We propose to develop and analyze water-soluble nanomaterials as reporters in FRET optical bioassay technologies to probe conformational dynamics for nucleo-protein interactions. The ability to interrogate bimolecular interactions in the context of binding and dissociation kinetics and thermodynamics is essential to obtain a molecular understanding of the interplay between structure and function. Using variations on existing organic dye based bioassays, we propose to develop the chemistry, probe the properties, and demonstrate the applicability of nanomaterials in the bio-nano conjugates, while providing a foundation for interfacing more complex and diverse protein-DNA-systems. The selected protein """"""""systems"""""""" include the well studied EcoRV endonuciease (R.ECORV), the EcoRi DNA methyltransferase (M.ECORI), and the Papl regulon that controls the expression of pill-related proteins in pathogenic E.coli bacteria. Each of the three systems involves dramatic conformation changes, ranging from base flipping (M.ECORi) and localized DNA bending (M.EcoRi and R.EcoRV) to large scale DNA looping (Papl regulon). The use of nanoscale materials in FRET based assays results in substantial enhanced photostabiiity in comparison to organic dyes (20x), and enhanced long range energy transfer (> 10 nm) for FRET applications. The nanomaterial bimolecular interface, which involves components of similar size scales, remains poorly understood and any application in bioassay technologies requires analysis of the biocompatibility of these materials. The development of long-range FRET assays based on nanomaterial bio-conjugates will allow a significant extension of the current technology into multiple protein binding assays for analysis of conformational changes in the nucleic acid structures. This information is needed for a basic understanding of protein ligand interactions, for the intelligent redesign of such systems, and for the development of modulators of such interactions (e.g., novel drugs). ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB000832-04
Application #
6887403
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Korte, Brenda
Project Start
2003-04-01
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
4
Fiscal Year
2005
Total Cost
$221,589
Indirect Cost
Name
Florida State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
790877419
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
Riskowski, Ryan A; Armstrong, Rachel E; Greenbaum, Nancy L et al. (2016) Triangulating Nucleic Acid Conformations Using Multicolor Surface Energy Transfer. ACS Nano 10:1926-38
Armstrong, Rachel E; Strouse, Geoffrey F (2014) Rationally manipulating aptamer binding affinities in a stem-loop molecular beacon. Bioconjug Chem 25:1769-76
Singh, Mani Prabha; Jennings, Travis L; Strouse, Geoffrey F (2009) Tracking spatial disorder in an optical ruler by time-resolved NSET. J Phys Chem B 113:552-8
Kogot, Joshua M; Parker, Alex M; Lee, Jihun et al. (2009) Analysis of the dynamics of assembly and structural impact for a histidine tagged FGF1-1.5 nm Au nanoparticle bioconjugate. Bioconjug Chem 20:2106-13
Mason, Brian P; Hira, Steven M; Strouse, Geoffrey F et al. (2009) Microcapsules with three orthogonal reactive sites. Org Lett 11:1479-82
Kogot, Joshua M; England, Hannah J; Strouse, Geoffrey F et al. (2008) Single peptide assembly onto a 1.5 nm Au surface via a histidine tag. J Am Chem Soc 130:16156-7
Lovingood, Derek D; Strouse, Geoffrey F (2008) Microwave induced in-situ active ion etching of growing InP nanocrystals. Nano Lett 8:3394-7
Peterson, Stacey N; Reich, Norbert O (2008) Competitive Lrp and Dam assembly at the pap regulatory region: implications for mechanisms of epigenetic regulation. J Mol Biol 383:92-105
Lovingood, Derek D; Oyler, Ryan E; Strouse, Geoffrey F (2008) Composition control and localization of S2- in CdSSe quantum dots grown from Li4[Cd10Se4(SPh)16]. J Am Chem Soc 130:17004-11
Washington 2nd, Aaron L; Strouse, Geoffrey F (2008) Microwave synthesis of CdSe and CdTe nanocrystals in nonabsorbing alkanes. J Am Chem Soc 130:8916-22

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