A fluorescent probe for all seasons; bright and sensitive

Researchers at McGill designed and synthesized the brightest fluorescent nucleobase analog (FBA) reported to date.

Understanding the nature and function of DNA requires tools with high sensitivity and precise positioning. FBAs are a powerful class of molecular probes which mimic natural nucleobases. “However, insufficient brightness of all previously reported FBAs has limited their applications such as single-molecule studies”, says Dr. Nathan Luedtke, a professor of chemistry at McGill who has developed many new FBAs over the past 10 years. In 2019, when Luedtke moved from University of Zurich to McGill with Ph.D. student, Ashkan Karimi, they also brought with them an ongoing project aimed at creating the brightest FBA ever made. In collaboration with Prof. Richard Börner (University of Applied Sciences, Mittweida, Germany), the team integrated a well-known molecular rotor fluorophore, trans-stilbene into thymidine. The probe, ^tsT, was designed to not undergo fluorescence quenching via photo-induced electron transfer and therefore remain bright inside duplex DNA.

^tsT has ideal molecular rotor properties for detecting local dynamics of DNA. “It is like a smart mechanical arm”, says Karimi the first author of the paper, “it rotates and senses the microenvironment around it.” The amount of rotation depends on the rigidity of DNA and the rotation causes fluorescence quenching of the probe. The rotary behavior of ^tsT together with its unique brightness makes it the most sensitive base-pair mismatch reporter to date. This type of tool could eventually be used in point-of-care clinal detection of single polymorphisms (SNPs). In addition, ^tsT has provided some first insights into the fundamental dynamic behavior of duplex and single-stranded DNA. “We now know the dynamic motion of a base pair mismatch in duplex DNA are even greater than those present in single-stranded DNA”, says Luedtke the senior author of the paper, “^tsT can open a new window for nucleic acid research as it may enable other types of demanding applications, such as tracking of single-molecule dynamics.”

About the paper:

“A Highly Fluorescent Nucleobase Molecular Rotor”, by Ashkan Karimi, Richard Börner, Guillaume Mata, and Nathan W. Luedtke was published in the August 2020 issue of the Journal of the American Chemical Society.

DOI: https://pubs.acs.org/doi/10.1021/jacs.0c05180

The brightest fluorescent nucleobase analog to date is a molecular rotor which discriminates matched/mismatched base pairs.