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The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.

Candidatus phylum Eremiobacterota (formerly WPS-2) is an as-yet-uncultured bacterial clade that takes its name from Ca. Eremiobacter, an Antarctic soil aerobe proposed to be capable of a novel form of chemolithoautotrophy termed atmospheric chemosynthesis, that uses the energy derived from atmospheric H 2 -oxidation to fix CO 2 through the Calvin-Benson-Bassham (CBB) cycle via type 1E RuBisCO. To elucidate the phylogenetic affiliation and metabolic capacities of Ca. Eremiobacterota, we analysed 63 public metagenome-assembled genomes (MAGs) and nine new MAGs generated from Antarctic soil metagenomes. These MAGs represent both recognized classes within Ca. Eremiobacterota, namely Ca. Eremiobacteria and UBP9. Ca. Eremiobacteria are inferred to be facultatively acidophilic with a preference for peptides and amino acids as nutrient sources. Epifluorescence microscopy revealed Ca. Eremiobacteria cells from Antarctica desert soil to be coccoid in shape. Two orders are recognized within class Ca. Eremiobacteria: Ca. Eremiobacterales and Ca. Baltobacterales. The latter are metabolically versatile, with individual members having genes required for trace gas driven autotrophy, anoxygenic photosynthesis, CO oxidation, and anaerobic respiration. UBP9, here renamed Ca. Xenobia class. nov., are inferred to be obligate heterotrophs with acidophilic adaptations, but individual members having highly divergent metabolic capacities compared to Ca. Eremiobacteria, especially with regard to respiration and central carbon metabolism. We conclude Ca. Eremiobacterota to be an ecologically versatile phylum with the potential to thrive under an array of "extreme" environmental conditions.

Biology demonstrates how a near infinite array of complex systems and structures at many scales can originate from the self-assembly of component parts on the nanoscale. But to fully exploit the 2 benefits of self-assembly for nanotechnology, a crucial challenge remains: How do we rationally encode well-defined global architectures in subunits that are much smaller than their assemblies? Strain accumulation via geometric frustration is one mechanism that has been used to explain the self-assembly of global architectures in diverse and complex systems a posteriori. Here we take the next step and use strain accumulation as a rational design principle to control the length distributions of self-assembling polymers. We use the DNA origami method to design and synthesise a molecular subunit known as the PolyBrick, which perturbs its shape in response to local interactions via flexible allosteric blocking domains. These perturbations accumulate at the ends of polymers during growth, until the deformation becomes incompatible with further extension. We demonstrate that the key thermodynamic factors for controlling length distributions are the intersubunit binding free energy and the fundamental strain free energy, both which can be rationally encoded in a PolyBrick subunit. While passive polymerisation yields geometrical distributions, which have the highest statistical length uncertainty for a given mean, the PolyBrick yields polymers that approach Gaussian length distributions whose variance is entirely determined by the strain free energy. We also show how strain accumulation can in principle yield length distributions that become tighter with increasing subunit affinity and approach distributions with uniform polymer lengths. Finally, coarse-grained molecular dynamics and Monte Carlo simulations delineate and quantify the dominant forces influencing strain accumulation in a molecular system. This study constitutes a fundamental investigation of the use of strain accumulation as a rational design principle in molecular self-assembly.

Pseudogenes are abundant in the human genome and had long been thought of purely as nonfunctional gene fossils. Recent observations point to a role for pseudogenes in regulating genes transcriptionally and post-transcriptionally in human cells. To computationally interrogate the network space of integrated pseudogene and long non-coding RNA regulation in the human transcriptome, we developed and implemented an algorithm to identify all long non-coding RNA (lncRNA) transcripts that overlap the genomic spans, and specifically the exons, of any human pseudogenes in either sense or antisense orientation. As inputs to our algorithm, we imported three public repositories of pseudogenes: GENCODE v17 (processed and unprocessed, Ensembl 72); Retroposed Pseudogenes V5 (processed only), and Yale Pseudo60 (processed and unprocessed, Ensembl 60); two public lncRNA catalogs: Broad Institute, GENCODE v17; NCBI annotated piRNAs; and NHGRI clinical variants. The data sets were retrieved from the UCSC Genome Database using the UCSC Table Browser. We identified 2277 loci containing exon-to-exon overlaps between pseudogenes, both processed and unprocessed, and long non-coding RNA genes. Of these loci we identified 1167 with Genbank EST and full-length cDNA support providing direct evidence of transcription on one or both strands with exon-to-exon overlaps. The analysis converged on 313 pseudogene-lncRNA exon-to-exon overlaps that were bidirectionally supported by both full-length cDNAs and ESTs. In the process of identifying transcribed pseudogenes, we generated a comprehensive, positionally non-redundant encyclopedia of human pseudogenes, drawing upon multiple, and formerly disparate public pseudogene repositories. Collectively, these observations suggest that pseudogenes are pervasively transcribed on both strands and are common drivers of gene regulation.

