FRET to further reduce the background noise. To guide the design of molecular beacons, we have synthesized MBs with various molecular structures and performed in-solution thermodynamic and kinetic studies of MB-target binding. We have also studied the feasibility of detecting K-ras codon 12 mutant mRNA and survivin mRNA in pancreatic cell lines. To further develop the new dual FRET molecular beacons technology for clinical applications, in this Phase I STTR project, we will enhance the intracellular stability of molecular beacons by modifying the backbone with 2 -O-methyl and phosphorothioate (PS) chemistry. We will synthesize 5 types of molecular beacons with a random sequence and compare their stability in both cell lysates and living cells. We will also study the effect of such modifications on hybridization kinetics and thermodynamics. We will determine the signal-to-noise ratio and specificity of dual FRET molecular beacons by detecting mRNA expression in both cell lysates and living cells. We will synthesize dual FRET MBs targeting K-ras codon 12 mutations and survivin, deliver the MBs into pancreatic cancer cell lines and HDF cells and establish the detection specificity and signal-to-noise ratio using fluorescence imaging and spectroscopy. We will determine the detection sensitivity by systematically varying the relative ratios of normal and cancerous cells in a mixture in vitro and seek out the cancer cells based on MB-induced fluorescence using a confocal microscope and a FACS cell sorter. The goals are to develop the dual FRET molecular beacons technology for early cancer detection and diagnosis, and to commercialize this technology for a wide range of biomedical applications including medical research, cancer analysis, drug discovery, and in vivo detection of gene expression.
Keywords: fluorescence resonance energy transfer, fluorescent dye /probe, gene expression, neoplasm /cancer diagnosis, oligonucleotide, technology /technique development, biosensor, early diagnosis, messenger RNA, neoplasm /cancer genetics, thermodynamics, thiophosphate, cell line, confocal scanning microscopy, flow cytometry, polymerase chain reaction
Project start date: 2003-08-01
Project end date: 2004-07-31
1R41CA103103-01 (2003): $129731
SINGLE RNA SENSITIVE PROBES FOR STUDYING VIRAL REPLICATION AND BUDDING
Philip J Santangelo, Assistant Professor
Georgia Institute Of Technology, 505 10th St Nw, Atlanta, Ga 30332-0420
Grant 1R01GM094198-01 from National Institute Of General Medical Sciences
Abstract: Human respiratory syncytial virus (hRSV) is recognized as the most important viral agent of serious pediatric respiratory tract disease. Worldwide, acute respiratory tract disease is the leading cause of mortality due to infectious disease, and hRSV remains one of the pathogens deemed most important for vaccine and antiviral development, but the development of virus specific antiviral drugs is not easy. The difficulties of developing antivirals result, in part, from viral replication taking place inside the infected cell while utilizing the cell´s molecular machinery. In addition, due to the mutation rate of RNA viruses, it is essential to identify conserved virus specific mechanisms, involving only viral components, which are vital to their replication. In order for effective antiviral drugs to be discovered, a significant leap in our understanding of viral life cycles must be achieved. To do this, we need to be able to visualize at high-resolution, the dynamic spatio-temporal distribution of vRNAs and proteins within an infected cell. Fluorescent fusion protein technology currently enables the live-cell imaging of viral proteins, but no standard technology exists to image non-engineered RNA with single RNA sensitivity. In response, we´ve developed multiply-labeled tetravalent RNA imaging probes or MTRIPs, published recently in Nature Methods. In preliminary experiments, MTRIPs, when delivered via cell membrane permeabilization with streptolysin O (SLO), bound specifically and rapidly to RNA (<10 minutes) and allowed for single RNA imaging using widefield epifluorescence microscopy techniques in living cells. Target RNA was identified by the enhanced signal-to-background ratio achieved through binding of multiple probes per RNA. Therefore, our short term goal is, through optimization of the ligand affinity and probe core composition, to create a probe and methodology which will allow us to study RNA virus replication and budding of viral particles in time and space within a living cell with single molecule sensitivity. Our long term goals are to use the methodology to identify new targets for antiviral drugs, and use the new probes as part of drug screening assays for RSV but also to extend their application to other RNA viruses, such as influenza, in order to generate a significant leap in our fundamental understanding of RNA virus cellular biology. Human respiratory