Explore the vast range of titles available.

Due to its longevity and enormous information density, DNA is an attractive medium for archival storage. The current hamstring of DNA data storage systems—both in cost and speed—is synthesis. The key idea for breaking this bottleneck pursued in this work is to move beyond the low-error and expensive synthesis employed almost exclusively in today’s systems, towards cheaper, potentially faster, but high-error synthesis technologies. Here, we demonstrate a DNA storage system that relies on massively parallel light-directed synthesis, which is considerably cheaper than conventional solid-phase synthesis. However, this technology has a high sequence error rate when optimized for speed. We demonstrate that even in this high-error regime, reliable storage of information is possible, by developing a pipeline of algorithms for encoding and reconstruction of the information. In our experiments, we store a file containing sheet music of Mozart, and show perfect data recovery from low synthesis fidelity DNA. The current bottleneck for DNA data storage systems is the cost and speed of synthesis. Here, the authors use inexpensive, massively parallel light-directed synthesis and correct for a high error rate with a pipeline of encoding and reconstruction algorithms.

Fluorescence is an ideal tool to see and manipulate nucleic acids, and engage in their rich and complex biophysical properties. Labeling is the preferred approach to track and quantify fluorescence with nucleic acids and cyanine dyes are emblematic in this context. The fluorescent properties of cyanine dyes are known to be sequence-dependent, with purines in the immediate vicinity increasing the fluorescence intensity of Cy3 and Cy5 dyes, and the ability of nucleobases to modulate the photophysical properties of common fluorophores may influence fluorescence measurements in critical assays such as FISH, qPCR or high-throughput sequencing. In this paper, we comprehensively map the sequence-dependence of Cy3 and Cy5 dyes in 3ʹ-fluorescently labeled single-stranded DNA by preparing the complete permutation library of the 5 consecutive nucleotides immediately adjacent to the dye, or 1024 sequences. G-rich motifs dominate the high fluorescence range, while C-rich motifs lead to significant quenching, an observation consistent with 5ʹ-labeled systems. We also uncover GCGC patterns in the extreme top range of fluorescence, a feature specific to 3ʹ-Cy3 and Cy5 oligonucleotides. This study represents the final piece in linking nucleotide identity to fluorescence changes for Cy3, Cy5 and fluorescein in all 3ʹ, 5ʹ, single-stranded and double-stranded DNA formats.

Data storage on DNA has emerged as a molecular approach to safeguarding digital information. Microarrays are an excellent source of complex DNA sequence libraries and are playing a central role in the development of this technology. However, the amount of DNA recovered from microarrays is often too small, and a PCR amplification step is usually required. Primer information can be conveyed alongside the DNA library itself in the form of readable barcodes made of DNA on the array surface. Here, we present a synthetic method to pattern QR and data matrix barcodes using DNA photolithography, phosphoramidite chemistry and fluorescent labeling. Patterning and DNA library synthesis occur simultaneously and on the same surface. We manipulate the chemical composition of the barcodes to make them indelible, erasable or hidden, and a simple chemical treatment under basic conditions can reveal or degrade the pattern. In doing so, information crucial to retrieval and amplification can be made available by the user at the appropriate stage. The code and its data contained within are intimately linked to the library as they are synthesized simultaneously and on the same surface. This process is, in principle, applicable to any in situ microarray synthesis method, for instance, inkjet or electrochemical DNA synthesis.

The versatile and tunable self-assembly properties of nucleic acids and engineered nucleic acid constructs make them invaluable in constructing microscale and nanoscale devices, structures and circuits. Increasing the complexity, functionality and ease of assembly of such constructs, as well as interfacing them to the macroscopic world requires a multifaceted and programmable fabrication approach that combines efficient and spatially resolved nucleic acid synthesis with multiple post-synthetic chemical and enzymatic modifications. Here we demonstrate a multi-level photolithographic patterning approach that starts with large-scale in situ surface synthesis of natural, modified or chimeric nucleic acid molecular structures and is followed by chemical and enzymatic nucleic acid modifications and processing. The resulting high-complexity, micrometer-resolution nucleic acid surface patterns include linear and branched structures, multi-color fluorophore labeling and programmable targeted oligonucleotide immobilization and cleavage. Increasing the complexity of engineered nucleic acid constructs and interfacing the microscopic with the macroscopic requires a multifaceted and programmable fabrication approach. Here the authors demonstrate multi-level photolithographic patterning for highly complex patterns at micrometer resolution.

