Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism.

CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.

Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking.

In single-particle tracking, individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here we introduce a robust, parameter-free alternative to single-particle tracking: temporal analysis of relative distances (TARDIS). In TARDIS, an all-to-all distance analysis between localizations is performed with increasing temporal shifts. These pairwise distances represent either intraparticle distances originating from the same particle, or interparticle distances originating from unrelated particles, and are fitted analytically to obtain quantitative measures on particle dynamics. We showcase that TARDIS outperforms tracking algorithms, benchmarked on simulated and experimental data of varying complexity. We further show that TARDIS performs accurately in complex conditions characterized by high particle density, strong emitter blinking or false-positive localizations, and is in fact limited by the capabilities of localization algorithms. TARDIS' robustness enables fivefold shorter measurements without loss of information.

Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

Design Principles of DNA-Barcodes for Nanopore-FET Readout, Based on Molecular Dynamics and TCAD Simulations.

Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s-1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s-1, with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.

Body-size shifts in aquatic and terrestrial urban communities.

Body size is intrinsically linked to metabolic rate and life-history traits, and is a crucial determinant of food webs and community dynamics1,2. The increased temperatures associated with the urban-heat-island effect result in increased metabolic costs and are expected to drive shifts to smaller body sizes 3 . Urban environments are, however, also characterized by substantial habitat fragmentation 4 , which favours mobile species. Here, using a replicated, spatially nested sampling design across ten animal taxonomic groups, we show that urban communities generally consist of smaller species. In addition, although we show urban warming for three habitat types and associated reduced community-weighted mean body sizes for four taxa, three taxa display a shift to larger species along the urbanization gradients. Our results show that the general trend towards smaller-sized species is overruled by filtering for larger species when there is positive covariation between size and dispersal, a process that can mitigate the low connectivity of ecological resources in urban settings 5 . We thus demonstrate that the urban-heat-island effect and urban habitat fragmentation are associated with contrasting community-level shifts in body size that critically depend on the association between body size and dispersal. Because body size determines the structure and dynamics of ecological networks 1 , such shifts may affect urban ecosystem function.

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework.

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.

Urbanization drives cross-taxon declines in abundance and diversity at multiple spatial scales.

The increasing urbanization process is hypothesized to drastically alter (semi-)natural environments with a concomitant major decline in species abundance and diversity. Yet, studies on this effect of urbanization, and the spatial scale at which it acts, are at present inconclusive due to the large heterogeneity in taxonomic groups and spatial scales at which this relationship has been investigated among studies. Comprehensive studies analysing this relationship across multiple animal groups and at multiple spatial scales are rare, hampering the assessment of how biodiversity generally responds to urbanization. We studied aquatic (cladocerans), limno-terrestrial (bdelloid rotifers) and terrestrial (butterflies, ground beetles, ground- and web spiders, macro-moths, orthopterans and snails) invertebrate groups using a hierarchical spatial design, wherein three local-scale (200 m × 200 m) urbanization levels were repeatedly sampled across three landscape-scale (3 km × 3 km) urbanization levels. We tested for local and landscape urbanization effects on abundance and species richness of each group, whereby total richness was partitioned into the average richness of local communities and the richness due to variation among local communities. Abundances of the terrestrial active dispersers declined in response to local urbanization, with reductions up to 85% for butterflies, while passive dispersers did not show any clear trend. Species richness also declined with increasing levels of urbanization, but responses were highly heterogeneous among the different groups with respect to the richness component and the spatial scale at which urbanization impacts richness. Depending on the group, species richness declined due to biotic homogenization and/or local species loss. This resulted in an overall decrease in total richness across groups in urban areas. These results provide strong support to the general negative impact of urbanization on abundance and species richness within habitat patches and highlight the importance of considering multiple spatial scales and taxa to assess the impacts of urbanization on biodiversity.

Spatiotemporal Heterogeneity of κ-Carrageenan Gels Investigated via Single-Particle-Tracking Fluorescence Microscopy.

Hydrogels made of the polysaccharide κ-carrageenan are widely used in the food and personal care industry as thickeners or gelling agents. These hydrogels feature dense regions embedded in a coarser bulk network, but the characteristic size and behavior of these regions have remained elusive. Here, we use single-particle-tracking fluorescence microscopy (sptFM) to quantitatively describe κ-carrageenan gels. Infusing fluorescent probes into fully gelated κ-carrageenan hydrogels resulted in two distinct diffusional behaviors. Obstructed self-diffusion of the probes revealed that the coarse network consists of κ-carrageenan strands with a typical diameter of 3.2 ± 0.3 nm leading to a nanoprobe diffusion coefficient of ∼1-5 × 10-12 m2/s. In the dense network regions, we found a fraction with a largely decreased diffusion coefficient of ∼1 × 10-13 m2/s. We also observed dynamic exchange between these states. The computation of spatial mobility maps from the diffusional data indicated that the dense network regions have a characteristic diameter of ∼1 µm and show mobility on the second-to-minute timescale. sptFM provides an unprecedented view of spatiotemporal heterogeneity of hydrogel networks, which we believe bears general relevance for understanding transport and release of both low- and high-molecular weight solutes.

