Louis J. Maher - US grants

The proposed research involves understanding and manipulating the interplay of natural and artificial DNA-binding molecules. The long term objective of this research is to evaluate rationally-designed or affinity-selected compounds as modulators of DNA function by exploring the properties of such compounds at the molecular level. This strategy is intended to lay a foundation both for the eventual development of novel information-directed therapeutics, and for a broadened understanding of fundamental molecular genetic processes. The specific aims of this proposal concern a new approach for artificial regulation of gene expression by nucleic acid ligands. These proposed regulatory ligands are unique in that they are composed entirely of nucleic acids (DNA, or ultimately, RNA) rather than polypeptides. This proposal describes the design and implementation of an artificial repressor/operator system that is to be functional in E. coli. This system will be based on inducible synthesis of a stable repressor RNA capable of recognizing a homopurine operator in double-helical DNA via triple-helix formation. Preliminary studies have shown the feasibility of this approach using an in vitro model in which oligonucleotide regulation has been conferred upon a bacteriophage T7 promoter. The project has four specific aims to extend these results: 1. Analysis of additional RNA oligomers as transcriptional repressors using the in vitro T7 RNA polymerase repression system described above. 2. Identification of other potential sequence-specific repressor RNAs using multiple cycles of operator affinity selection and amplification. 3. Cloning and inducible in vivo synthesis of repressor RNAs in stable expression cassettes for gene regulation experiments in E. coli. 4. Assembly of an in vivo assay system for monitoring expression of a reporter gene driven by a bacteriophage T7 promoter under the control of an inducible RNA repressor. This project is significant for two key reasons. First, design and implementation of a simple transcriptional control interaction based on nucleic acid ligands will offer a model for creation of similar interactions for therapeutic control of target genes in eukaryotes (after delivery of repressor RNAs using viral vectors). Second, the proposed approach may shed light on the fascinating possibility that certain cases of natural gene regulation could involve recognition of double-helical DNA by RNA.

The genome is encoded within double-stranded DNA. The properties and interactions of this important macromolecule are dominated by its polyelectrolyte character. For example, the high negative charge density of DNA causes counterion condensation, a phenomenon that has implications for all protein/DNA interactions. Its high negative charge density of DNA also causes DNA to behave as a relatively stiff, worm-like polymer in dilute solution. In contrast to its behavior as a naked polymer, DNA can be highly bent in complexes with certain proteins. Such DNA bending is believed to be critical for DNA packaging and for biological functions such as transcriptional regulation. We are interested in the role of electrostatic (charge-charge) interactions in DNA bending by proteins and ions. In particular, we have been testing the hypothesis that laterally-asymmetric phosphate neutralization by cationic proteins can induce DNA to collapse toward its neutralized surface because of unbalanced interphosphate repulsions within the double helix. Results of our previous studies of chemically-modified DNA strands and charge variants of DNA binding proteins tend to support this simple hypothesis. The present proposal seeks to refine and extend this model, ultimately placing our studies of DNA bending in a more biological context. Four specific aims will be undertaken: First, we will attempt to corroborate our prior observation that DNA bending results when derivatives of the yeast bZIP DNA binding protein GCN4 contain cationic or anionic amino acid substitutions near their basic regions. Second, we will determine the role of asymmetric phosphate charge neutralization in DNA bending by the E. coli CAP protein and the mammalian histone octamer. Third, we will tether multivalent cations or anions at a single DNA site and observe effects on DNA bending. Fourth, we will study the importance of DNA bending in a biological context by exploring how the flexibility of DNA affects transcription activation using a eukaryotic transcription system.

There is growing appreciation that small, non-coding RNAs can participate in gene regulation. Can small RNAs inhibit DNA-binding proteins? We have developed an artificial example by performing in vitro genetic selection experiments identifying a small RNA aptamer that competitively inhibits human transcription factor NF-kappaB binding to DNA in vitro. Optimization by yeast in vivo genetic selections resulted in an RNA that inhibits NF-kappaB in living yeast cells. We have solved the X-ray co-crystal structure of this unusual RNA/NF-I