syncytial virus (hRSV), an RNA virus, is the leading cause of viral pneumonia, bronchiolitis, respiratory failure, mechanical ventilation, and viral death in infants in the USA and worldwide, and causes nine times as many infant deaths as influenza virus. Currently, there are no effective vaccines for hRSV disease, and new techniques and targets for antiviral screening are badly needed. In this grant application, through the collaboration of a probe developer and well established virologist, we will develop, optimize, and validate our single RNA-sensitive, live-cell imaging probes and methodology, allowing for the identification of viral replication sites and quantification of replication and budding of the virus without interfering with viral processes; this will lead to a more accurate spatio-temporal view of RNA virus replication and the ability to screen molecules that inhibit essential virus-specific processes
Keywords: Acute; Affect; Affinity; Antiviral Agents; Antiviral Drugs; Antivirals; Applications Grants; Area; Assay; Binding; Binding (Molecular Function); Binding Sites; Bioassay; Biochemical; Biologic Assays; Biological Assay; Biology; Bronchiolitis; Cell Communication and Signaling; Cell Signaling; Cell membrane; Cells; Cellular Inclusions; Cellular biology; Cessation of life; Childhood; Chimera Protein; Chimeric Proteins; Collaborations; Combining Site; Communicable Diseases; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Membrane; Death; Development; Disease; Disorder; Drug Evaluation, Preclinical; Drug Screening; Drug usage; EC 2.7.7.48; Engineering; Engineerings; Evaluation Studies, Drug, Pre-Clinical; Evaluation Studies, Drug, Preclinical; Filament; Fluorescence; Fusion Protein; Gene Probes, RNA; Gene Products, RNA; Gene Transcription; Generalized Growth; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Genomics; Goals; Grant Proposals; Grants, Applications; Grippe; Growth; Human respiratory syncytial virus; Image; Inclusion Bodies; Infant; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Influenza; Influenza Virus; Intervening Sequences; Intracellular Communication and Signaling; Introns; Journals; Kinetic; Kinetics; Knowledge; Label; Laboratories; Lead; Life; Life Cycle; Life Cycle Stages; Ligands; Light; Location; Magazine; Mechanical ventilation; Messenger RNA; Method LOINC Axis 6; Methodology; Methods; Methods and Techniques; Methods, Other; Microinjections; Microscopy; Moab, Clinical Treatment; Molecular; Molecular Interaction; Monoclonal Antibodies; Mortality; Mortality Vital Statistics; Mutation; Nature; Nucleic Acids; Nucleoside-triphosphate[{..}]RNA nucleotidyltransferase (RNA-directed); Nucleotides; Passive Immunization; Pathogenesis; Pb element; Photoradiation; Plasma Membrane; Plasmids; Play; Position; Positioning Attribute; Preclinical Drug Evaluation; Process; Proteins; Publications; Publishing; RNA; RNA Expression; RNA Probes; RNA Replicase; RNA Transport; RNA Viruses; RNA, Messenger; RNA, Non-Polyadenylated; RNA, Viral; RNA-Binding Proteins; RNA-Dependent RNA Polymerase; RNA-Directed RNA Polymerase; RSV Vaccines; Reactive Site; Research; Resolution; Respiratory Failure; Respiratory Syncytial Virus Vaccines; Respiratory Tract Diseases; Respiratory syncytial virus; Ribonucleic Acid; Ribonucleic Acid Transport; Role; Science of Virology; Scientific Publication; Screening procedure; Signal Transduction; Signal Transduction Systems; Signaling; Site; Speed; Speed (motion); Techniques; Technology; Time; Tissue Growth; Trans-Acting Factors; Trans-Activators; Transactivators; Transcription; Transcription, Genetic; Transfection; Translations; UTRs; Untranslated Regions; Vaccines; Viral; Viral Burden; Viral Diseases; Viral Gene Products; Viral Gene Proteins; Viral Load; Viral Load result; Viral Pneumonia; Viral Proteins; Virion; Virology; Virus; Virus Diseases; Virus Particle; Virus Replication; Viruses, General; Work; base; biological signal transduction; cell biology; cell imaging; cell type; cellular imaging; design; designing; disease/disorder; drug development; drug use; experiment; experimental research; experimental study; flexibility; flu infection; gene product; genome mutation; granule; heavy metal Pb; heavy metal lead; imaging; imaging probe; infant death; influenza infection; influenzavirus; influenzavirus (unspecified); interest; life course; mRNA; mechanical respiratory assist; ontogeny; overexpression; particle; pathogen; pediatric; plasmalemma; public health relevance; research study; respiratory insufficiency/failure; response; screening; screenings; single molecule; social role; stoichiometry; streptolysin O; trans acting factor (genetic); viral RNA; viral infection; virology; virus RNA; virus development; virus infection; virus multiplication; virus protein
