Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results.

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.

Three-dimensional nanolithography guided by DNA modular epitaxy.

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.

Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA.

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.

Precise pitch-scaling of carbon nanotube arrays within three-dimensional DNA nanotrenches.

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%.

Programmable autonomous synthesis of single-stranded DNA.

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.

A DNA nanoscope via auto-cycling proximity recording.

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.

Biophysical induction of cell release for minimally manipulative cell enrichment strategies.

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.

Performance of a population of independent filaments in lamellipodial protrusion.

Actin polymerization is responsible for moving a wide variety of loads, from the protrusion of membrane-bound filopodia and lamellipodia of immune, cancer, and other motile cells, to the propulsion of some intracellular pathogens. A universal explanation of the forces and velocities generated by these systems has been hampered by a lack of understanding in how a population of independent filaments pushes these loads. Protrusion of a lamellipodium by the very filaments supporting the membrane load is thought to operate by the Brownian ratchet mechanism, with overall organization governed by the dendritic-nucleation/array-treadmilling model. We have incorporated these two models into a two-dimensional, stochastic computer model of lamellipodial protrusion, and studied how force and velocity generation varied under different assumptions. Performance is very sensitive to the extent to which the work of protrusion is shared among individual polymerization events within the filament population. Three identified mechanisms promote this "work-sharing": 1), Most systems, including lamellipodia, utilize a self-organizing distribution of filament-load distances which serves to decrease the effective size of a monomer and dramatically improve performance. 2), A flexible membrane allows for consistent performance over wide leading edges. 3), Finally, very flexible filaments are capable of sharing work very uniformly, and therefore, of near-perfect theoretical performance. Transient tethering to the lamellipodial membrane limits their efficacy, however, and mandates a minimum filament stiffness. Overall, we estimate lamellipodia to operate with 40-nm bending-length filaments and low characteristic tether forces. Modeled lamellipodia exhibit sigmoidal force-velocity relationships and share the work of protrusion only moderately well among filaments, performing at approximately one-half of theoretical force and velocity maximums. At this level of work-sharing, the natural monomer size is optimal for generating velocity.

Intrinsic dynamic behavior of fascin in filopodia.

Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s(-1) and that it undergoes diffusion at moderate rates with a coefficient of 6 microm(2)s(-1). This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction-diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25-60 actin monomers.

Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model.

The dendritic-nucleation/array-treadmilling model provides a conceptual framework for the generation of the actin network driving motile cells. We have incorporated it into a 2D, stochastic computer model to study lamellipodia via the self-organization of filament orientation patterns. Essential dendritic-nucleation submodels were incorporated, including discretized actin monomer diffusion, Monte-Carlo filament kinetics, and flexible filament and plasma membrane mechanics. Model parameters were estimated from the literature and simulation, providing values for the extent of the leading edge-branching/capping-protective zone (5.4 nm) and the autocatalytic branch rate (0.43/sec). For a given set of parameters, the system evolved to a steady-state filament count and velocity, at which total branching and capping rates were equal only for specific orientations; net capping eliminated others. The standard parameter set evoked a sharp preference for the +/-35 degree filaments seen in lamellipodial electron micrographs, requiring approximately 12 generations of successive branching to adapt to a 15 degree change in protrusion direction. This pattern was robust with respect to membrane surface and bending energies and to actin concentrations but required protection from capping at the leading edge and branching angles >60 degrees. A +70/0/-70 degree pattern was formed with flexible filaments approximately 100 nm or longer and with velocities < approximately 20% of free polymerization rates.