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Abstract RNA-driven phase separation is emerging as a promising approach for engineering biomolecular condensates with diverse functionalities. Condensates form thanks to weak yet specific RNA–RNA interactions established by design via complementary sequence domains. Here, we demonstrate how RNA condensates formed by star-shaped RNA motifs, or nanostars, can be dynamically controlled when the motifs include additional linear or branch-loop domains that facilitate access of regulatory RNA molecules to the nanostar interaction domains. We show that condensates dissolve in the presence of RNA “invaders” that occlude selected nanostar bonds and reduce the valency of the nanostars, preventing phase separation. We further demonstrate that the introduction of “anti-invader” strands, complementary to the invaders, makes it possible to restore condensate formation. An important aspect of our experiments is that we demonstrate these behaviors in one-pot reactions, where RNA nanostars, invaders, and anti-invaders are simultaneously transcribed in vitro using short DNA templates. Our results lay the groundwork for engineering RNA-based assemblies with tunable, reversible condensation, providing a promising toolkit for synthetic biology applications requiring responsive, self-organizing biomolecular materials. 

Recent discoveries in biology have highlighted the importance of protein and RNA-based condensates as an alternative to classical membrane-bound organelles. Here, we demonstrate the design of pure RNA condensates from nanostructured, star-shaped RNA motifs. We generate condensates using two different RNA nanostar architectures: multi-stranded nanostars whose binding interactions are programmed via linear overhangs, and single-stranded nanostars whose interactions are programmed via kissing loops. Through systematic sequence design, we demonstrate that both architectures can produce orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. Successful co-transcriptional formation of condensates from single-stranded nanostars suggests that they may be genetically encoded and produced in living cells. We provide a library of orthogonal RNA condensates that can be modularly customized and offer a route toward creating systems of functional artificial organelles for the task of compartmentalizing molecules and biochemical reactions. 

Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate fluorophores through embedded fluorescent light-up aptamers. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with a controlled number and relative size, and showing the ability to capture proteins using selective protein-binding aptamers. The affinity between otherwise orthogonal nanostructures can be modulated by introducing dedicated linker constructs, enabling the production of bi-phasic RNA condensates with a prescribed degree of interphase mixing and diverse morphologies. The in situ expression of programmable RNA condensates could underpin the spatial organization of functionalities in both biological and synthetic cells. 

Functional balance theory predicts that plants will allocate less carbon belowground when the availability of nutrients is elevated. We tested this prediction in two successional northern hardwood forest stands by quantifying fine root biomass and growth after 5–7 years of treatment in a nitrogen (N) x phosphorus (P) factorial addition experiment. We quantified root responses at two different levels of treatment: the whole-plot scale fertilization and small-patch scale fertilization of ingrowth cores. Fine root biomass was higher in plots receiving P, and fine root growth was highest in plots receiving both N and P. Thus, belowground productivity did not decrease in response to long-term addition of nutrients. We did not find conclusive evidence that elevated availability of one nutrient at the plot scale induced foraging for the other nutrient at the core scale, or that foraging for nutrients at the core scale responded to addition of limiting nutrients. Our observations suggest NP co-limitation of fine root growth and indicate complex interactions of N and P affecting aboveground and belowground production in early successional northern hardwood forest ecosystems. 

The Multiple Element Limitation in Northern Hardwood Ecosystems (MELNHE) project studies N , P, and Ca acquisition and limitation of forest productivity through a series of nutrient manipulations in northern hardwood forests. This data set includes data testing effects of elevated N and P availability on fine root growth (using ingrowth cores) and biomass in the MELNHE project. Subsets of ingrowth cores were treated with nutrients differing from the plot-scale nutrient treatments to test fine root foraging. Additional detail on the MELNHE project, including a datatable of site descriptions and a pdf file with the project description and diagram of plot configuration can be found in this data package: https://portal.edirepository.org/nis/mapbrowse?scope=knb-lter-hbr&identifier=344 These data were gathered as part of the Hubbard Brook Ecosystem Study (HBES). The HBES is a collaborative effort at the Hubbard Brook Experimental Forest, which is operated and maintained by the USDA Forest Service, Northern Research Station. 

Abstract Soil respiration in 15 stands across 3 sites within the White Mountain National Forest was measured between 2008 and 2020. Stands included in the dataset are part of the Multiple Element in Northern Hardwood Ecosystems (MELNHE) study, a full-factorial NxP fertilization experiment. Pre- and post-treatment data are included, with treatment beginning in 2011. Soil temperature, soil moisture, and relative air humidity at the time of measurement were also recorded next to or above the soil respiration collar at the time of the soil respiration measurement. Having been cut between 1883 and 1990, stands are representative of different successional stages.