Fig. 6: Example of potential SsnA/NTS biotechnological applications with or without coupling rolling circle amplification (RCA).
From: Discovery of the widespread site-specific single-stranded nuclease family Ssn
In this figure, the left panels in each sub-figure illustrate the experimental setup, while the right panels illustrate the results. a SsnA cleavage of an NTS75 concatemer amplified by RCA at different time points. Semi-digested concatemers of various sizes, containing multiple NTS75 sequences, are incrementally digested, ultimately yielding a single band matching the original oligonucleotide size (120 nt) but corresponding to its reverse sequence. b SsnA activity on two ssDNA oligonucleotides, each containing half of the NTS stem without the loop. After hybridization with sequences containing the complementary half of the palindrome, cleavage is observed via post-electrophoresis staining (ssGreen-Lumiprobe). The “1–38” sequence includes the first 38 nucleotides of NTS75, while “39–75” includes the second half of NTS75. “39–75polyA” represents 39–75 with added polyA (in red), “HemiF” consists of the 1–38 sequence with random ends (yellow), and “LT” contains the 39-75 part of NTS with a complementary HemiF sequence to increase stem size. An asterisk (*) indicates 5′ 6-FAM labeling, and “HI” corresponds to heat-inactivated samples. c Cleavage of an RCA-produced ssDNA concatemer containing the first half of the NTS. HemiR circularization and amplification produce HemiF. Hybridization with the 39–75 oligo enables specific cleavage using an ssDNA guide. d Quantification assay via fluorescence quenching. An ssDNA sequence (*1–38Q) containing the first half of the NTS with 5′ 6-FAM and a 3′ quencher (3′ BHQ1) was hybridized with the 39–75 sequence and cleaved by SsnA to release fluorescence, quantifiable based on the amount of 39–75 oligo. e Detection of the 39–75 region via RCA amplification, hybridization with *1–38Q, and cleavage by SsnA over 150 min. The RCA with a correctly oriented 39–75 concatemer is detected, while the reverse concatemer is not. The indicated concentrations represent the amount of DNA originally used for RCA. Histograms represent the mean, and orange bars represent the standard deviation. Each point represents an independent reaction mix. Three replicates were done with DNA and protein, two without protein. Experiments were repeated with SsnA from two purification batches. Schematic representation Created in BioRender. Veyrier, F. (2025) https://BioRender.com/x76q784 and https://BioRender.com/y23w368. Source data are provided as a Source Data file76.
