DNA Condensation: from simple ions to complex proteins

In nature, DNA exists primarily in a highly condensed state.  DNA packaging in the cell is typically protein mediated using, for example, histones (in eukaryotic nuclei) or protamines (in sperm cells).  The scale of this compaction is immense.  For a typcial human chromosome consisting of a single DNA approx. 5 cm in length, every cell thus has almost 2 meters of DNA compacted within its roughly 10 µm size. We work to address fundamental biophysical questions of DNA condensation by integrating x-ray scattering and osmotic stress experiments to investigate how cations mediate DNA-DNA intermolecular forces while extending from in vitro to in vivo packaging, such as mammalian sperm cells.  Our long-term goal is to identify the biochemistry that underlies DNA packaging and mispackaging and understand the interrelationship to DNA damage, disease, and reproductive health.

Small molecule drug interactions with DNA

Many small molecules (such as drugs, ligands, etc.) are able to recognize and bind to single-or double-stranded nucleic acids, often inducing structural alterations.  The unique double helix structures of DNA allows for binding through various modes including covalent binding (DNA-adducts), major or minor groove binding, and intercalation.  Currently, little is known about small molecule drug interactions with highly packaged DNA that is typically found within cells.  The reactivity of drugs and mutagens toward packaged DNA is presumably quite different from dilute DNA, and dependent on both accessibility and changes in DNA-DNA interaction energies.  We are working on measuring directly the forces induced by these DNA drug interactions on DNA molecules in various states of condensation and correlating these forces to DNA packaging, DNA damage and disease.

Extracellular Gene Delivery: Nanoparticle Transport in Complex Media

In order to reach target cells, gene complexes must traverse the extracellular matrix (ECM), a crowded, interacting environment of biomacromolecules.  Before accessing cells, a particle or a virus must also navigate through even messier milieus such as mucus or bacterial biofilms before successful delivery is possible.  In these cases, one often finds that not only charged and uncharged polysaccharides and fibrous proteins of the ECM but even foreign DNA and bacteria can be entrapped in these gels.  One can easily imagine the difficulties in interpreting diffusion data through systems which could have many specific and nonspecific interactions such as electrostatics, chemical binding, immobile barriers, and binding sites.  For in vivo applicability, both transport properties and efficient delivery of intact gene complexes to the target tissue are crucial.  Our group is involved in the study of fundamental questions using fluorescent techniques including fluorescence correlation spectroscopy (FCS) to gain insight into the transport and interactions of gene therapy contructs in complex biological systems. 

Intracellular Gene Delivery: Understanding rate-limiting steps to efficient transfection

Endocytosis is the cellular process by which molecules, such as proteins, are absorbed from outside the cell membrane by engulfing within a membrane-bound vesicle.  Once compartmentalized, these endosomes are either recycled back to the surface or are sorted for degradation through acidification.  Since most non-viral carriers of DNA depend on endocytosis for their import into cells; endosomal release and the subsequent DNA release therefore corresponds to two of the most important barriers to efficient  transfection.  We are actively involved in using physical techniques including SAXS and FCS to better understand these mechanisms and ultimately engineer better optimized gene delivery systems.