Nucleic acid Structure and Recognition - L.J. Maher

Meet Our Students

Yeng Her (2011-present)

Understanding cancer caused by defects in energy metabolism

Born in a refugee camp in Thailand, Yeng Her escaped with his family to the United States, where he went on to receive his undergraduate degree in chemistry from the University of Wisconsin at Madison. Following two years of post-baccalaureate research experience in Dr. Mahers lab, Yeng was accepted into Mayo Clinic’s M.D./Ph.D. Medical Scientist Training Program (MSTP). For the Ph.D. portion of his work, Yeng is studying familial paraganglioma (PGL). This rare tumor is particularly intriguing because it can run in families.

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Estefania Mondragon (2011-present)

New research tools built from RNA and DNA

A native of Oaxaca, Mexico, Estefania Mondragon was first exposed to the exploding world of small RNA molecules during her undergraduate research training in structural biology at the University of Colorado in Boulder. Estefania's interests in RNA structure and function remain even as she has broadened her curiosity into the field of neurobiology at Mayo Graduate School.

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Justin Peters (2008–present)

Getting Back to Basics with DNA

Each human cell is crammed with 2 meters of DNA, meaning that there is enough of the thread-like DNA in a single human body to reach to the moon and back more than 100 times. The genetic recipes for the microscopic machines of life are all encoded in DNA, and genetic engineering has made DNA familiar to many researchers and students. Surprisingly, although the double-helical structure of DNA has been known for 55 years, the origins of the distinct physical properties of the DNA molecule remain unknown. Justin Peters, a recent graduate of Wartburg College in Waverly, Iowa, is getting back to basics with the DNA molecule by probing its essential features.

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John-paul Bida (2006–2011)

Predicting how RNA molecules will fold

With training in mathematics and computer science from Johns Hopkins University, John-paul Bida entered Mayo Graduate School in 2006 as a perfect candidate to tackle a thesis project involving the holy grail of computational biology: predicting how macromolecules fold. His quantitative thinking and engineering aptitude made the transition fast and fascinating. John-paul's project involves a bold proposition: the unknown folded structures of RNA molecules can be accurately predicted by computer simulations.

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Susan Wurster (2005–2009)

Searching Vast Random Libraries of Small RNAs for New Inhibitors

Arriving at Mayo Graduate School in 2004 from her undergraduate engineering background at Oral Roberts University in Oklahoma, Susan Wurster was curious about molecular biology projects that linked engineering principles and drug discovery. In the Maher lab she initiated a study involving the creation and characterization of RNA "aptamers," small folded and unnatural RNA molecules whose shapes allow them to stick tightly to specific molecular targets in cells.

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Emily Smith (2003–2008)

Linking Metabolic Defects and Cancer

After graduating from Saint Peters College in New Jersey, Emily Smith entered Mayo Graduate School in 2002 with an interest in cellular metabolism. She was a perfect fit to fill a pioneering role in a new project in the Maher lab. Unlike other nucleic acids work in the lab, Emily wished to understand the peculiar role of cell metabolism in the origin of human tumors called pheochromocytomas (PHEO) and paragangliomas (PGL). As a Mayo PGL patient, Maher had become intrigued in the unusual genetic inheritance pattern of such tumors. There was an obvious need for a new biochemical perspective with model organisms, and Emily Smith accepted the challenge. PGL and PHEO tumors run in families.

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Kasandra Jean-Louise Riley (2002–2007)

Surprises from the "guardian of the genome"

While the p53 tumor suppressor protein is famous as an established barrier to the development of cancer, much remains to be understood of its biochemistry and detailed function. p53 is a transcription factor that binds double-stranded DNA. Kasandra Riley first came to Mayo as a Summer Undergraduate Research fellow from Wartburg College. Kasandra returned to undertake a Ph.D. thesis project that investigated an unexpected feature of p53: the ability of the protein to bind to RNA. This phenomenon was encountered in the Maher lab by accident when p53 was expressed in yeast cells as a negative control for experiments performed by Ph.D. Student Laura Cassiday. Kasandra Riley pursued this puzzle and carefully investigated the p53-RNA interaction in yeast, in vitro, and in mammalian cells. Her work suggested that the basic carboxyl terminus of p53 is responsible for RNA binding, and that this interaction is not sequence specific. Moreover, Kasandra's work suggested that the normal array of post-translational modifications present at the p53 C terminus prevent RNA interactions within living cells.

