By leveraging fluorescence blinking, researchers from Osaka University have developed a complementary method to FRET for studying biomolecular dynamics at the single-molecule level.
When an otherwise fluorescent molecule stops emitting light for a brief period of time, it is called fluorescence blinking. In a study published recently in Angewandte Chemie, the Osaka team used cyclooctatetraene (COT) to modulate fluorescence blinking in biochemical assays.
"COT suppresses fluorescence blinking, and so increases fluorescence, by coming into physical contact with the fluorophore," explains Jie Xu, lead author. "In contrast, modulating emission by a widely used technique known as fluorescence resonance energy transfer, FRET, works over only much longer distances—in the region of 1 to 10 nanometers—and only on a nanosecond timescale."
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The researchers first tested their setup on double-stranded DNA containing an internal spacer. When COT was on one end of the spacer and the fluorophore at the other end, there was more fluorescence than when COT was not present. However, fluorescence blinking wasn't eliminated entirely. The researchers exploited this fact by testing how the chemical architecture of the spacer modulates blinking.
"Increasing spacer length and increasing pi-stacking interactions—noncovalent interactions between aromatic rings—in the spacer increased the fluorophore's time in the 'off' state," says Kiyohiko Kawai, senior author. "FRET can't provide information on biomolecular dynamics over these subnanometer distances."
The researchers next detected ultrasmall concentrations of an RNA molecule that is a biomarker for many cancers. They first affixed a fluorescent probe containing COT to a glass slide. The probe was designed such that binding to the RNA biomarker would increase fluorescence from the probe. "Binding to the target RNA decreased the probe's time in the off state by half," says Xu. "This provides a clear means to detect a cancer biomarker."