The potential metabolism and ecological roles of many microbial taxa remain unknown because insufficient genomic data are available to assess their functional potential. Two such microbial “dark matter” taxa are the Candidatus bacterial phyla Cloacimonadota and Omnitrophota, both of which have been identified in global anoxic environments, including (but not limited to) organic-carbon-rich lakes. Using 24 metagenome-assembled genomes (MAGs) obtained from an Antarctic lake (Ace Lake, Vestfold Hills), novel lineages and novel metabolic traits were identified for both phyla. The Cloacimonadota MAGs exhibited a capacity for carbon fixation using the reverse tricarboxylic acid cycle driven by oxidation of hydrogen and sulfur. Certain Cloacimonadota MAGs encoded proteins that possess dockerin and cohesin domains, which is consistent with the assembly of extracellular cellulosome-like structures that are used for degradation of polypeptides and polysaccharides. The Omnitrophota MAGs represented phylogenetically diverse taxa that were predicted to possess a strong biosynthetic capacity for amino acids, nucleosides, fatty acids, and essential cofactors. All of the Omnitrophota were inferred to be obligate fermentative heterotrophs that utilize a relatively narrow range of organic compounds, have an incomplete tricarboxylic acid cycle, and possess a single hydrogenase gene important for achieving redox balance in the cell. We reason that both Cloacimonadota and Omnitrophota form metabolic interactions with hydrogen-consuming partners (methanogens and Desulfobacterota, respectively) and, therefore, occupy specific niches in Ace Lake.

Supplementary Note 1 -Search algorithm for locating minimally-strained crossovers.Determination of DNA backbone coordinates. The backbone of a DNA double helix was modelled geometrically with a parametric equation for helices:where r is the radius of an ideal DNA helix (1.0 nm), g is the minor groove angle of the DNA (135°) and is positive for one strand of DNA and negative for the complementary strand, and ℎ 45 is the helical pitch of a DNA helix (3.55 nm). The coordinates of C3' carbon atoms along the helical path were defined by the helical pitch of DNA on a double helix in base pair units ( ℎ 78 = 10.5 / ) at points where = F × GH 8FIJK LM° where i is the base number and ranges from 0 to the length of the DNA helix in base pair units.Thus, a set of coordinates for C3' carbon atoms and NEMids could be determined for a DNA helix. This set of coordinates was transformed to construct an arrangement of three helices with the desired internal angle (helix α, helix β and helix α+1), (Figure 1D and 1E) and an inter-helix distance of 2.25 nm. This corresponds to a geometry where perfectly-aligned crossovers should have an ideal bond-distance. For a given internal angle and number of helices in the nanotube, the minimally strained crossover locations were determined for all combinations of q a and q b angles ( Figure 1D and E), which were sampled every 1°.Identification of crossover locations. From these geometric models, all inter-NEMid and inter-C3' carbon distances and strain scores can be determined. To reduce the number of calculations and ensure an average staple crossover density of one crossover every 1.5 turns of a DNA helix, it was useful to first approximate the location of staple crossovers. This was done as follows: Template crossovers are placed at ends of DNA helices in the nanotube. At the 'bottom' end of the nanotube, the algorithm first determines whether the template crosses over between helix α and helix β, or between helix β and helix α+1 by assessing the strain score of each. The location of the template crossover at the bottom of helix β then sets a reference point from which the location of staple crossovers that form the first length unit can be approximated. If the first template

Living systems achieve robust self-assembly across length scales. Meanwhile, nanofabrication strategies such as DNA origami have enabled robust self-assembly of submicron-scale shapes.However, erroneous and missing linkages restrict the number of unique origami that can be practically combined into a single supershape. We introduce crisscross polymerization of DNA-origami slats for strictly seed-dependent growth of custom multi-micron shapes with user-defined nanoscale surface patterning. Using a library of ~2000 strands that can be combinatorially assembled to yield any of ~1e48 distinct DNA origami slats, we realize five-gigadalton structures composed of >1000 uniquely addressable slats, and periodic structures incorporating >10,000 slats. Thus crisscross growth provides a generalizable route for prototyping and scalable production of devices integrating thousands of unique components that each are sophisticated and molecularly precise.