A new tool could reduce the costs of diagnosing infectious diseases.
Biomedical researchers at the University of Texas at Austin have developed a new, less expensive way to detect nuclease digestion, one of the critical steps in many nucleic acid detection applications, such as those used to identify COVID-19 and other infectious diseases. .
A new study published in the journal Nanotechnology from nature shows that this low-cost tool, called Subak, is effective at determining when nucleic acid cleavage occurs, which occurs when an enzyme called nuclease breaks down nucleic acids, such as DNA or RNA, into smaller fragments.
The traditional way to identify nuclease activity, the fluorescence resonance energy transfer (FRET) probe, costs 62 times more than the Subak reporter.
“To make diagnostics more accessible to the public, we have to reduce costs,” said Soonwoo Hong, Ph.D. student in the lab of Tim Yeh, an associate professor in the Department of Biomedical Engineering at the Cockrell School of Engineering, who led the work. “Any improvements in nucleic acid detection will strengthen our testing infrastructure and facilitate widespread detection of diseases like COVID-19.”
The research team, which also included Jennifer Brodbelt, a professor of chemistry in the College of Natural Sciences at UT Austin, and MinJun Kim, a professor of mechanical engineering in the Lyle School of Engineering at Southern Methodist University, replaced the FRET probe traditional with the Subak reporter in a test called DETECTR (DNA endonuclease-directed CRISPR trans reporter).
Subak's reporters are based on a special class of fluorescent nanomaterials known as silver nanoclusters. They are made up of 13 silver atoms wrapped within a short chain of DNA. This organic/inorganic composite nanomaterial is too small to be visible to the naked eye and ranges in size from 1 to 3 nanometers (one billionth of a meter).
Nanomaterials in this length scale, such as semiconductor quantum dots, can be highly luminescent and exhibit different colors. Fluorescent nanomaterials have found applications in television displays and biosensors, such as Subak reporters.
“We have very clear evidence from mass spectrometry that the transformation of Ag13 to Ag10 “underlines the green-to-red color conversion observed in the sample after digestion of the DNA template,” Brodbelt said.
Subak reporters, which can be synthesized at room temperature in a one-pot reaction, cost just $1 per nanomole. In contrast, producing the FRET probe, which uses complex steps to label a donor dye and a quencher, costs $62 per nanomole.
“These highly luminescent silver nanoclusters can be called quantum dots, as they show strong fluorescence emission of tunable size due to the quantum confinement effect,” Yeh said. “No one can precisely tune the size of the cluster (and the corresponding emission color) until our Subak demonstration,” highlighting the innovation of this research.
In addition to further testing the Subak reporter for nuclease digestion, the team also wants to investigate whether it can be a probe for other biological targets.
The work is supported by a National Science Foundation grant to Yeh and Brodbelt.