RNA catalytic and binding interactions with proteins and small molecules are fundamental elements of cellular life processes as well as the basis for RNA therapeutics and molecular engineering. In the absence of quantitative predictive capacity for such bioaffinity interactions, high throughput experimental approaches are needed to sufficiently sample RNA sequence space. Here we report on a simple and highly accessible approach to convert commercially available customized DNA microarrays of any complexity and density to RNA microarrays via a T7 RNA polymerase-mediated extension of photocrosslinked methyl RNA primers and subsequent degradation of the DNA templates. RNA microarrays have many potential applications, but are difficult to produce. Here, the AUs present a method for converting commercial, customizable DNA microarrays into RNA microarrays using an accessible three-step process involving primer photocrosslinking, extension, and template degradation.

Nitrogen availability frequently limits photosynthetic production in Sphagnum moss-dominated high-latitude peatlands, which are crucial carbon-sequestering ecosystems at risk to climate change effects. It has been previously suggested that microbial methane-fueled fixation of atmospheric nitrogen (N 2 ) may occur in these ecosystems, but this process and the organisms involved are largely uncharacterized. Peat mosses of the genus Sphagnum are ecosystem engineers that frequently predominate over photosynthetic production in boreal peatlands. Sphagnum spp. host diverse microbial communities capable of nitrogen fixation (diazotrophy) and methane oxidation (methanotrophy), thereby potentially supporting plant growth under severely nutrient-limited conditions. Moreover, diazotrophic methanotrophs represent a possible “missing link” between the carbon and nitrogen cycles, but the functional contributions of the Sphagnum -associated microbiome remain in question. A combination of metagenomics, metatranscriptomics, and dual-isotope incorporation assays was applied to investigate Sphagnum microbiome community composition across the North American continent and provide empirical evidence for diazotrophic methanotrophy in Sphagnum -dominated ecosystems. Remarkably consistent prokaryotic communities were detected in over 250 Sphagnum SSU rRNA libraries from peatlands across the United States (5 states, 17 bog/fen sites, 18 Sphagnum species), with 12 genera of the core microbiome comprising 60% of the relative microbial abundance. Additionally, nitrogenase ( nifH ) and SSU rRNA gene amplicon analysis revealed that nitrogen-fixing populations made up nearly 15% of the prokaryotic communities, predominated by Nostocales cyanobacteria and Rhizobiales methanotrophs. While cyanobacteria comprised the vast majority (>95%) of diazotrophs detected in amplicon and metagenome analyses, obligate methanotrophs of the genus Methyloferula (order Rhizobiales ) accounted for one-quarter of transcribed nifH genes. Furthermore, in dual-isotope tracer experiments, members of the Rhizobiales showed substantial incorporation of 13 CH 4 and 15 N 2 isotopes into their rRNA. Our study characterizes the core Sphagnum microbiome across large spatial scales and indicates that diazotrophic methanotrophs, here defined as obligate methanotrophs of the rare biosphere ( Methyloferula spp. of the Rhizobiales ) that also carry out diazotrophy, play a keystone role in coupling of the carbon and nitrogen cycles in nutrient-poor peatlands. IMPORTANCE Nitrogen availability frequently limits photosynthetic production in Sphagnum moss-dominated high-latitude peatlands, which are crucial carbon-sequestering ecosystems at risk to climate change effects. It has been previously suggested that microbial methane-fueled fixation of atmospheric nitrogen (N 2 ) may occur in these ecosystems, but this process and the organisms involved are largely uncharacterized. A combination of omics (DNA and RNA characterization) and dual-isotope incorporation approaches illuminated the functional diversity of Sphagnum -associated microbiomes and defined 12 bacterial genera in its core microbiome at the continental scale. Moreover, obligate diazotrophic methanotrophs showed high nitrogen fixation gene expression levels and incorporated a substantial amount of atmospheric nitrogen and methane-driven carbon into their biomass. Thus, these results point to a central role for members of the rare biosphere in Sphagnum microbiomes as keystone species that couple nitrogen fixation to methane oxidation in nutrient-poor peatlands.

