Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in an E. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a ‘bio-orthogonal’ hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devices in vivo and of the intracellular delivery of therapeutic nanostructures, as well as the in vitro and in vivo detection of different DNA/RNA targets.

Candidiasis is a group of opportunistic infections caused by yeast of the genus Candida. The appearance of drug resistance and the adverse effects of current antifungal therapies require the search for new, more efficient therapeutic alternatives. Killer yeasts have aroused as suitable candidates for mining new antifungal compounds. Killer strains secrete antimicrobial proteins named killer toxins, with promissory antifungal activity. Here we found that the killer yeast Wickerhamomyces anomalus Cf20 and its cell-free supernatant (CFS) inhibited six pathogenic strains and one collection strain of Candida spp. The inhibition is mainly mediated by secreted killer toxins and, to a lesser extent, by volatile compounds such as acetic acid and ethyl acetate. A new large killer toxin (>180 kDa) was purified, which exerted 70–74% of the total CFS anti-Candida activity, and the previously described glucanase KTCf20 was inhibitory in a lesser extent as well. In addition, we demonstrated that Cf20 possesses the genes encoding for the β-1,3-glucanases WaExg1 and WaExg2, proteins with extensively studied antifungal activity, particularly WaExg2. Finally, the 10-fold concentrated CFS exerted a high candidacidal effect at 37°C, completely inhibiting the fungal growth, although the nonconcentrated CFS (RCF 1) had very limited fungistatic activity at this temperature. In conclusion, W. anomalus Cf20 produces different low and high molecular weight compounds with anti-Candida activity that could be used to design new therapies for candidiasis and as a source for novel antimicrobial compounds as well.

DNA origami enables the programmable self-assembly of nucleic acids into precisely defined nanostructures, yet the influence of primary base sequence on folding reliability remains incompletely understood. In particular, off-target interactions between scaffold and staple strands may introduce kinetic traps and reduce assembly yield, even when the intended Watson-Crick complementarity is preserved. Here we show that scaffold sequence strongly affects DNA origami assembly through the prevalence of off-target binding reactions implicit in the chosen base sequence. We developed a multi-objective computational framework that scores candidate scaffold sequences according to four classes of off-target interactions and selects variants predicted to minimise these effects for a given origami design. Using this approach, we identified both favourable and unfavourable scaffold regions from biological and synthetic sequences and tested them experimentally across 2D and 3D DNA origami structures. Atomic force microscopy showed that scaffolds predicted to have fewer off-target interactions consistently folded with higher yield, whereas off-target-prone scaffolds largely failed despite having fully complementary staple sets. Single-molecule optical tweezers further revealed that scaffold variants with fewer predicted off-target interactions assemble into more mechanically uniform origami structures. These results establish off-target sequence effects as a major determinant of origami folding and we provide a software tool to select scaffold sequences that minimise off-target reactions for any DNA origami design.

Designs for scaffolded DNA origami nanostructures are commonly and minimally published as the list of DNA staple and scaffold sequences required. In nearly all cases, high-level editable design files (e.g. caDNAno) which generated the low-level sequences are not made available. This de facto ‘raw sequence’ exchange format allows published origami designs to be re-attempted in the laboratory by other groups, but effectively stops designs from being significantly modified or re-purposed for new future applications. To make the raw sequence exchange format more accessible to further design and engineering, in this work we propose the first algorithmic solution to the inverse problem of converting staple/scaffold sequences back to a ‘guide schematic’ resembling the original origami schematic. The guide schematic can be used to aid the manual re-input of an origami into a CAD tool like caDNAno, hence recovering a high-level editable design file. Creation of a guide schematic can also be used to double check that a list of staple strand sequences does not have errors and indeed does assemble into a desired origami nanostructure prior to costly laboratory experimentation. We tested our reverse algorithm on 36 diverse origami designs from the literature and found that 29 origamis (81%) had a good quality guide schematic recovered from raw sequences. Our software is made available at https://revnano.readthedocs.io .