Fluorescence applications penetrate nearly every field of biological research. At all scales, investigations into biological systems rely heavily on the availability and performance of high-quantum yield fluorescent probes, most important and widespread of which are low molecular weight organic compounds that emit light in distinct, separable regions of the visible spectrum. Despite their demonstrated utility in serving as critical reagents in a myriad of diagnostic tools and advancing our understanding of biological mechanisms, the overall performance of organic fluorophores is limited by their photostability and phototoxicities in complex biological environments. Critically, the photon budgets of all fluorophore classes are limited by undesirable photophysical properties that limit both the photon flux as well as the total time interval over which photon emission events can be observed. Such phenomena, which include both transient (blinking) and irreversible (photobleaching) dark state excursions, add key uncertainties to all fluorescence applications. Issues of this nature are particularly limiting for single-molecule fluorescence studies, including super-resolution techniques, where relatively high levels of illumination intensity must be employed. During the current funding period, we have demonstrated the capacity to engineer and effectively utilize cyanine fluorophores spanning the visible spectrum that exhibit 3-100-fold increases in brightness and photostability, respectively. These critical advances have enhanced the time resolution and signal-to-noise ratio of single-molecule imaging investigations in both in vitro and in vivo settings to shed unprecedented functional insights on a variety of distinct biological systems that could not have been otherwise achieved. This increase in fluorophore performance directly correlates with reductions in photo-induced generation of reactive oxygen species that can impart harmful phototoxic effects in the biological systems in which they are utilized. Here we propose to build on the quantitative understanding of fluorophore performance established during the initial funding period to develop and implement predictably tunable intra-molecular photostabilization strategies. In so doing, we will generate a suite of chemically distinct organic fluorophores widely used in fluorescence applications that exhibit up to 200-fold increases in photostability across the visible spectrum. Such technologies are anticipated to revolutionize the investigation of biological processes in vitro, in living cells and ultimately in animals.

Public Health Relevance

The proposed research seeks to develop and predictably tune the performance of ultra-stable, non-toxic fluorescent probes for biological research. To do so, we will leverage and build upon mechanistic insights gained through the prior funding period to gain deeper insights into the fundamental limits to brightness and photostability. Progress on this front will be benchmarked and tangible milestones will be established through collaborative and internal in vitro and in vivo fluorescence imaging investigations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM098859-09
Application #
9975542
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sammak, Paul J
Project Start
2012-09-10
Project End
2021-04-30
Budget Start
2019-06-01
Budget End
2021-04-30
Support Year
9
Fiscal Year
2019
Total Cost
Indirect Cost
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
Terry, Daniel S; Kolster, Rachel A; Quick, Matthias et al. (2018) A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport. Nat Commun 9:230
Flis, Julia; Holm, Mikael; Rundlet, Emily J et al. (2018) tRNA Translocation by the Eukaryotic 80S Ribosome and the Impact of GTP Hydrolysis. Cell Rep 25:2676-2688.e7
VuĊĦurovi?, Nikola; Altman, Roger B; Terry, Daniel S et al. (2017) Pseudoknot Formation Seeds the Twister Ribozyme Cleavage Reaction Coordinate. J Am Chem Soc 139:8186-8193
Zheng, Qinsi; Jockusch, Steffen; Zhou, Zhou et al. (2017) Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging. Chem Sci 8:755-762
Dyla, Mateusz; Terry, Daniel S; Kjaergaard, Magnus et al. (2017) Dynamics of P-type ATPase transport revealed by single-molecule FRET. Nature 551:346-351
Alejo, Jose L; Blanchard, Scott C (2017) Miscoding-induced stalling of substrate translocation on the bacterial ribosome. Proc Natl Acad Sci U S A 114:E8603-E8610
Gregorio, G Glenn; Masureel, Matthieu; Hilger, Daniel et al. (2017) Single-molecule analysis of ligand efficacy in ?2AR-G-protein activation. Nature 547:68-73
Herschhorn, Alon; Gu, Christopher; Moraca, Francesca et al. (2017) The ?20-?21 of gp120 is a regulatory switch for HIV-1 Env conformational transitions. Nat Commun 8:1049
Herschhorn, Alon; Ma, Xiaochu; Gu, Christopher et al. (2016) Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the HIV-1 Envelope Glycoproteins. MBio 7:
Dyla, Mateusz; Andersen, Jacob Lauwring; Kjaergaard, Magnus et al. (2016) Engineering a Prototypic P-type ATPase Listeria monocytogenes Ca(2+)-ATPase 1 for Single-Molecule FRET Studies. Bioconjug Chem 27:2176-87

Showing the most recent 10 out of 35 publications