Canonical nucleic acids (DNA and RNA) naturally store genetic information with high density and programmability, making them promising candidates for molecular data storage. However, their susceptibility to degradation under harsh conditions, such as extreme pH, nuclease activity, and chemical attack, limits practical applications. In contrast, non-canonical nucleic acids (ncNAs) with natural or synthetic structural modifications exhibit enhanced stability and unique functional potential. This review systematically summarizes the fundamental properties of ncNAs, evaluates their suitability for molecular data storage, and discusses how their distinctive advantages may overcome the intrinsic limitations of canonical nucleic acids while addressing challenges in next-generation storage systems. Canonical nucleic acids, with high storage density, are promising media for data storage but are limited by instability. Here, authors summarise the unique properties and advantages of non-canonical nucleic acids in data storage and highlight key challenges for their practical application.

Numerous Gram-negative bacteria possess N-acyl-L-homoserine lactone (AHL)-mediated quorum-sensing (QS) systems that regulate the activation of specific genes. Burkholderia plantarii causes rice seedling blight by producing the phytotoxin tropolone. In this study, we investigated multiple AHL-type QS systems in B. plantarii MAFF 301723T and their involvement in virulence regulation. MAFF 301723 harbors three AHL-mediated QS systems, designated plaI1/plaR1, plaI2/plaR2, and plaI3/plaR3. The plaI1/plaR1 system, which produces N-octanoyl-l-homoserine lactone, is functional and essential for swarming motility. When forced expression of plaI2 induces the biosynthesis of 3-OH-C10-HSL, it was suggested that expression is rarely observed in wild-type MAFF 301723. The plaI3 gene directs the synthesis of the putative C16:2-HSL, which is a rare AHL bearing two double bonds in the hexadecanoyl chain that has not been previously reported in Burkholderia spp. The plaI3/plaR3-QS system is crucial for tropolone production. These findings suggest that multiple QS systems collectively contribute to the complex virulence regulation of B. plantarii, thereby providing new insights into the development of QS-targeted biocontrol strategies for agriculture.

Photoreceptor proteins regulate fundamental biological processes such as vision, photosynthesis and circadian rhythms. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions. The exact molecular mechanism of B12 photoreception and how this differs from the thermal pathways remains unknown. Here we provide a detailed description of photoactivation in the prototypical B12 photoreceptor CarH from nanoseconds to seconds, combining time-resolved and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. Building on the crystal structures of the initial tetrameric dark and final monomeric light-activated states, our structural snapshots of key intermediates in the truncated B12-binding domain illustrate how photocleavage of a cobalt–carbon (Co–C) bond within the B12 chromophore adenosylcobalamin triggers a series of structural changes that propagate throughout CarH. Breakage of the photolabile Co–C5′ bond leads to the formation of a previously unknown adduct that links the C4′ position of the adenosyl moiety to the Co ion and can subsequently be cleaved thermally over longer timescales to allow release of the adenosyl group, ultimately causing tetramer dissociation. This adduct, which differentiates CarH from thermally activated B12 enzymes, steers the photoactivation pathway and acts as the molecular bridge between photochemical and photobiological timescales. The biological relevance of our study is corroborated by kinetic data on full-length CarH in the presence of DNA. Our results offer a spatiotemporal understanding of CarH photoactivation and pave the way for designing B12-dependent photoreceptors for optogenetic applications. Spatiotemporal insight into photoactivation of the prototypical B12 photoreceptor CarH is revealed across nine orders of magnitude in time, identifying a transient adduct that distinguishes it from thermally activated B12 enzymes.

