Novel development of heat treatment techniques for seed surface sterilisation

Heat treatment to prevent seed borne diseases, e.g. in the form of hot water or warm humid air, will normally heat up the entire seed. Heat treatment of the seed embryo will always have a negative side-effect on seed vigour and the duration of several minutes are difficult to implement in seed plans treating huge volumes of seed. However, in the case of common bunt in wheat and similar diseases, where the inoculum is transmitted as fungal spores on the surface of the seed, an uniform and efficient surface sterilisation will be sufficient to prevent the transmission of the disease. To test the effect of surface heat sterilisation of seed, an equipment was developed, where seeds were exposed to a combination of steam and ultrasound. The principle is that the ultrasound will create a fluctation of the air molecules in the chamber, and thereby increase the access of the hot steam to the surface of the seed. In this equipment, common bunt in wheat was eliminated after 4 sec. treatment and in spelt after 8 seconds. A 16 times increase of the duration of the treatment did not decrease germination speed of the seed, tested in a cold sand test. This demonstrates that surface transmitted diseases can be controlled efficiently and environmentally friendly by equipment adaptable to commercial seed plans

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low

A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3′-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity