Grant Abstracts 2011   Carnegie Mellon University Adrien Treuille, Rhiju Das Pittsburgh, PA $1,000,000 December 2011 Computational models cannot yet robustly design RNAs that fold correctly in vitro, slowing the development of vital antibacterial, antitumor, and antiviral therapies.  The research team proposes a novel strategy to rapidly advance RNA bioengineering:  Internet-scale RNA design competitions rigorously scored by wet-lab feedback and organized through an online game.  Launched in early 2011, EteRNA hosts a community of over 25,000 citizen scientists who outperform existing RNA design methods.  The research team proposes to formalize this community’s insights into the first extensively validated automated RNA design algorithm, EteRNAbot.  They also propose to expand the game’s scale to thousands of hypotheses per month, enabling a new scale of public involvement in hypothesis-driven research.  Finally, they propose to extend the EteRNA interface from secondary structure design to fully 3D engineering of RNA-based biomedical sensors and scaffolds.  They will evaluate success via high-throughput experimentation; by the publication of tools and principles generated by EteRNA players; and by the widespread adoption of EteRNA’s novel high-throughput experimentation paradigm into future citizen science projects.   Columbia University Dirk Englund, Jonathan Owen, Rafael Yuste New York, NY $1,000,000 December 2011 The research team proposes to develop a novel method for direct, real-time imaging of neuronal voltage signals.  Neuroscience has traditionally relied on electrodes to measure the activity of neurons, one at a time.  It is, however, the interaction of many cells in neuronal circuits that generates behavior, and tools to monitor such ensemble activity, while still preserving single-cell resolution, are lacking.  To tackle this technical challenge, the team will use color centers in diamond nanocrystals, together with techniques derived from atomic physics, to sense electric fields with unprecedented resolution.  Diamond is well suited for use in biological preparations because it is chemically inert, cytocompatible and ideal for coupling to biological molecules.  The team proposes to harness these properties in diamond nanoprobes to measure electrical signals from neurons in real-time, in neuronal cell cultures, in neuronal circuits in brain slices and in awake mice.   Purdue University Joseph Irudayaraj, Feng Zhou, Sophie Lelièvre, Ann Kirchmaier West Lafayette, IN $1,000,000 December 2011 It is becoming increasingly clear that genetic mutations alone cannot fully account for devastating chronic adult-onset diseases, neurological disorders and cancer.  Rather, environmental assaults also contribute significantly to disease etiology and pathology.  Environmental factors act on epigenetic processes in a cell to alter gene expression without causing a genetic mutation.  This gives the cell plasticity to respond to the environment without causing permanent damage to its DNA.  Understanding how epigenetic marks govern gene expression at the single cell level will foster development of intervention strategies to change cell fate by inducing or reversing epigenetic modifications.  However, current knowledge of how these marks function is limited by existing technologies.  To break through this critical limitation, the research group will create, refine and validate a set of technologies that will enable researchers to identify, examine and even alter epigenetic marks in a locus-specific manner at the single cell level.  They will test this system in a neural stem cell differentiation model and validate it in a 3D breast epithelial tissue culture system.  This toolkit will be designed to enable users to customize strategies to identify and alter epigenetic processes at a single gene in individual cells to reset key events during neural differentiation or tumor formation.   Texas Children's Hospital Huda Zoghbi (Howard Hughes Medical Institute), Daoun Ji, Jianrong Tang, Yuri Dabaghian, Daniel Curry, Akash Patel Houston, TX $1,000,000 December 2011 Intellectual disabilities and autism spectrum disorders (lD/ASDs) represent a huge health care burden.  Dozens of genes that perform diverse molecular functions have been shown to cause these disorders when mutated, deleted or duplicated.  The challenge is to understand how such disparate molecular changes result in similar phenotypes and how we can treat these disorders given their genetic heterogeneity.  The investigators’ overarching hypothesis is that diverse molecular changes alter neural network activity and synaptic homeostasis; that such alterations in network activity lead to the overlapping cognitive and motor phenotypes; and that manipulating the aberrant activity, through modalities such as deep brain stimulation (DBS), will prove effective in treating this broad class of disorders.  They will investigate neural circuit dysfunction and treatment response in genetically-accurate models of ID/ASDs.  Specifically, they will determine the neural circuit activity in genetic models of Rett and Angelman syndromes using in vivo recordings of single units, synaptic plasticity, and network oscillations in freely-moving mice for each disease model.  They will then relate the circuit abnormalities to the cognitive deficits of these mice, and finally, will use DBS to explore possible therapeutic intervent.   University of California, Irvine H. Kumar Wickramasinghe, Kavita Arora, Rahul Warrior, Edward Nelson, Arthur Lander, Alice Yamada Irvine, CA $1,000,000 December 2011 The team proposes a unique instrument (the Single Cell Analyzer, SCA) for analysis of mRNA levels (at copy numbers of 5 to 2,000 molecules) in space and time within a living cell.  Current understanding of disease and developmental processes is largely based on biochemical or gene expression studies in the whole organ or tissue, coupled with phenotypic analysis of mutations in the context of the whole organism.  The SCA quantitatively queries gene expression at the single cell level, permitting examination of biological processes at much greater resolution.  The team has demonstrated that an active atomic force microscope (AFM) probe chemically modified with DNA primers can be inserted into a cell, without destruction, to selectively capture mRNA via an applied attractive dielectrophoretic force, and subsequently quantified.  Since mRNA levels can be assessed without affecting cell viability, the same cell can be monitored over time.  The proposed instrument will have applications in areas ranging from developmental and systems biology to personalized medicine, cancer diagnosis and stem cell research, with the potential to transform cell biology much as PCR has revolutionized molecular biology.  While transcript levels can be measured in fixed samples, no technology exists today that can dynamically quantify mRNA expression in individual living cells.   