[unreadable] DESCRIPTION (provided by applicant): "Student Development" defines the mission of the Mayo Clinic College of Medicine Initiative for Minority Student Development (IMSD). The goal of the Mayo Clinic College of Medicine IMSD is to stimulate interest and train minority students in patient-oriented basic, translational and clinical research. During the first 7 years of this program, qualitative research has revealed common characteristics among students who ultimately persist into PhD, MD/PhD and clinical research careers to improve student selection process. By focusing on patient-oriented research students are better able to see the value of their research, and see how they can contribute to important research questions during their careers as scientists and/or physicians. With this vision they are most likely to include research as all or part of their careers. [unreadable] [unreadable] The IMSD has two closely coordinated sequences to assist students toward laboratory and clinical research careers. For laboratory research, the IMSD provides: (1) 10-week summer undergraduate research program; (2) linkage to the Mayo postbaccalaureate research program; (3) a new 2-year sequence for PhD and MD/PhD students to enhance professional development. For clinical research, the IMSD provides: (1) 8-week summer research program for medical students; (2) a 1-year Certificate or 2-year Masters Clinical Research following their third or fourth year of medical school. [unreadable] [unreadable] The summer programs provide a brief but deep research experience that mimics the exciting elements of scientific discovery; seminars to introduce them to the full range of research options (from bench to bedside and back); and a weekly group meeting to learn important research skills. The Clinical Research Training Program and PhD programs give students the skills they need to become independent. Extensive guidance and advising helps each student identify and excel in their unique career goals. The Mayo IMSD serves to increase the diversity of its own degree programs and prepare students for success around the U.S. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): DNA is a locally inflexible polymer. Nonetheless, the intrinsic inflexibility of DNA is somehow overcome in cells, allowing the constant folding and looping of DNA for storage and gene expression. It is fundamentally important to understand how DNA flexibility is enhanced in healthy, and diseased cells. HMGB proteins are abundant non-histone proteins in eukaryotic chromatin. HMGB proteins are thought to function as architectural factors that enhance DNA bending and twisting. We seek to better understand, both the molecular mechanism of HMGB proteins, and the functional role of HMGB proteins in gene expression in living cells. Four specific aims are proposed. 1. Characterization of HMGB mechanism by single molecule experiments. We will test if HMGB proteins cause transient flexible hinges in DNA. 2. Analysis of DNA collapse by cationic protein domains. We will test if HMGB proteins can use cationic domains to cause DNA collapse. 3. Analysis of facilitated DNA looping in bacteria. We will test if HMGB proteins can stabilize bacterial repression loops and substitute for bacterial architectural DNA binding proteins. 4. Analysis of the effect of HMGB proteins on transcription activator position effects in yeast. We will test if HMGB proteins stabilize DNA regulatory loops in yeast. Lav description: The instructions for building living things are coded in recipes (genes) within the cell cookbook (DNA). Very long DNA molecules are difficult to sharply bend and twist, but must nonetheless be folded into many shapes in order to function in cells. By understanding the cell proteins that make DNA flexible, we will learn about tools that may be used to artificially regulate disease genes.

DESCRIPTION (provided by applicant): The majority of research training programs for individuals from underrepresented groups focus only on basic science research. Although critically important, these programs often fail to provide the motivational link between basic science research and improved health. The goal of the Mayo Post-Baccalaureate Research Education Program (PREP) is to increase the number of individuals from underrepresented groups who choose biomedical research careers, especially disease-oriented research. The program assists both those likely to succeed and those for whom the PREP experience may truly be pivotal in their career paths. Our hypothesis is that students from underrepresented groups increasingly choose disease-directed basic or translational research careers when they clearly see the value of this research in improving human health. Mayo's PREP is successful and mature, having been a unique cornerstone of institutional education diversity efforts since Mayo pioneered this program nationally in 1996. Mayo's ongoing commitment to diversity is supported from the highest executive level and has resulted in significant positive changes at the institution. The unique, highly-integrated clinical and biomedical research programs at Mayo provide an ideal environment to foster the career goals of apprentice scientists. Mayo's PhD graduate school, medical school, MSTP training site, and residency program are parts of the famed Mayo Clinic tertiary care center. Sustained funding of the Mayo PREP positively impacts diversity across the Mayo campus. The PREP synergizes with other ongoing Mayo diversity programs, including Mayo's NIH IMSD pre-doctoral program, URM summer research programs, and other institutionally-supported diversity education initiatives directed through Mayo's newly-formed Education Office for Diversity. Mayo's successful PREP and IMSD programs celebrate peer mentoring and mutual support between URM students across the summer undergraduate, clinical research, PREP, PhD, and MD/PhD research spectrum. This synergy is critical for student self-efficacy, identity as biomedical research scientists, and elucidation of the training path. PREP progress during the prior funding cycle, together with longitudinal data derived from 13 cohorts of prior PREP students, provides objective evidence of success in promoting graduate training leading to the PhD degree. During the next funding cycle we will maintain our successful program while increasing program evaluation, mentor training, and recruitment. Specific aims: 1. Recruit to Mayo 9 new post-baccalaureate apprentice scientists each year (total of up to 36 over 4 years) for a program of research with intensive enrichment toward individualized student academic and professional development. PREP apprentice scientists may receive one or two years of training, depending on their needs. 2. Maintain Mayo's PREP program where > 75% of post-baccalaureate apprentice scientists enter PhD or MD/PhD programs with a primary interest in laboratory-based or translational research. 3. Enrich Mayo's post-baccalaureate apprentice scientists with skills important for successful initiation and completion of the PhD degree. Each aspect of the Mayo PREP program is designed to enhance students' creativity, self-efficacy, commitment, and purpose as scientists. The program offers activities specifically designed to encourage student interest in research areas related to health disparities. 4. Improve long-term contact and ongoing mentoring of Mayo post-baccalaureate apprentice scientists through graduate school and beyond. We will achieve this goal in synergy with Mayo's NIH-funded IMSD program for minority pre-doctoral students, and with Mayo's Education Office for Diversity. 5. Continue internal and external evaluation with semi-structured interviews ensuring increased utility, feasibility, propriety, and accuracy of Mayo's PREP activities.

