SMU nanotechnology expert MinJun Kim helped a team of researchers at the University of Texas at Austin develop a low-cost way to detect nuclease digestion—one of the critical steps in many nucleic acid sensing applications used to identify Covid-19.
Nucleic acid detection is the primary method for identifying pathogens that cause infectious diseases. As millions of PCR tests are conducted every day around the world during the COVID-19 pandemic, it is important to reduce the cost of these tests.
A study published in the journal Nature Nanotechnology found that this low-cost tool, called Subaq, is useful in telling when nuclease digestion has occurred, when an enzyme called nuclease breaks nucleic acids such as DNA or RNA into smaller pieces. .
The traditional way to identify nuclease activity, the fluorescence resonance energy transfer (FRET) assay, is 62 times more expensive than the Subak reporter.
„The Subak reporter is cost-effective and simpler than FRET-based systems, providing an alternative method for detecting nuclease activity,Robert C. Lyle School of Engineering at SMU. said Kim, principal investigator of the Womack Chair and BAST Laboratory. „Many nucleic acid detection methods today, such as PCR and DETECTR, still rely on the use of FRET probes in their final steps.”
Unlike PCR, DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) is a simple assay or test that relies on CRISPR-Cas nuclease for pathogen DNA detection. Kim and researchers at UT Austin have successfully converted the FRET probe into the DETECTR assay with a subaqueous reporter, thereby significantly reducing the cost of the assay.
Subak reporters are based on a special class of fluorescent silver nanoclusters. They are composed of 13 silver atoms wrapped around a short strand of DNA – an organic/inorganic composite nanomaterial that is too small to be seen by the naked eye and ranges from 1 to 3 nanometers (one billionth of a meter) in size. .
Nanomaterials at this length scale are highly fluorescent like quantum dots and exhibit different colors. Fluorescent nanomaterials have found applications in biosensing such as TV displays and subaqueous reporter.
Lead researcher Tim Yeh, an associate professor of biomedical engineering at UT Austin's Cockrell School of Engineering, and his team programmed the Chubak reporters to emit a different color when digested by nucleases.
„These DNA-patterned silver nanoclusters initially emit green fluorescence, but undergo a significant color shift to bright red when fragmented by DNA nucleases.” Kim said. „The color change of the positive reporters is easily visualized under UV light,” Although the actual device is small.
Subaqueous reporters cost just $1 per nanomolecule to produce. In contrast, FRET — which requires the use of different fluorescent dyes to produce results that are more demanding — costs $62 per nanomolecule to produce, Kim said.
Kim and Madhav L. Kimire, a dean's postdoctoral fellow at SMU's Moody School of Graduate and Advanced Studies, worked with Ye to develop and characterize the DNA/AGNC silver nanoclusters. This involves increasing the intensity of green and red fluorescence before and after fragmentation by nuclei.
Characterization involves confirming the size, structure, and stability of nanoclusters in specific environments.
„Improving these low-cost detectors is necessary to monitor their fluorescence properties, to ensure the stability of the nanocluster, to control the size and structure, and most importantly to improve their sensitivity and selectivity in different environmental conditions, and to make them more reliable for sensing purposes.” Kimire said.
In addition to further testing for nuclear digestion of the Subak reporter, the team also wants to investigate whether this could be a probe for other biological targets.
Source: https://www.smu.edu