University of Florida Laura Ranum, Maurice Swanson Gainesville, FL $1,000,000 December 2011 Well-established rules of translational initiation have been used as a cornerstone in molecular biology to understand gene expression and to frame fundamental questions about what proteins a cell synthesizes, how proteins work and to predict the consequences of mutations.  For a group of neurological diseases caused by the abnormal expansion of short segments of DNA (e.g. CAG/CTG repeats), mutations within or outside of predicted coding and non-coding regions are thought to cause disease by protein gain or loss-of-function or RNA gain-of-function mechanisms.  The research team unexpectedly discovered the canonical rules of protein translation do not apply for these repeats and in the absence of an ATG initiation codon, expanded CAG and CTG often express homopolymeric proteins in all three frames (e.g. polyGln, polyAla, polySer from CAG repeats).  This Repeat Associated Non-ATG translation (RAN translation) depends on RNA structure and repeat length and occurs when expansion constructs are integrated into mammalian brain.  Additionally, they showed that RAN translation occurs in two human CAG/CTG expansion diseases: DM1 and SCA8.  The project will pursue critical questions:  How does RAN translation work?  Is RAN translation a key, previously unrecognized cellular process?  Are repetitive sequences throughout the genome translated into proteins and if so, what is their function?   Washington University in St. Louis Robi Mitra, Stephen Johnson, Mark Johnston St. Louis, MO $1,000,000 December 2011 There is still a great deal to learn about the design principles that guide vertebrate development.  Since all cells in an organism contain the same genes, transcribing different sets of genes confers a cell’s specialized role.  Which genes get turned on or off to create a particular cell type at the right time, in the right place during the development of an organism?  This is one of the pivotal questions in developmental biology.  The research team has developed a technology, Transposon “Calling Cards,” to attack this question in a novel way, and has shown that it works in yeast and mammalian cell cultures.  They propose to develop this technology, which allows them to mark every place in the genome where genes are active, for use in a living animal.  If successful, they will be able to record gene expression along different cell lineages throughout the development of a living vertebrate.  The team will be able to produce a complete record of gene activation at different stages of vertebrate development, watching as cells and their progeny specialize and form organs.  These data would contribute to the field of developmental biology by providing a blueprint for the generation of all cell types, and could ultimately guide the reprogramming of embryonic or induced pluripotent stem cells to produce specific cell types for personalized transplants, such as pancreatic beta cells to treat diabetes.   Salk Institute for Biological Studies Joseph Ecker, Terry Sejnowski, Margarita Behrens La Jolla, CA $1,000,000 June 2011 The human genome sequence lists every DNA base of the roughly 3 billion bases that makes up the genome, but it doesn’t tell us about how its function is regulated.  That job belongs to the epigenome, the layer of genetic control beyond the regulation inherent in the sequence of the genes themselves.  Methylation of DNA at the carbon 5 position of cytosines constitutes an important epigenetic layer that contributes to the definition of transcriptional and regulatory potential of the genome.  DNA methylation in cells dynamically changes during aging and is susceptible to environmental influences.  To shed light on the dynamic variation of these marks in normal and diseased cells, novel technologies must be developed for the creation of base resolution maps of DNA methylation in single cells.  The interdisciplinary team proposes to develop an approach to “read the epigenome” information from single chromosomes within single neurons.  Being able to study DNA methylation in its entirety in single cells will greatly increase our understanding of how gene expression is influenced by the environment, including the impact of diet and stress, as well as how genome function is regulated in health but dysfunctional in disease states such as schizophrenia and other mental disorders.   Thomas Jefferson University Isidore Rigoutsos Philadelphia, PA $1,000,000 June 2011 Despite very significant advances in the field of non-coding RNAs (ncRNAs), much remains to be uncovered.  This project will study a novel and currently under-characterized class of non coding RNA transcripts (ncRNAs) that are linked to pyknons.  Pyknons are a group of DNA motifs that were discovered several years ago using a data agnostic pattern discovery approach and which exhibit a number of intriguing properties.  To date the pyknon framework has correctly anticipated several discoveries in the RNA field, including: the existence of novel classes of short ncRNAs with specific lengths; the role of messenger RNAs (mRNAs) as a source of regulatory ncRNAs; and the targeting of the introns of unspliced mRNAs by exon-derived ncRNAs.  The planned work will combine computational analyses with modern experimental techniques to study samples from several diverse human conditions in order to determine the rules governing the pyknons’ biogenesis, processing and mechanisms of regulatory action.   University of California, Davis David Jay Segal, Anne Knowlton, Jan Nolta, Scott Simon, David Rocke Davis, CA $1,000,000 June 2011 One of the greatest challenges facing biomedical research since the sequencing of the human genome has been to understand the role of genetic variation in human disease.  Many genetic variants have been associated with common diseases.  However, determining the functional consequences of these variants has been difficult.  Several variants are often inherited together in tightly linked blocks, making it difficult to determine the causative variant.  Additionally, people have millions of other genetic differences, making it difficult to correlate cellular phenotypes with a particular variant.  Different gene sets are expressed in different cells, but it is difficult to extract disease-relevant cells from large numbers of patients.  Here the PIs propose a new method for the functional analysis of genetic variation, using custom nucleases to genetically modify individual variants in induced pluripotent stem cells.  They will focus on variants at the 9p2l region of the genome that have been associated with coronary artery disease.  The methods developed should provide a new way to unlock the wealth of data from genome wide association studies, and to probe the genetic architecture of common diseases.