Methods and compositions for altering photophysical properties of fluorophores via proximal quenching
The invention is directed to fluorophore-containing compositions and configurations wherein proximity between the fluorophore and one or more protective agents (PAs) modifies the lifetime of fluorescent and/or dark states, their frequency of occurrence, and the total lifetime of fluorescence in order to appropriately modify the photophysical characteristics of the fluorophore. The invention is also directed to methods that utilize these compositions and configurations.
1. A composition comprising a fluorophore having attached thereto at least one protective agent moiety selected from cyclooctatetraene (COT), Trolox, p-nitrobenzyl alcohol (NBA), chloramphenicol, n-propyl gallate, and 1,4-diazabicyclo[2.2.2]octane (DABCO), wherein the at least one protective agent moiety is attached covalently to the fluorophore either directly or via a linker.
2. The composition of claim 1 , wherein at least two protective agent moieties are attached to said fluorophore.
3. The composition of claim 1 , wherein said linker has a size of less than 1,000 Daltons.
4. The composition of claim 1 , wherein said composition is conjugated to a biomolecule or a surface of a bulk solid by either a direct bond or by a conjugating group connecting said biomolecule or surface and at least one of the fluorophore or said at least one protective agent moiety.
5. The composition of claim 1 , wherein said fluorophore exhibits an emission wavelength greater than 594 nm.
6. A method for performing single-molecule Fluorescence Resonance Energy Transfer (FRET), the method comprising measuring the FRET efficiency of a labeled biomolecule comprised of a biomolecule attached to a donor fluorophore and acceptor fluorophore, wherein said donor and/or acceptor fluorophores are attached covalently either directly or via a linker to one or more protective agent moieties selected from cyclooctatetraene (COT), Trolox, p-nitrobenzyl alcohol (NBA), chloramphenicol, n-propyl gallate, and 1,4-diazabicyclo[2.2.2]octane (DABCO).
7. A method for detecting a cellular process in an organism using fluorescence, the method comprising (i) administering to said organism a targeting probe comprising a biomolecule possessing a targeting portion thereon, at least one fluorophore, and at least one protective agent moiety selected from cyclooctatetraene (COT), Trolox, p-nitrobenzyl alcohol (NBA), chloramphenicol, n-propyl gallate, and 1,4-diazabicyclo[2.2.2]octane (DABCO), wherein said protective agent moiety is attached covalently either directly or via a linker to said fluorophore such that the protective agent moiety enhances the photophysical properties of said fluorophore, and ii) detecting said targeting probe using fluorescence in said organism.