DESCRIPTION (provided by applicant): The goal of the Mayo Clinic College of Medicine (MCCOM) Initiative for Maximizing Student Diversity (IMSD) is to prepare underrepresented minority (URM) pre-doctoral students to become future national leaders in disease-related basic and translational research. Most URM student research training programs focus primarily on basic science research. Although critically important, this focus does not adequately address the need for URM investigators to appreciate how basic science research translates to improved health. This perspective is a unique attribute of MCCOM. Since 1997 the MCCOM IMSD has successfully combined 1) supplemental training of URM pre-doctoral students;2) training of summer URM medical students;and 3) training of URM medical students for one or two years through the Clinical Research Training Program (CRTP). These MCCOM IMSD activities have been fully integrated with the MCCOM Post-baccalaureate Research Education Program (PREP) funded by NIGMS. The MCCOM IMSD program proposed here will now focus exclusively on enhancing the experience of URM students in the Mayo Ph.D. and M.D./Ph.D. programs. Synergy between the IMSD and PREP funded programs will remain a major strength. Over the past eleven years 87% of URM pre-doctoral students supported by the MCCOM IMSD have completed their doctoral degree or are on track to do so. The MCCOM IMSD has also improved the skills of Mayo faculty in addressing the unique needs of URM students. The next period of proposed MCCOM IMSD support is critical to continue this highly successful project that began in 1997, maintaining Mayo Clinic as a national leader in addressing the needs of URM trainees. The following specific aims will be accomplished: 1. Support MCCOM URM Ph.D. and M.D. /Ph.D. students by providing 2 years of fellowship support and professional development skills at the outset of Ph.D. training. 2. Engage participating students in rigorous oral presentation and grant writing workshops to improve communication skills. 3. Provide continuous counseling within the MCCOM IMSD program and periodic subsequent progress reviews. 4. Counsel pre-doctoral URM students in the process of obtaining competitive post-doctoral fellowship positions. 5. Evaluate the MCCOM IMSD through an expert external reviewer and internal advisory committee. Public health relevance: This research education program will provide 2 years of fellowship support and professional skills development at the outset of Ph.D. training. The goal is to help students from diverse backgrounds (e.g. ethnic/racial minorities, individuals with disabilities, and individuals from disadvantaged backgrounds) to advance to successful biomedical research careers at the PhD level thus contributing to the diversification of the biomedical research workforce.

DESCRIPTION (provided by applicant): Cancer cells have been likened to speeding cars. Mutated oncogenes hold down accelerator pedals; mutated tumor suppressor genes ruin brakes. Though often helpful, this analogy completely fails in the fascinating case of familial paraganglioma (PGL). Amazingly, PGL is a neuroendocrine cancer caused by loss of succinate dehydrogenase in the tricarboxylic acid (TCA) cycle of central metabolism. This is equivalent to severely disabling the engine of a car! How can loss-of-function mutations in a metabolic enzyme possibly be oncogenic, and why only in neuroendocrine cells? These irresistible biochemical questions drive this proposal, and answers will have significance for cancer therapy far beyond PGL. The central hypothesis is that succinate accumulation due to loss of SDH triggers neuroendocrine cell transformation by epigenetic effects resulting from inhibition of at least three different 2-ketoglutarate- dependent dioxygenase enzymes that produce succinate as a byproduct. It is as if the speeding car of cancer loses control because the driver is intoxicated by fumes from a faulty engine! We hypothesize that dioxygenase inhibition alters gene expression by novel epigenetic effects including (i) inappropriate activation of Hypoxia Inducible Factor (HIF), (ii) accumulation of methylated histones, and (iii) depletion of genomic 5-hydroxymethylcytosine. The strategy is to characterize PGL tumors and mammalian cells lacking SDH function, and to develop a nematode model of PGL for drug screening in Caenorhabditis elegans. Aim 1 will seek evidence for dioxygenase inhibition in primary human PGL tumor samples. Aim 2 will explore dioxygenase inhibition in cultured human and mouse cells lacking SDH. Aim 3 will monitor PGL tumorigenesis in mice with conditional SDH disruption. Finally, Aim 4 will develop a C. elegans model of PGL to uncover new therapeutics.