Relevance: Narrative Human respiratory syncytial virus (hRSV), an RNA virus, is the leading cause of viral pneumonia, bronchiolitis, respiratory failure, mechanical ventilation, and viral death in infants in the USA and worldwide, and causes nine times as many infant deaths as influenza virus. Currently, there are no effective vaccines for hRSV disease, and new techniques and targets for antiviral screening are badly needed. In this grant application, through the collaboration of a probe developer and well established virologist, we will develop, optimize, and validate our single RNA-sensitive, live-cell imaging probes and methodology, allowing for the identification of viral replication sites and quantification of replication and budding of the virus without interfering with viral processes; this will lead to a more accurate spatio-temporal view of RNA virus replication and the ability to screen molecules that inhibit essential virus-specific processes
Project start date: 2010-07-01
Project end date: 2015-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
1R01GM094198-01 (2010): $303045
QUANTIFYING NATIVE RNA DYNAMICS DURING REGULATION
Philip J Santangelo, Assistant Professor
Georgia Institute Of Technology, 505 10th St Nw, Atlanta, Ga 30332-0420
Grant 1R21EB009455-01A2 from National Institute Of Biomedical Imaging And Bioengineering
Abstract: Gene regulation is a fundamental process necessary for normal cell function, cell differentiation, and for responding to environmental stimuli. In addition, it plays a significant role in many important pathologies, such as cancer and viral infections. Regulation at the post-transcriptional level has become extremely important in cancer, linked with the early stages of tumorigenesis and with metastasis. On the subcellular level, it has been directly linked to mRNA localization and dynamics, mRNA-protein and mRNA-miRNA interactions. Currently, there have been no studies of native mRNA dynamics on the single RNA or granule level in living mammalian cells. The reason for this has been the lack of methods to image native RNAs on the single RNA or granule level. To address this issue, the Santangelo lab has developed a single RNA sensitive imaging strategy recently published in Nature Methods. Our methodology consists of two parts; the probes, which bind to native RNA via Watson-Crick pairing, and efficient cytosolic delivery via streptolysin O. Our probe design consists of four high-affinity, nuclease resistant, linear nucleic acids, labeled with multiple, high quantum-yield fluorophores linked together by streptavidin, via the biotin-streptavidin linkage, where streptavidin is the probe core. Target RNA is identified by the enhanced signal-to-background ratio achieved through binding of multiple probes per RNA. In this way, the identification of target is achieved in an analogous way to that of GFP-RNA binding protein or peptide systems, but native target sequences are utilized and significantly fewer binding sites. In preliminary experiments, probes bound rapidly to native, cytosolic target RNA (<10 minutes), and allowed for the first demonstrations of single RNA sensitivity, RNA granule trafficking, and dynamic RNA- protein colocalization studies, when used in conjunction with fluorescent fusion proteins. Therefore, the short term goal of this study is to fully validate our single molecule-sensitive imaging approach for the study of RNA trafficking, and quantify the dynamics of native 2-actin mRNA on the single RNA or granule level while trafficking to translation sites, a known regulatory process, highly associated with cell motility. We will also compare these results with those from plasmid-derived RNAs. Our long term goals are two-fold, 1) to develop the ability to use mRNA dynamics measurements to indirectly measure mRNA function, and 2) to utilize this imaging methodology in conjunction with siRNA, fluorescent antibody staining and fusion proteins, to determine the roles of trans-acting factors, such as RNA binding proteins and miRNAs, in the regulation of gene expression on the post-transcriptional level during a range of regulatory processes. This should enable new methods to intervene and control these processes, especially as applied to preventing cancer cell metastasis or making these cells more susceptible to treatment. Gene regulation plays a critical role in many important biological problems and processes, such as cancer pathogenesis and stem cell differentiation, viral infections, and the assembly of neural circuits. Recently, RNA dynamics have been strongly linked to post-transcriptional gene regulation through the mechanisms of local translation and through the dynamic trafficking of mRNA between polysomes, P-bodies, and stress granules. In this grant we will fully validate a single molecule sensitive, fluorescent, native RNA imaging methodology, quantify the dynamics of native RNAs while trafficking to local translation sites, and compare these results with previous plasmid-derived RNA dynamics, in order to either confirm the use of plasmid-derived RNA as a model of native RNA dynamics or identify differences in motion, possibly due to variations in cis-acting sequences, RNA structure or as a consequence of traveling through a different biogenesis pathway
