bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.04.10.036558

Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection

Bao, M.·Jensen, E.·Chang, Y.·Korensky, G.·Du, K.
View original source ↗Export metadata ↓

Abstract

We have developed a novel detection system which couples CRISPR-Cas recognition of target sequences, Cas mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for instrument-free detection of viral nucleic acid targets. After target recognition and Cas mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic particles. A complimentary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to un-cleaved probes on the magnetic beads. After separation of hybridized from unhybridized quantum dot conjugates by magnetic sequestration, the signal is measured fluorometrically to provide a signal proportional to the cleaved probes and therefore the amount of target nucleic acid. To demonstrate the power of this assay, a 250 bp DNA target sequence matching a portion of the African swine fever virus (ASFV) genome is used and a detection limit of ~0.5 nM is achieved without target amplification using a simple portable ultraviolet flashlight. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple, instrument free assay for rapid nucleic acid screening in both hospitals and point-of-care settings.

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Bao, M., Jensen, E., Chang, Y., Korensky, G., Du, K.. 2020-04-12. Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection. https://doi.org/10.1101/2020.04.10.036558

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The structures of two intermediates en route to DNA repair within a (6-4) photolyase

Pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are mutagenic DNA lesions produced when UV light crosslinks two adjacent pyrimidine bases. These lesions must be repaired to maintain genome integrity, a function executed by the (6-4) photolyase enzymes. This repair chemistry is complex, involving the transfer of a hydroxyl group from one base to another, accompanied or followed by cleavage of an interbase bond. It remains unclear how the enzyme orchestrates this reaction. Typically, models invoke a stepwise process in which an oxetane intermediate is transiently populated, but despite over thirty years of investigation, such an intermediate has never been definitively observed. Conducting dose-controlled X-ray crystallography on the (6-4) photolyase from Vibrio cholerae, we observed the transformation of 6-4PP into an oxetane species. Furthermore, we identified a previously unanticipated hemi-cleaved intermediate. The progressive transformation of 6-4PP into oxetane, hemi-cleaved, and fully repaired species requires a reexamination of the existing models of DNA repair by (6-4) photolyases.

biochemistry↗
bioRxiv2026

Endothelial protein C receptor is an entry receptor for Langya virus

Langya virus (LayV) was recently discovered in infected individuals with febrile illness and classified as a Parahenipavirus, a group of poorly understood pathogens. LayV is distantly related to Nipah henipavirus which causes recurring outbreaks with case fatality rates reaching up to 90%. No vaccines or therapeutics are approved for use in humans for any parahenipavirus or henipavirus despite their broad geographic distribution and pandemic potential. Here, we discovered that human endothelial protein C receptor (EPCR) is an entry receptor for LayV and that the tetrameric LayV attachment glycoprotein (G) binds EPCR avidly. We reveal the molecular basis of LayV G recognition of the EPCR receptor and provide a blueprint of the determinants of receptor ortholog usage. We found that human and shrew EPCR orthologs promote cell entry of LayV and of the closely related Mojiang pseudoviruses. These results along with the detection of several Parahenipaviruses in shrews set these pathogens apart from Henipaviruses, which use Pteropid fruit bats as reservoir hosts. Our data support future surveillance efforts, by enabling prediction of EPCR utilization among newly discovered Parahenipaviruses, and the development and evaluation of countermeasures targeting this emerging group of human pathogens.

biochemistry↗
bioRxiv2026

RNA-targeted aminopyridine compounds provide a new antifungal strategy

While RNA is an emerging therapeutic target class, progress is limited by a shortage of well-defined targets in which ligand binding is coupled to measurable function. The group I intron, a self-splicing ribozyme essential in fungal mitochondria but absent from humans, offers both a tractable platform for exploring first principles of small molecule-RNA targeting and a selective antifungal target. We recalibrated an intron-splicing assay to approximate cellular conditions and performed an extensive, cryo-EM-guided structure-activity relationship campaign against the Candida albicans mtLSU intron. Ligands containing a flexible diamine outperformed conformationally constrained analogues, suggesting a general principle for engaging dynamic RNA surfaces and yielding lead aminopyridine 56 (IC 21.6 nM). Compound 56 synergizes with amphotericin B against C. albicans, suggesting a path for combination therapies. RT-qPCR confirmed direct target engagement in cells. This work establishes the fungal group I intron as a viable, mechanistically distinct antifungal target and a model platform for RNA-targeted discovery.

biochemistry↗