Continuous discoveries in bioengineering and more efficient molecular biology methods have allowed scientists to create new designer biomolecules with unique and distinct properties. In that regard, bio- nanotechnology and nanoscale analysis are becoming increasingly prevalent, raising the demand for techniques with high sensitivity that can detect biomolecules in biological samples. Bioluminescent photoproteins, such as aequorin, possess great potential to provide with a solution to this new challenge because they can be detected at low concentrations, and have different emission wavelengths depending on the protein variant used, a property that can be exploited in multiplex analysis. We now propose to prepare new aequorin variants to broaden the scope of their use in bioanalysis, thus allowing for detection of biomolecules that are not detectable by other technologies. Protein molecular switches with optical properties are another type of designer biomolecules that, in the presence of a target ligand, demonstrate an altered response manifested by an "on/off" signal. These molecules can be useful in a variety of applications, such as in the development of nanosensors for in vitro and in vivo detection. To that end, we plan to design and develop bioluminescent molecular switches that incorporate the recognition properties of binding proteins with the bioluminescence afforded by the aequorin variants. The hypotheses formulated for the proposed work are based on knowledge gained during our current funding period, and investigate the use of a series of computational and synthetic approaches along with genetic engineering strategies targeting the alteration of the electronic environment of the chromophore that should lead to new bioluminescent proteins and molecular switches with a wide range of spectral properties. These photoproteins will be employed in the development of assays for important biomolecules. Finally, we will investigate the use of the newly prepared bioluminescent molecular switches in the multiplex analysis of biomolecules and in the simultaneous analysis of molecules in single cells. We anticipate that the new photoproteins will provide with new enabling technologies for in vitro and in vivo biosensing, imaging, and multiplex analysis that have a number of advantages over existing methods.

Public Health Relevance

NARRATIVE The increasing importance of nanoscale analysis has raised the demand for highly sensitive systems that can detect biomolecules in biological samples. Bioluminescent photoproteins, such as aequorin, possess great potential to provide a solution to this new challenge because they can be detected at very low concentrations in physiological fluids. In that regard, we plan to design and prepare genetically modified photoproteins that form the basis of enabling technologies for the detection of relevant biomolecules and panels of biomarkers for in vitro and in vivo applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047915-18
Application #
8291039
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Edmonds, Charles G
Project Start
1995-01-01
Project End
2014-08-31
Budget Start
2011-12-01
Budget End
2014-08-31
Support Year
18
Fiscal Year
2012
Total Cost
$344,978
Indirect Cost
$116,517
Name
University of Miami School of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
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