Chemosensory impairment is an outstanding symptom of SARS-CoV-2 infections. We hypothesized that measured sensory impairments are accompanied by transcriptomic changes in the foliate papillae area of the tongue. Hospital personnel with known SARS-CoV-2 immunoglobulin G (IgG) status completed questionnaires on sensory perception ( n  = 158). A subcohort of n  = 141 participated in forced choice taste tests, and n  = 43 participants consented to donate tongue swabs of the foliate papillae area for whole transcriptome analysis. The study included four groups of participants differing in IgG levels (≥ 10 AU/mL = IgG + ; < 10 AU/mL = IgG - ) and self-reported sensory impairment (SSI ± ). IgG + subjects not detecting metallic taste had higher IgG + levels than IgG + participants detecting iron gluconate ( p  = 0.03). Smell perception was the most impaired biological process in the transcriptome data from IgG + /SSI + participants subjected to gene ontology enrichment. IgG + /SSI + subjects demonstrated lower expression levels of 166 olfactory receptors (OR) and 9 taste associated receptors (TAS) of which OR1A2, OR2J2, OR1A1, OR5K1 and OR1G1, as well as TAS2R7 are linked to metallic perception. The question raised by this study is whether odorant receptors on the tongue (i) might play a role in metal sensation, and (ii) are potential targets for virus-initiated sensory impairments, which needs to be investigated in future functional studies.

The essential micronutrient zinc is known to inhibit gastric acid secretion (GAS), where its homeostasis is strictly regulated. We hypothesized that the gastric bitter taste receptors, TAS2Rs, regulate the following: (i) zinc-modulated proton secretory activity (PSA) as a key mechanism of GAS and (ii) zinc homeostasis in immortalized parietal cells. To confirm this hypothesis, human gastric tumor cells (HGT-1) were exposed to 100–1000 µM of zinc salts for 30 min in order to quantitate their TAS2R-dependent PSA and intracellular zinc concentration using a fluorescence-based pH sensor and ICP-MS, respectively. Thereby, we identified TAS2R43 as a key player in parietal cell PSA and zinc homeostasis, with both conclusions being verified by a CRISPR-Cas9 knockout approach. Moreover, by regulating the zinc importer protein ZIP14, TAS2R43 proved to perform a protective role against excessive zinc accumulation in immortalized parietal cells.

Uracil-DNA glycosylase (UDG) is a critical DNA repair enzyme that is well conserved and ubiquitous in nearly all life forms. UDG protects genomic information integrity by catalyzing the excision from DNA of uracil nucleobases resulting from misincorporation or spontaneous cytosine deamination. UDG-mediated strand cleavage is also an important tool in molecular biotechnology, allowing for controlled and location-specific cleavage of single- and double DNA chemically or enzymatically synthesized with single or multiple incorporations of deoxyuridine. Although the cleavage mechanism is well-understood, detailed knowledge of efficiency and sequence specificity, in both single and double-stranded DNA contexts, has so far remained incomplete. Here we use an experimental approach based on the large-scale photolithographic synthesis of uracil-containing DNA oligonucleotides to comprehensively probe the context-dependent uracil excision efficiency of UDG.

DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3′ terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly efficient reverse synthesis of complex DNA arrays using a photolithographic approach. The method is analogous to conventional solid phase synthesis but makes use of phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group. The use of BzNPPOC, with more than twice the photolytic efficiency of the 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) previously used for 5′→3′ synthesis, combined with additional optimizations to the coupling and oxidation reactions results in an approximately 3-fold improvement in the reverse synthesis efficiency of complex arrays of DNA oligonucleotides. The coupling efficiencies of the reverse phosphoramidites are as good as those of regular phosphoramidites, resulting in comparable yields. Microarrays of DNA surface tethered on the 5′ end and with free 3′ hydroxyl termini can be synthesized quickly and with similarly high stepwise coupling efficiency as microarrays using conventional 3′→5′ synthesis.