New experimental insights allow researchers to probe protein-DNA interactions with greater precision
A monomolecular imaging technique, called protein-induced fluorescence enhancement (PIFE), has gained popularity in recent years as a tool for observing DNA-protein interactions with nanometric accuracy. Yet, according to a new KAUST study, research laboratories have not used this technique to its full potential.
The PIFE test is based on the idea that DNA labelled with a fluorescent dye will shine brighter when proteins are bound in the vicinity. In many cases, this is true - leading many scientists to adopt the PIFE rather than other labour-intensive techniques based on the dual marking of proteins and DNA.
But the graduate students of Samir Hamdan, Fahad Rashid, Manal Zaher and Vlad-Stefan Raducanu, realized that protein binding to DNA-dye complexes can sometimes have the opposite effect. Instead of improving the fluorescent signal, protein interactions can sometimes reduce the glow, depending on certain system properties.
Hamdan credits his students' curiosity for making this observation and explaining in detail how it works. Inspiration from Rashid's previous work led the team to the phenomenon they call protein-induced fluorescence extinction (PIFQ). And as Rashid explains, "We have undertaken to better define the conditions that lead to dams or fluorescent busts.
Through a combination of experimental and computer analyses, the KAUST team has shown that the initial fluorescence state of the DNA-dye complex determines whether the PIFE or PIFQ will be obtained after protein binding. Without this knowledge, the probability of either event becomes equivalent to a coin toss, which can compromise the mechanistic interpretation of laboratory results.
"When fluorescence and structural work allow us to understand this initial state, the anticipation of one or the other of these effects becomes experimentally possible," explains Raducanu.
Factors such as DNA sequence and dye position could tip the balance in favour of PIFE or PIFQ; the KAUST team became so good at interpreting the molecular code that they were able to accurately predict what would happen simply by measuring how these parameters influence the initial fluorescence state of the DNA-dye system.
"We turned each measure into a game," says Zaher, "and we are happy to say that our hypothesis predicted the result more than 90% of the time!
These new discoveries are expected to significantly expand the scope and experimental promise of this powerful monomolecular imaging tool, Raducanu predicts. "By introducing the PIFQ, we are offering field researchers the opportunity to address several biological issues for which the PIFE may not have been observed," he says.
Scientists may also choose to combine PIFE and PIFQ to decipher multi-step and multi-protein processes with a single DNA-dye construct.
"Taking into account the contextual nature of fluorescence modulation in the DNA-dye system opens the door to many possibilities in experimental design that could be adapted to the needs of researchers," explains Dr. Zaher.
"We now anticipate that the interpretation of data and the attribution of molecular events from monomolecular data will become easier and more accurate," adds Rashid.
