Biology subjects

Kamionskaya, M. V.

Publications and source records attributed to Kamionskaya, M. V..

2 recordsLinked to original sources

bioRxiv2026

G-quadruplex folding uncouples cis-activation from collateral trans-cleavage by Cas12a

The CRISPR/Cas12a system is widely used in nucleic-acid diagnostics because Cas12a couples guide-directed recognition of a cis-target with indiscriminate collateral trans-cleavage of ssDNA. Controlling these activities through DNA structure can tune diagnostic signals. G-quadruplexes (G4s) are strong structural regulators, yet their ability to modulate Cas12a activation and collateral cleavage remains undefined. Here we focused on the G4 scaffold (TGGG)n and tested it with LbCas12a and AsCas12a by real-time cleavage assays, circular dichroism, FRET and denaturing PAGE. In the cis position, compact (TGGG)n G4s activated Cas12a; for the most stable (TGGG)5, kcat/KM was 5.9x104 and 8.9x104 M-1 s-1 for LbCas12a and AsCas12a, respectively. K+ reduced cis-activation rates by approximately 10-fold, depending on scaffold and temperature. FRET and denaturing PAGE showed that productive cis-recognition involves G4 unfolding followed by target-strand cleavage. In the trans position, compact G4s fully resisted collateral reporter cleavage. Core disruption and G-rich non-G4 controls restored reporter cleavage, showing that resistance depends on G4 architecture rather than guanine content. These data define a compact G4 scaffold that remains functionally trans-resistant while retaining guide-dependent cis-target competence. Thus, compact G4 folding provides a programmable structural mechanism for separating Cas12a activation from reporter cleavage, opening a route to signal-gated diagnostic designs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/740342v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cb80c1org.highwire.dtl.DTLVardef@17200d9org.highwire.dtl.DTLVardef@195b9c3org.highwire.dtl.DTLVardef@d6e23b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification

CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have notable limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 minutes. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 minutes. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.

biochemistry↗