The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.

Lithographic scaling of periodic three-dimensional patterns is critical for advancing scalable nanomanufacturing. Current state-of-the-art quadruple patterning or extreme-ultraviolet lithography produce a line pitch down to around 30 nm, which might be further scaled to sub-20 nm through complex post-fabrication processes. Herein, we report the use of three-dimensional (3D) DNA nanostructures to scale the line pitch down to 16.2 nm, around 50% smaller than state-of-the-art results. We use a DNA modular epitaxy approach to fabricate 3D DNA masks with prescribed structural parameters (geometry, pitch and critical dimensions) along a designer assembly pathway. Single-run reactive ion etching then transfers the DNA patterns to a Si substrate at a lateral critical dimension of 7 nm and a vertical critical dimension of 2 nm. The nanolithography guided by DNA modular epitaxy achieves a smaller pitch than the projected values for advanced technology nodes in field-effect transistors, and provides a potential complement to the existing lithographic tools for advanced 3D nanomanufacturing. Epitaxially grown 3D DNA masks with prescribed geometry, pitch and size improve the resolution of reactive ion etching-based nanolithography, scaling the line pitch down to 16.2 nm and the critical dimension size to 7.2 nm.

The SARS-CoV-2 pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to SARS-CoV-2 research and diagnostic development, leading to large-scale production of nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids, but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants in SARS-CoV-2 testing samples results in anomalous test results. The impact of these stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in quarantine, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both use and development of diagnostics. These experiences highlight a neglected aspect of the test, trace, isolate public health strategy for managing COVID-19: we cannot easily identify and diagnose an erroneous test result. To prevent undue personal and economic strain and maintain faith in the testing process, we propose: a test, isolate, verify, and trace approach to COVID-19 diagnosis in research and clinical diagnostic workplaces.

Precise fabrication of semiconducting carbon nanotubes (CNTs) into densely aligned evenly spaced arrays is required for ultrascaled technology nodes. We report the precise scaling of inter-CNT pitch using a supramolecular assembly method called spatially hindered integration of nanotube electronics. Specifically, by using DNA brick crystal-based nanotrenches to align DNA-wrapped CNTs through DNA hybridization, we constructed parallel CNT arrays with a uniform pitch as small as 10.4 nanometers, at an angular deviation <2° and an assembly yield >95%.

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 L reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 μL reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

AbstractThe COVID-19 pandemic has strained testing capabilities worldwide. There is an urgent need to find economical and scalable ways to test more people. We presentTapestry, a novel quantitative nonadaptive pooling scheme to test many samples using only a few tests. The underlying molecular diagnostic test is any real-time RT-PCR diagnostic panel approved for the detection of the SARS-CoV-2 virus. In cases where most samples are negative for the virus, Tapestry accurately identifies the status of each individual sample with a single round of testing in fewer tests than simple two-round pooling. We also present a companion Android applicationBYOM Smart Testingwhich guides users through the pipetting steps required to perform the combinatorial pooling. The results of the pooled tests can be fed into the application to recover the status and estimated viral load for each individual sample.NOTE: This protocol has been validated with in vitro experiments that used synthetic RNA and DNA fragments and additionally, its expected behavior has been confirmed using computer simulations. Validation with clinical samples is ongoing. We are looking for clinical collaborators with access to patient samples. Please contact the corresponding author if you wish to validate this protocol on clinical samples.

Techniques that can both spatially map out molecular features and discriminate many targets would be highly valued for their utility in studying fundamental nanoscale processes. In spite of decades of development, no current technique can achieve both nanoscale resolution and discriminate hundreds of targets. Here, we report the development of a novel bottom-up technology that: (a) labels a sample with DNA barcodes, (b) measures pairwise-distances between labeled sites and writes them into DNA molecules, (c) reads the pairwise-distances by sequencing and (d) robustly integrates this noisy information to reveal the geometry of the underlying sample. We demonstrate our technology on DNA origami, which are complex synthetic nanostructures. We both spatially localized and uniquely identified over a hundred densely packed unique elements, some spaced just 6 nm apart, with an average spatial localization accuracy (RMS deviation) of ~2 nm. The bottom-up, sequencing-enabled mechanism of the DNA nanoscope is fundamentally different from top-down imaging, and hence offers unique advantages in precision, throughput and accessibility.### Competing Interest StatementN.G., T.S. and P.Y. have filed a provisional patent covering aspects of this work. P.Y. is co-founder and director of Ultivue Global, NuProbe Inc. and Torus Biosystems

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

The use of autologous cells harvested and subsequently transplanted in an intraoperative environment constitutes a new approach to promote regeneration. Usually cells are isolated by selection methods such as fluorescence- or magnetic- activated cell sorting with residual binding of the antibodies or beads. Thus, cell-based therapies would benefit from the development of new devices for cell isolation that minimally manipulate the target cell population. In the clinic, 5 to 10 percent of fractures do not heal properly and CD31+ cells have been identified as promising candidates to support bone regeneration. The aim of this project was to develop and prototype a simple system to facilitate the enrichment of CD31+ cells from whole blood. After validating the specificity of a commercially available aptamer for CD31, we combined this aptamer with traditional magnetic bead strategies, which led to enrichment of CD31+ cells with a purity of 91±10%. Subsequently, the aptamer was attached to agarose beads (Ø = 100-165 um) that were incorporated into a column-based system to enable capture and subsequent release of the CD31+ enriched cells. Different parameters were investigated to allow a biophysical-based cell release from beads, and a simple mixing was found sufficient to release initially bound cells from the optimized column without the need for any chemicals that promote disassociation. The system led to a significant enrichment of CD31+ cells (initial population: 63±9%, released: 87±3%) with excellent cell viability (released: 97±1%). The composition of the released CD31+ fraction indicated an enrichment of the monocyte population. The angiogenic and osteogenic potential of the released cell population were confirmed in vitro. These results and the simplicity of this system highlight the potential of such approach to enable cell enrichment strategies in intraoperative settings.

Analysis of the spatial arrangement of molecular features enables the engineering of synthetic nanostructures and the understanding of natural ones. The ability to acquire a comprehensive set of pairwise proximities between components would satisfy an increasing interest in investigating individual macromolecules and their interactions, but current biochemical techniques detect only a single proximity partner per probe. Here, we present a biochemical DNA nanoscopy method that records nanostructure features in situ and in detail for later readout. Based on a conceptually novel auto-cycling proximity recording (APR) mechanism, it continuously and repeatedly produces proximity records of any nearby pairs of DNA-barcoded probes, at physiological temperature, without altering the probes themselves. We demonstrate the production of dozens of records per probe, decode the spatial arrangements of 7 unique probes in a homogeneous sample, and repeatedly sample the same probes in different states.The spatial organisation of nanostructures is fundamental to their function. Here, the authors develop a non-destructive, proximity-based method to record extensive spatial organization information in DNA molecules for later readout.

Cascade reactions can be used to carry out logic operations on the surface of cells and identify the presence of particular collections of cell surface markers.

L'invention concerne des systemes et des procedes de sequestration spatiale d'elements dans des circuits d'acides nucleiques.