Fig. 2: Chemically modified DNA enables CRISPR-Cas-mediated urinary readout for in vivo sensing.

a, Schematic of urine testing in a murine model and the study time course. Urine samples were collected from mice injected with 1 nmol of ssDNA activator after 1 h of intravenous administration. DNA fragment activates the collateral nuclease activity of Cas12a upon binding to crRNA. Such activity can be tracked by the dequenching of fluorescence from a fluorophore (F, 5′-FAM)–quencher (Q, 3′-IABkFQ) paired oligonucleotide. The initial reaction velocity (V₀) is determined from the slope of the curve at the beginning of a reaction. b, Length optimization of ssDNA activator in vitro and in vivo, by quantifying the trans-cleavage rate of Cas12a upon activation of native or modified ssDNA in solution (4 nM) or mouse urine (1 nmol per injection). The trans-cleavage rates (initial reaction velocity, V₀) were normalized to that of a 24-mer. c, Schematic showing set-up of paper-based lateral-flow assay. When Cas12a is activated by the DNA fragments in mouse urine, it cleaves the fluorescein (FAM)–biotin paired oligonucleotide reporter and frees the FAM molecule, which can be detected on the ‘sample band’. Uncleaved reporters are trapped on the ‘control band’ via binding of biotin to streptavidin. d, Different bands are visible on paper strips. Band intensities were quantified using ImageJ, and each curve was aligned below the corresponding paper strip. The top peak of the curve shows the freed FAM molecule in cleaved reporter samples, and the bottom peak shows the presence of the uncleaved FAM–biotin reporter. e, Michaelis–Menten plot of LbaCas12a-catalysed ssDNA trans-cleavage upon a representative DNA–crRNA pairing (complementary sequences are shown) on paper. Data were plotted with the quantified band intensity of cleaved reporter on paper strips. Km is determined as the DNA activator concentration at which the reaction rate is half of its maximal value.

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