DESCRIPTION (provided by applicant): The goal of the Mayo Clinic College of Medicine (Mayo) Initiative for Maximizing Student Development (IMSD) is to prepare underrepresented (UR) pre-doctoral students to become future national leaders in disease-related basic and translational research and education. Our approach is to enhance the self-identification of talented UR pre-doctoral students toward medically relevant research careers, and to increase the numbers of these students entering and persisting in these careers. The Mayo IMSD is distinguished by its long and successful track record, Mayo's outstanding basic science research training environment, and Mayo's reputation as a national health-care leader. The Mayo IMSD addresses the stated needs of UR pre-doctoral students to appreciate how basic science research translates into improved health. Mayo IMSD program leaders continuously assess the Mayo Clinic research training environment, including Mayo Graduate School student and faculty demographics, UR and non-UR student PhD completion rates, and outcomes in competitive postdoctoral training productive research careers. Challenges and impediments to PhD degree completion at Mayo Graduate School are analyzed. As a result of our past experience training IMSD students and our comprehensive assessment, the Mayo IMSD proposes renewed funding to achieve the following three specific aims: Aim 1: Recruit and matriculate new UR PhD students for each of 5 years (alternating 5 and 4 new students per year in grant years 17-21) to participate in IMSD for the first 2 years of their PhD program. Achievement of this aim will ensure that UR students will comprise 15-20% of each incoming Mayo Graduate School class. Aim 2: Facilitate the research career success and productivity of IMSD students through a proven enrichment curriculum emphasizing professional writing and presentation skills, intensive career and academic counseling, and social support. In parallel, preparation for future leadership careers will be supported by student achievement of specific milestones. Aim 3: Continue to assess and continuously improve the Mayo IMSD program through internal mechanisms, intense tracking of participants for at least 10 years, and professional program evaluation. Aggressive goals have been set deliberately. The culture of the Mayo IMSD is to encourage trainees to aim high in developing skills and accomplishments for later career success. IMSD trainees will graduate with PhD degrees, but will also understand and achieve concrete academic and career development metrics. In this way, the Mayo IMSD will facilitate acquisition of skills and accomplishments necessary for persistence of trainees to careers as leaders in research and education.

Cell-penetrating aptamers targeting sub-cellular compartments ABSTRACT: An unmet need in modern nanomedicine is a method for ef?cient delivery of nucleic acids and related cargo into relevant sub-cellular compartments in living tissues. Upon exposure of cells to nucleic acids or nucleic acid/cationic lipid complexes, conventional methods result in uptake into membrane- bound vesicles (still topologically outside of the cell) or indiscriminate membrane fusion. Moreover, many carrier lipid formulations are toxic and of limited use in vivo. While the power of in vitro selection (SELEX) has been previously applied to select nucleic acid sequences that bind cells or gain preferential vesicular uptake into speci?c cells or tissues, vesicular escape with intracellular targeting has not been envisioned. We have developed a novel approach to this goal. We apply in vitro selection to identify nucleic acid aptamers that ef?ciently enter speci?c sub-cellular compartments by selecting for sequences that undergo enzymatic modi?cation dependent on speci?c intracellular enzyme activities. We show that this reward approach can identify naked DNA aptamers with substantially improved delivery to the cell nucleus (?karyophilic? aptamers). The method will be extended to identify cell-penetrating aptamers speci?c for different tissues in living mice. Rewarding aptamer sequences capable of vesicle escape and sub-cellular compartment delivery opens a new ?eld of opportunities. In the future it may be possible to extend this reward approach to identify sequences that target other sub-cellular compartments. Four speci?c aims are proposed to test the hypotheses that cell-penetrating DNA aptamers targeting speci?c sub-cellular compartments can be identi?ed by selecting molecules modi?ed by organelle-speci?c enzyme activities, and that such homing aptamers can ef?ciently deliver cargo to cells and tissues. Aim 1 will continue our selection of karyophilic (nucleus- homing) DNA aptamers, setting the stage for the targeting of other sub-cellular compartments. Aim 2 will seek to understand the mechanism of nuclear delivery of karyophilic DNA aptamers. Aim 3 will explore the ability of karyophilic DNA aptamers to direct cargo delivery into cells. Aim 4 will extend this reward approach in vivo to develop a library of tissue-speci?c karyophilic DNA aptamers in mice.