Freshwater fish diversity hotspots for conservation priorities in the Amazon Basin.

Conserving freshwater habitats and their biodiversity in the Amazon Basin is a growing challenge in the face of rapid anthropogenic changes. We used the most comprehensive fish-occurrence database available (2355 valid species; 21,248 sampling points) and 3 ecological criteria (irreplaceability, representativeness, and vulnerability) to identify biodiversity hotspots based on 6 conservation templates (3 proactive, 1 reactive, 1 representative, and 1 balanced) to provide a set of alternative planning solutions for freshwater fish protection in the Amazon Basin. We identified empirically for each template the 17% of sub-basins that should be conserved and performed a prioritization analysis by identifying current and future (2050) threats (i.e., degree of deforestation and habitat fragmentation by dams). Two of our 3 proactive templates had around 65% of their surface covered by protected areas; high levels of irreplaceability (60% of endemics) and representativeness (71% of the Amazonian fish fauna); and low current and future vulnerability. These 2 templates, then, seemed more robust for conservation prioritization. The future of the selected sub-basins in these 2 proactive templates is not immediately threatened by human activities, and these sub-basins host the largest part of Amazonian biodiversity. They could easily be conserved if no additional threats occur between now and 2050.

Puntos Calientes de Diversidad de Peces de Agua Dulce para las Prioridades de Conservación en la Cuenca del Amazonas Resumen Cada día, la conservación de los hábitats de agua dulce y su biodiversidad en la cuenca del Amazonas es un reto creciente de cara a los rápidos cambios antropogénicos. Usamos la base de datos de presencia de peces más completa que existe (2,355 especies válidas; 21,248 puntos de muestreo) y tres criterios ecológicos (carácter irremplazable, representatividad y vulnerabilidad) para identificar los puntos calientes de biodiversidad con base en seis patrones de conservación (tres proactivos, uno reactivo, uno representativo y uno balanceado) y así proporcionar un conjunto de soluciones alternativas para la planeación de la protección de peces de agua dulce en la cuenca del Amazonas. Identificamos para cada patrón de manera empírica el 17% de las subcuencas que deberían conservarse y realizamos un análisis de priorización identificando amenazas actuales y a futuro (2050) (es decir, grado de deforestación y fragmentación del hábitat causado por presas). Dos de nuestros tres patrones proactivos tuvieron alrededor del 65% de su superficie cubierta por áreas protegidas; niveles altos de carácter irremplazable (60% de especies endémicas) y de representatividad (71% de la fauna ictiológica del Amazonas); y una vulnerabilidad baja actual y a futuro. Entonces, estos dos patrones parecen estar más completos para la priorización de la conservación. El futuro de las subcuencas en estos dos patrones proactivos no está amenazado por las actividades humanas a corto plazo. Además, estas subcuencas albergan la mayor parte de la biodiversidad amazónica. Se podrían conservar fácilmente si ninguna amenaza adicional sucede entre ahora y el 2050.

Novel Cardinium strains in non-marine ostracod (Crustacea) hosts from natural populations.

Endosymbiotic bacteria are known from many metazoan taxa, where they manipulate host biology and reproduction. Here, we used classic PCR amplification and direct DNA sequencing with universal primers for four different endosymbionts to test for their presence in more than 300 specimens of three recent non-marine ostracod superfamilies from different geographic areas and aquatic habitats. We verified these results with "high throughput" amplicon sequencing of 16S of nine selected specimens and evolutionary placement algorithms. The phylogenetic position of endosymbionts detected in ostracod hosts was compared to known endosymbionts from other metazoans. While Wolbachia, Spiroplasma and Rickettsia are absent, we find evidence for the general presence of Cardinium bacteria in natural populations of various non-marine ostracod species. Phylogenetic reconstructions based on Cardinium 16S data and estimates of genetic distances both indicate that Cardinium from ostracods are distantly related to Cardinium from Diptera and Nematoda but represent novel strains with a monophyletic origin. Cardinium bacteria from different ostracod hosts have genetic distances of up to 3.8%, providing evidence against recent and frequent horizontal transmissions amongst the three ostracod superfamilies. High throughput sequencing reveals more than 400 different 16S amplicon sequence variants in the investigated ostracods as well as the presence of different Cardinium strains within individual Eucypris virens and Heterocypris hosts. These results call for future, more in-depth investigations. Mapping Cardinium infections on COI trees of non-marine ostracod hosts shows that the occurrence of these endosymbionts is not linked to genetic species identity or phylogenetic host groups and, except for one ostracod morphospecies, prevalence never reaches 100%.

Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis.

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.

Manipulation of Recrystallization and Network Formation of Oil-Dispersed Micronized Fat Crystals.

A detailed investigation was carried out on the modulation of the coupling between network formation and the recrystallization of oil-dispersed micronized fat crystal (MFC) nanoplatelets by varying oil composition, shear, and temperature. Sunflower (SF) and bean (BO) oils were used as dispersing media for MFC nanoplatelets. During MFC dispersion production at high shear, a significant increase in the average crystal thickness (ACT) could be observed, pointing to recrystallization of the MFC nanoplatelets. More rapid recrystallization of MFC occurred in the SF dispersion than in the BO dispersion, which is attributed to higher solubility of MFC in the SF oil. When the dispersions were maintained under low shear in narrow gap Couette geometry, we witnessed two stages of recrystallization (measured via rheo-SAXD) and the development of a local yield stress (measured via rheo-MRI). In the first stage, shear-enabled mass transfer induces rapid recrystallization of randomly distributed MFC nanoplatelets, which is reflected in a rapid increase in ACT (rheo-SAXD). The formation of a space-filling weak-link MFC network explains the increase in yield stress (assessed in real time by rheo-MRI). In this second stage, recrystallization slows down and yield stress decreases as a result of the formation of MFC aggregates in the weak link network, as observed by confocal Raman imaging. The high fractal dimension of the weak-link network indicates that aggregation takes place via a particle-cluster mechanism. The effects of oil type and shear on the recrystallization rate and network strength could be reproduced in a stirred bowl with a heterogeneous shear stress field, which opens perspectives for the rational manipulation of MFC thickness and network strength under industrial processing conditions.

Aquatic long-distance dispersal and vicariance shape the evolution of an ostracod species complex (Crustacea) in four major Brazilian floodplains.

Cladogenesis is often driven by the interplay of dispersal and vicariance. The importance of long-distance dispersal in biogeography and speciation is increasingly recognised, but still ill-understood. Here, we study faunal interconnectivity between four large Brazilian floodplains, namely the Amazon, Araguaia, Pantanal (on Paraguay River) and Upper Paraná River floodplains, investigating a species complex of the non-marine ostracod genus Strandesia. We use DNA sequence data from the mitochondrial COI and the nuclear Elongation Factor 1 alpha genes to construct molecular phylogenies and minimum spanning networks, to identify genetic species, analyse biogeographic histories and provide preliminary age estimates of this species complex. The Strandesia species complex includes five morphological and eleven genetic species, which doubles the known diversity in this lineage. The evolutionary history of this species complex appears to comprise sequences of dispersal and vicariance events. Faunal and genetic patterns of connectivity between floodplains in some genetic species are mirrored in modern hydrological connections. This could explain why we find evidence for (aquatic) long-distance dispersal between floodplains, thousands of kilometres apart. Our phylogenetic reconstructions seem to mostly indicate recent dispersal and vicariance events, but the evolution of the present Strandesia species complex could span up to 25 Myr, which by far exceeds the age of the floodplains and the rivers in their current forms.

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs.

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 × 106 localizations per second to be calculated on a standard multi-core central processing unit with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function to two phase vectors (phasors) by calculating the first Fourier coefficients in both the x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

On Callistocypris thailandensis sp. nov. (Ostracoda, Crustacea) from Thailand.

Callistocypris thailandensis sp. nov. is here described from terrestrial habitat in Khao Yai National Park, Nakhon Ratchasima province, Thailand. The new species differs from the other three Callistocypris species in the shape of carapace, the presence of strong longitudinal ridges on the ventral part of the valves and the chaetotaxy of several limbs. Zoogeography and ecology of the genus are briefly discussed.

Contribution to the knowledge of the genus Vestalenula Rossetti & Martens, 1998 (Crustacea, Ostracoda, Darwinulidae), with the description of a new species, V. carinata n. sp., from the island of Florianópolis, Brazil.

