Structure-Function Studies of Biopolymers

Project Background:  Oligonucleotides (which include DNA and RNA) and protein are two of the three biological polymers found inside all living organisms (carbohydrates being the third).  Most science-minded students know that proteins are important for catalytic function, but DNA and RNA can also serve as biological catalysts in addition to their functions as repositories for genetic information. What allows these biopolymers to perform these unique functions are their molecular shapes.

Project Overview:  Projects in this category utilize the unique shapes that biopolymers adopt to afford them unique functions.  The unique function we are interested in is an ability to selectively and sensitively bind sucralose (SplendaTM).  The goal is to use these methods to detect levels of sucralose in environmental and biological samples.

Specific Aims:

  • Development of an antibody-based assay for the detection of sucralose.   In this project, an immunogenic form of sucralose is used to generate antibodies in rabbits.  These antibodies are purified using fast protein liquid chromatography (FPLC)  and conjugated to a 96-well plate.  Using a reporter protein also conjugated to sucralose,  competitive ELISA (enzyme-linked immunosorbent assay) can be used to detect sucralose levels in aqueous samples.
  • Identify a DNA sequence (or set of sequences) capable binding to sucralose.  Systematic evolution of ligands by exponential enrichment (SELEX) is used in combination with magnetic bead technology to identify sequences within a library of DNA sequences that fold into a shape that binds sucralose.  Amplification of these sequences is accomplished using PCR (polymerase chain reaction).  Several rounds of this process yield a DNA population enriched with sequences capable of specific and selective binding to sucralose.

Environmental Biochemistry

Project Background:  Much like is highlighted in the projects on structure-function studies of biopolymers, the roles (functions) that chemicals play in our bodies are largely a function of their structures and chemical functionality.  Few chemicals made by nature tend to have a long “shelf-life” – DNA happens to be an exception, with a half-life under physiological conditions of just about 1 million years.  This is consistent, though, with the important role DNA plays in the storage of genetic information.  In this way, most natural products have reasonable breakdown times in the environment.  Chemicals made by man can be another story – particularly when one considers substances falling into categories of food additives and drugs.  Food additives designed to limit caloric value in foods sometimes function by being metabolically unreactive; this is the case with sucralose (SplendaTM), a trichlorinated variant of sucrose.  Molecules that are metabolically inert may be deemed “safe” because of this inability to be broken down.  However, this lack of chemical reactivity also allows them to exit the body in the same form that they entered, and generally persistent in the environment for lengthy periods of time.

Project Overview:  Many synthetic compounds are beginning to environmentally accumulate with uncertain consequences.  Projects within this area are focused on determining the cause and effect relationship between chemicals of concern in our environment and the negative consequences in aquatic vertebrates, which include tumor formation and interference in the sexual dimorphism in fish.

Specific Aims:

  • Examination of the role that endocrine disrupters play in skin lesion formaton in Lake Erie brown bullhead.   Damage to DNA can be caused through a variety of means and can include immune suppression by endocrine disrupters.  A comet assay will be used to quantitatively examine cellular levels of DNA damage in brown bullhead from Lake Erie presenting with skin lesions.
  • Examination of the effects that increased sucralose levels may have on the foraging behavior of aquatic organisms in Lake Erie.