Journal: bioRxiv

Article Title: ProPER: Programmable, multiplexed detection of molecular proximities and RNA life-cycle stages in situ

doi: 10.64898/2026.04.24.718482

Figure Lengend Snippet: (a) Principle of proximity detection by in situ PER (left) and reaction mechanism (right). The docking sites for primer and hairpin need to be in close physical proximity for the catalytic primer extension to take place. Extended DNA concatemer is detected by fluorescently labeled imager oligos. When a cognate primer and hairpin pair are in close proximity, PER – takes place. The polymerase extends the primer with copies of sequence a and generates a linear ssDNA concatemer through autonomous and isothermal cycles of: (i) primer-hairpin binding, (ii) extension of the primer by a strand-displacing polymerase (in this work BST large fragment) using the hairpin’s a* sequence as a template, (iii) branch migration, (iv) dissociation of the primer from the hairpin. (b) Applications for proximity detection by ProPER. Primer and hairpin are docked to RNA by in situ hybridization of DNA probes or to proteins via DNA-labeled antibodies to detect proximity of the molecules or coincidence of probes on the same target. Bottom: In detection cascades different sequences are attached in a programmed order. Right: The life cycle of an RNA can be revealed by multiplexed detection of proximity to life-cycle markers. (c) In situ RNA FISH and ProPER for CBX5 gene in HeLa cells under limiting probe numbers. SABER-FISH was performed with in vitro pre-extended concatemers, whereas for ProPER primers were extended in situ after docking to a single site (ProPER medium) or multiple sites on a pre-extended primary probe (ProPER high). Representative images show maximum projections of FISH/ProPER signal (orange to white gradient) and DAPI (cyan). (d) Negative controls for the ProPER medium condition, where primer, hairpin dock or polymerase were left out. I Raw signal intensity of the spots (in arbitrary fluorescence units, a.u.) and (f) spot counts per cell showed higher values for ProPER compared to SABER-FISH with a small number of FISH probes (5-6 oligo probes). Signal to background ratio (S/B) is noted above each curve (see Methods ). Mann-Whitney-Wilcoxon test, two-sided, * p<0.05, *** p<0.001 (exact p-values in Supplementary Table S3 ). N=105-782 cells per condition. Scale bars 10 μm.

Article Snippet: Final concentrations in 200 μL reaction volume were 1× PBS, 480U/ml Bst LF polymerase (NEB, M0275M), 10 mM MgSO4, 0.6 mM dNTPs (dATP, dCTP, dTTP), 0.1 μM Clean.G hairpin (IDT), 0.6-1.0 μM hairpin and 0.75 μM primer.

Techniques: In Situ, Labeling, Sequencing, Binding Assay, Migration, RNA In Situ Hybridization, In Vitro, Fluorescence, MANN-WHITNEY