Keywords: 1H-Thieno(3, 4-d)imidazole-4-pentanoic acid, hexahydro-2-oxo-, (3aS-(3aalpha, 4beta, 6aalpha))-; Actins; Address; Affect; Affinity; Applications Grants; Binding; Binding (Molecular Function); Binding Sites; Biogenesis; Biological; Biotin; Cancer Biology; Cancers; Cell Communication and Signaling; Cell Differentiation; Cell Differentiation process; Cell Function; Cell Locomotion; Cell Migration; Cell Movement; Cell Process; Cell Signaling; Cell physiology; Cells; Cellular Function; Cellular Migration; Cellular Physiology; Cellular Process; Cellular biology; Chickens; Chimera Protein; Chimeric Proteins; Cis-Acting Locus; Cis-Acting Sequence; Combining Site; Coon`s Technic; Coon`s Technique; Cytoplasmic Granules; Diffusion; Disease; Disorder; Embryo; Embryonic; Epithelial Cells; Fibroblasts; Fluorescence Microscopy; Fluorescent Antibody Technic; Fluorescent Antibody Technique; Fluorescent Antinuclear Antibody Test; Fusion Protein; Gallus domesticus; Gallus gallus; Gallus gallus domesticus; Gene Action Regulation; Gene Expression; Gene Expression Regulation; Gene Products, RNA; Gene Regulation; Gene Regulation Process; Genes; Goals; Grant; Grant Proposals; Grants, Applications; Image; Immunofluorescence Technic; Immunofluorescence Technique; Individual; Intracellular Communication and Signaling; Label; Laboratories; Life; Life Cycle; Life Cycle Stages; Link; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Mammalian Cell; Measurement; Measures; Messenger RNA; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Method LOINC Axis 6; Methodology; Methods; Methods and Techniques; Methods, Other; Metric; Micro RNA; MicroRNAs; Microscopy, Fluorescence; Microscopy, Light, Fluorescence; Modeling; Molecular Interaction; Motility; Motility, Cellular; Motion; Nature; Neoplasm Metastasis; Neurobiology; Normal Cell; Nucleic Acids; Oncogenesis; Origin of Life; Pathogenesis; Pathology; Pathway interactions; Peptides; Plasmids; Play; Polyribosomes; Polysomes; Process; Proteins; Publishing; RNA; RNA, Messenger; RNA, Non-Polyadenylated; RNA, Small Interfering; RNA-Binding Proteins; Reactive Site; Regulation; Resistance; Ribonucleic Acid; Role; Science of Virology; Secondary Neoplasm; Secondary Tumor; Signal Transduction; Signal Transduction Systems; Signaling; Site; Small Interfering RNA; Staging; Staining method; Stainings; Stains; Stimulus; Strepavidin; Streptavidin; Stress; Structure; Subcellular Process; System; System, LOINC Axis 4; Techniques; Time; Trans-Acting Factors; Trans-Activators; Transactivators; Translations; Travel; Tumor Cell Migration; Validation; Variant; Variation; Viral Diseases; Virology; Virus Diseases; Vitamin H; actin 2; actin2; biological signal transduction; cancer cell; cancer metastasis; cell biology; cell motility; coenzyme R; design; designing; disease/disorder; experience; experiment; experimental research; experimental study; fluorescent antibody; fluorophore; gene product; granule; imaging; imaging modality; imaging probe; life course; mRNA; mRNP; malignancy; messenger ribonucleoprotein; miRNA; neoplasm/cancer; neural circuit; neural circuitry; neurobiological; nuclease; particle; pathway; prevent; preventing; public health relevance; quantum; research study; resistant; siRNA; single molecule; social role; stem cell differentiation; streptolysin O; therapeutic target; trafficking; trans acting factor (genetic); tumorigenesis; viral infection; virology; virus infection
Relevance: Narrative Gene regulation plays a critical role in many important biological problems and processes, such as cancer pathogenesis and stem cell differentiation, viral infections, and the assembly of neural circuits. Recently, RNA dynamics have been strongly linked to post-transcriptional gene regulation through the mechanisms of local translation and through the dynamic trafficking of mRNA between polysomes, P-bodies, and stress granules. In this grant we will fully validate a single molecule sensitive, fluorescent, native RNA imaging methodology, quantify the dynamics of native RNAs while trafficking to local translation sites, and compare these results with previous plasmid-derived RNA dynamics, in order to either confirm the use of plasmid-derived RNA as a model of native RNA dynamics or identify differences in motion, possibly due to variations in cis-acting sequences, RNA structure or as a consequence of traveling through a different biogenesis pathway
Project start date: 2010-04-01
Project end date: 2012-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: PA-06-418
1R21EB009455-01A2 (2010): $221855
Philip J Santangelo
Georgia Institute Of Technology
Project start date: 2011-08-01
Project end date: 2014-07-31