The genus Vestalenula is the most species rich in the putative ancient asexual ostracod Family Darwinulidae. Several new Recent species were described from various continents, mostly based on carapace shape and structure. These species were found in a variety of aquatic and (semi-) terrestrial habitats, including springs, streams, interstitial waters, leaf litter in forests and in splash zones of waterfalls. Here, we describe V carinata n. sp. from moist leaf litter, collected from the Brazilian island of Florian6polis. The species belongs to the danielopoli-lineage within the genus because of its elongated internal tooth in the left valve and elongated external keel on the right valve. It can be distinguished from all other species in this group by its size, its L/H ratio and the relative length of the keel. The relationship of this new species to the enigmatic, putative marine species Senidarwinula terraenuxforna Choe, 1988, is discussed. An identification key to species of the genus is provided.

Redescription of the type species of the genus Cypretta (Ostracoda, Crustacea), with notes on the taxonomy of the genus.

With 53 formally described species, the genus Cypretta is one of the most common freshwater ostracod genera in the world. It has a mainly circumtropical distribution. The type species, Cypretta tenuicauda (Vávra, 1895), was described from Zanzibar (Africa) in a superficial way. Therefore, the morphology and identity of this species and of the genus remained problematic until today. Here, we redescribe Cypretta tenuicauda from the original type material and discuss the morphology of the species and the diagnosis of the genus. The species is characterized by the presence of anterior marginal septa in both valves, the sub-triangular carapace shape in lateral view, the right valve overlapping the left valve, the generally wide carapace and the presence of a serrated posteroventral inner list in the right valve. In addition, both α and ß setae on the mandibular palp are long and thin, claws Ga and Gp on the caudal ramus are elongated and seta-like, while the caudal ramus itself is equally slender. The caudal ramus attachment is reduced to a simple branch. The present redescription of the type species will assist in creating order in what is now a taxonomically confused genus.

On a new genus and four new species of the subfamily Cyprettinae (Crustacea, Ostracoda) from Brazilian floodplains.

We describe the new genus Triangocypretta gen. nov. and four new species from Brazilian floodplains. Triangocypretta angustus gen. et spec. nov. and Triangocypretta labiata gen. et spec. nov. were described from the Amazon floodplain only, while Triangocypretta nates gen. et spec. nov. was described from Amazon, Araguaia, and Paran River floodplains. Triangocypretta hirsuta gen. et spec. nov. was recorded from all four floodplains: Amazon, Araguaia, Pantanal and Paran. The new genus is characterized by the triangular shape of the carapace in lateral view, the absence of teeth on the posteroventral inner list in the right valve and the presence of anterior marginal septa in both valves, as well as by the relatively short and thin and -setae on the mandibular palp. All populations found were asexual. Owing to the clear differences in valve anatomy and limb chaetotaxy as compared to species of Cypretta s.s., the four species were allocated to a new genus in the subfamily Cyprettinae.

Unraveling the impact of nano-scaling on silicon field-effect transistors for the detection of single-molecules.

Electrolyte-gated silicon field-effect transistors (FETs) capable of detecting single molecules could enable high-throughput molecular sensing chips to advance, for example, genomics or proteomics. For solid-gated silicon FETs it is well-known that nano-scaled devices become sensitive to single elementary charges near the silicon-oxide interface. However, in electrolyte-gated FETs, electrolyte screening strongly reduces sensitivity to charges near the gate oxide. The question arises whether nano-scaling electrolyte-gated FETs can entail a sufficiently large signal-to-noise ratio (SNR) for the detection of single molecules. We enhanced a technology computer-aided design tool with electrolyte screening models to calculate the impact of the FET geometry on the single-molecule signal and FET noise. Our continuum FET model shows that a sufficiently large single-molecule SNR is only obtained when nano-scaling all FET channel dimensions. Moreover, we show that the expected scaling trend of the single-molecule SNR breaks down and no longer results in improvements for geometries approaching the decananometer size. This is the characteristic size of the FET channel region modulated by a typical molecule. For gate lengths below 50 nm, the overlap of the modulated region with the highly conductive junctions leads to saturation of the SNR. For cross-sections below 10-30 nm, SNR degrades due to the overlap of the modulated region with the convex FET corners where a larger local gate capacitance reduces charge sensitivity. In our study, assuming a commercial solid-state FET noise amplitude, we find that a suspended nanowire FET architecture with 35 nm length and 5 × 10 nm2 cross-section results in the highest SNR of about 10 for a 15-base DNA oligo in a 15 mM electrolyte. In contrast with typical silicon nanowire FET sensors which possess micron-scale gate lengths, we find it to be key that all channel dimensions are scaled down to the decananometer range.