Cellobiose (4-O-β-D-Glucopyranosyl-D-glucopyranose) is an important disaccharide utilized, for example in food and cosmetics. It can be enzymatically synthesized involving two steps from sucrose and glucose, where first the sucrose undergoes phosphorolysis by sucrose phosphorylase, yielding glucose 1-phosphate and fructose. Glucose 1-phosphate is then combined with glucose into cellobiose, releasing phosphate in a reaction catalyzed by cellobiose phosphorylase. To better control and reuse the enzymes in the two main reaction steps, immobilization on Bacillus subtilis spores is a promising approach due to the ease of production and recyclability.  Here we describe the display of a sucrose phosphorylase and a cellobiose phosphorylase on B. subtilis spores through fusion with the crust protein CotY, to our knowledge, marking the first use of multiple enzymes directly displayed on the spore surface during sporulation in a reaction cascade. While immobilization had no effect on thermostability, we demonstrate the recyclability of the individual spore variants over four reaction cycles at 45 °C with sucrose phosphorylase maintaining 35% of its initial activity and cellobiose phosphorylase maintaining 65%. Both spore variants were used together to catalyse a reaction cascade in a separated two-pot, as well as in a one-pot reaction. The one-pot reaction achieved a 90% yield with respect to the initially available 40 mM of glucose. The one-pot cascade maintained activity after being recycled five times over the course of 120 hours. Furthermore, we report on improving the reaction yield in the two-pot reaction from 60% to 80% by using calcium to precipitate excess phosphate.  In this study we demonstrate that spores are a suitable immobilization platform for multistage reaction cascades. The spores displaying biocatalysts can be recovered and reused over multiple reaction cycles. The immobilization of glycosylic enzymes on spores enables cost-effective, scalable enzyme production on a temperature-resistant carrier that facilitates purification. The potential modularity of this approach adds to the adaptability of the system to different requirements in terms of substrate and product. 

Bacteria use diverse mechanisms to protect themselves against phages. Many antiphage systems form large oligomeric complexes, but how oligomerization is regulated during phage infection remains mostly unknown. Here we demonstrate that the bacterial immunity protein ring-activated zinc-finger RNase (RAZR) assembles into an active, 24-meric ring around the circumference of large ring structures formed by two unrelated phage proteins: a putative recombinase and a portal protein. Each multi-layered, megadalton-scale complex enables RAZR to cleave RNA nonspecifically to inhibit translation and restrict phage propagation. The recognition of unrelated phage proteins that form rings with similar diameters indicates that these proteins not only bind to RAZR but also enforce a geometry crucial to activation. The lack of large ring structures in the host probably prevents auto-immunity and RAZR activation before infection. The infection-triggered oligomerization of RAZR mirrors pathogen-induced oligomerization in eukaryotic innate immune complexes, underscoring a common principle of immunity across biology. An antiphage defence system has an activation mechanism that relies on the sensing of phage-encoded proteins that enforce geometry crucial to activation and are not typically present in non-infected cells.

Synthetic gene circuits are powerful tools for precisely programming gene expression and introducing novel cellular functions. However, their development and application in plants has lagged behind other systems, due mainly to the limited availability of modular genetic parts. We recently developed a CRISPR interference (CRISPRi)-based synthetic gene circuit system for programming gene expression in plants. Using a robust and high-throughput protoplast-based dual luciferase assay, we demonstrated the development, testing and functionality of these circuits in various plant species. Here we detail the key design principles and considerations for building and testing programmable and reversible CRISPRi-based gene circuits in plants. We also provide detailed procedures for isolating protoplasts from multiple plant species, including Arabidopsis thaliana, Brassica napus, Triticum aestivum and Physcomitrium patens. Furthermore, we provide step-by-step instructions for the 96-well plate-based protoplast transfection assay for testing genetic parts and synthetic circuits, using a dual luciferase assay. The detailed descriptions of these developed systems will enhance the efficiency and reproducibility of the construction, testing, and implementation of synthetic gene circuits in a variety of plant species. This protocol enables the design and testing of CRISPRi-based gene circuits in plants within ~4 weeks. This protocol details the design principles and procedures for building programmable CRISPRi-based gene circuits, and for testing individual components and the assembled circuits in various plant species using a high-throughput protoplast-based assay.