Robert McDonald (2002–2006)

Making new proteins that bend DNA

After graduating from the University of Minnesota - Twin Cities, Bob McDonald entered Mayo's M.D./Ph.D. program and undertook a Ph.D. thesis project to extend our understanding of how DNA can be bent by proteins. McDonald worked with purified recombinant proteins based on small, natural yeast DNA binding proteins Gcn4p and Pho4p. These proteins bind DNA as dimers, using recognition of the major grooves of DNA by alpha-helices. Previous students Julie Soukup and Phil Hardwidge in the laboratory had shown that small proteins of these kinds could be engineered to alter their electric charge in regions adjacent to the DNA contact surface. Such changes have the potential to alter the electrostatic attraction and repulsion forces experienced by the DNA, and to create asymmetric charge neutralization. Bob studied the extent to which these electrostatic changes could induce bending. Bob's work used both conventional electrophoretic gel mobility assays of DNA shape, and the application of fluorescence resonance energy transfer spectroscopy to demonstrate the direction and extent of DNA bending by charged proteins. He also developed methods for investigation of these problems by nuclear magnetic resonance spectroscopy.

Tessa Davis (2002–2006)

What are the functions of HMGB proteins within living cells?

A graduate of Caltech, Tessa Davis did her MS work in the Maher lab studying possible roles of high mobility group B (HMGB) proteins in chromatin structure and function. An important class of HMGB proteins binds DNA without sequence specificity. These molecules strongly bend DNA, changing its apparent physical properties by endowing the otherwise locally stiff double helix with "hinges." Tessa studied living yeast cells engineered to lack the yeast HMGB proteins Nhp6Ap and Nhp6Bp. These cells show some growth defects, and Tessa investigated approaches to creating yeast cell lines engineered to determine effects of HMGB proteins on the way genes are activated. Her work explored how assays might be created to determine if the ability of upstream activator proteins to activate genes from a distance might be dependent on HMGB protein function.

Laura Cassiday (1999–2002)

Structural and functional studies of RNA aptamers selected as transcription factor inhibitors

A graduate of Colorado State University, Laura Cassiday first came to Mayo as a Summer Undergraduate Research Fellow, and then returned to do Ph.D. thesis research in the artificial gene control by RNA aptamers selected in vitro for their ability to bind transcription factors. Laura Cassiday advanced work initiated in the lab by Lori Lebruska. Cassiday applied the yeast three-hybrid system to characterize and improve features of a small anti-NF-kappaB RNA aptamer that binds and inhibits the p50 homodimer form of this important transcription factor in vitro. Laura's work showed that the interaction can also be engineered to occur in living yeast, and this opened up powerful new approaches to selecting and optimizing RNA aptamers for function in the nucleus using genetic selections in yeast. Laura's work also included collaborative biophysics and structural biology experiments, culminating in the high-resolution X-ray crystal structure of the anti-NF-kappaB p50 RNA aptamer in complex with its protein target.

Phil Hardwidge (1998–2002)

New methods and studies of DNA intrinsic curvature and protein-induced DNA bending by electrostatic effects

Though his long-term interests remained in microbiology and infectious disease, Phil Hardwidge came to Mayo from the University of Illinois- Champaign/Urbana to explore biophysics using DNA/protein interactions as a research area. Phil's Ph.D. thesis research included projects related to improved quantitative electrophoretic and spectroscopic assays of intrinsic DNA curvature. He devised new methods to study DNA bending induced by purified proteins and variants of these proteins with altered charge.

Paula Hoyne (1997–2001)

Potential for unusual DNA structures in model systems and living bacteria

A native of New Jersey, Paula came to Mayo Graduate School and did Ph.D. thesis research in the area of unusual DNA structures. Paula used biophysical, biochemical, molecular biological, and bioinformatic methods to investigate the structures and stabilities of nucleic acid intramolecular triple helices. This class of structures has mysterious roles in biological systems. Paula's research focused on structures involving the "intra-strand purine motif," a group of triple-helix structures thought more likely to be stable under physiological conditions. In addition to measuring the formation and stabilities of these unusual DNA triple helices in the test tube, a collaboration allowed Paula to search bacterial genome sequences in search of sequences with the symmetry features necessary for potential triplex formation. A mysterious class of potential intrastrand triplex ("PIT") elements were identified in several bacteria, and molecular biological properties of representative PIT elements were investigated in vitro and within bacteria.

Eric Ross (1996–2001)

Importance of DNA flexibility in promoter architecture

After graduating from Yale University, Eric Ross came to the laboratory for Ph.D. work to explore fundamental features of transcription activation mechanisms in mammalian cells. Eric studied the hypothesis that the length and structure of DNA intervening between a transcription factor binding site and the basal promoter (TATA box) could influence gene activation. It was considered possible that activation occurs by DNA looping, and that some loop geometries might be favorable. Eric created a large number of templates altering activator spacing and the presence of intrinsically-curved elements. He used in vitro transcription in HeLa cell nuclear extract with addition of recombinant transcription activation proteins to explore these problems. Eric's work revealed independence of in vitro gene activation from details of the spacer DNA, pointing to linear distance as the main factor in activator strength. This surprising result led to a rediscovery of the profound ability of heat-resistant factors in mammalian nuclei to enhance the apparent flexibility of DNA. The implicated high mobility group B (HMGB) proteins are sequence nonspecific, but abundant and powerful in their ability to change DNA physical properties. Eric also contributed important theoretical work to improved in vitro assays of DNA curvature.

Nicole Becker (1995–1999)

In vivo footprinting approaches to searching for unusual DNA structures in living mammalian cells

Nicole Becker received her BS degree in biotechnology from St. Cloud State University and came to Mayo Graduate School to study the potential for monitoring the formation of unusual DNA structures within living cells. A number of interesting variations to conventional double-helical DNA structure have been detected in vitro (e.g. triple-helical DNA, Z-form DNA, G-quadruplex DNA) but the in vivo biology of such putative structures remains obscure at best. These structures share the property of having unpaired DNA bases within or adjacent to their location if embedded in B-form DNA. Nicole traveled to the City of Hope National Medical Center to learn from Gerd Pfeiffer techniques for in vivo footprinting by ligation-mediated PCR for application to monitoring DNA structure in vivo. Her work focused on a long homopurine-homopyrimidine element upstream of the murine metallotheionein-I gene in cultured cells. This element forms an unusual triplex structure at low pH in vitro, and is representative of many similar elements that are over-represented in mammalian genomes. Nicole carefully studied the structure of the MT-I homopurine/homopyrimidine element and other promoters where structural polymorphisms had been suggested. Her work detected no unusual DNA structures in vivo at these sites, raising important questions about the prevalence of unusual DNA structures in vivo.

Lori Lebruska (1995–1999)

In vitro selection of RNAs that bind and inhibit DNA-binding proteins

After receiving a degree in nutrition from Arizona State University, and service in the military, Lori Lebruska joined Mayo Graduate School and did her Ph.D. thesis training in the area of in vitro selection of RNA aptamers from vast random RNA libraries. Aptamers are small folded RNAs whose shapes are selected on the basis of their ability to bind to a target of interest. The resulting RNA "antibodies" are small and can be expressed within living cells as possible tools in research or therapy. Lori's worked opened an area of research asking if RNAs could mimic DNA and act as competitive inhibitors of DNA binding proteins, specifically transcription factors. Lori selected the important NF-kappaB family of transcription factors as targets for her selections. These proteins regulate development, inflammatory gene expression, HIV replication, and tumor cell resistance to chemotherapy and radiation. Lori performed in vitro selection experiments with recombinant NF-kappaB p50 homodimer protein, isolating an aptamer that binds the target protein with nanomolar affinity, and acts as a decoy for the transcription factor in vitro. Biochemical characterization set the stage for later structural studies of this molecule that eventually demonstrated that Lori had found a remarkable RNA that is able to mimic the structure of DNA.

Julie Strauss-Soukup (1993–1997)

Understanding the role of electrostatics in DNA structure and flexibility

Julie Strauss-Soukup graduated from Creighton University in Omaha, Nebraska, and joined the Maher lab (then at the University of Nebraska Medical Center) to do pioneering work to explore electrostatic effects in DNA bending by proteins. In particular, Julie developed methods to create DNA molecules with laterally asymmetric electrical charge, combining chemical synthesis with neutral methylphosphonate internucleotide linkages and enzymatic ligation and electrophoretic shape assays. This work led to the first test of a key theory that DNA will bend if the electrostatic forces along the backbone are unbalanced. Julie went on to pioneer methods to engineer DNA bending proteins with modified charge and test these proteins as DNA bending agents in laboratory experiments. This work has been influential in the field of DNA biophysics.

Garrett Soukup (1993–1997)

In vitro selection methods to identify RNAs that bind duplex DNA

Garrett Soukup graduated from Northwest Missouri State University and did his Ph.D. thesis research in the Maher lab (then at the University of Nebraska Medical Center), introducing the in vitro selection (SELEX) process after a training interval in the laboratory of Andrew Ellington, one of the inventors of the technology. Garrett performed in vitro and in vivo genetic selections to understand prospects for use of SELEX methods in finding novel RNA molecules with novel inhibitory functions. A key area of interest was the potential application of SELEX to identify RNAs that bind double-stranded DNA. Garrett's work included in vitro selections to optimize DNA recognition by RNA using triple helix formation in libraries that maximized display if the interacting macromolecules, and in techniques for reversible immobilization of duplex DNA on solid supports.

Wendy Olivas (1992–1996)

Tools for application of oligonucleotide-directed DNA triple helix formation in molecular biology

A graduate of Nebraska Wesleyan University, Wendy Olivas did her Ph.D. work in the Maher lab (then at the University of Nebraska Medical Center). Wendy's thesis project involved application of oligonucleotide-directed DNA triple helix formation to practical problems in genetic assays and artificial gene regulation. Wendy's contributions included mutation detection in native DNA samples, and studies of triple-helix formation by guanine-rich oligonucleotides in the purine triple helix motif. This work established potassium ion-dependent self-equilibria as daunting challenges for such oligonucleotides under in vivo conditions, and introduced chemical modifications that ameliorated these problems.