A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

A systematic review on cross-cultural validations and psychometric solidity of the orthotics and prosthetics user survey.

Research is essential to reflect patients' satisfaction with their devices in the field of Prosthetics and Orthotics, record their performance and health-related quality of life. This requires culturally adapted questionnaires for each country. Periodic assessment of validity and test fit are essential elements for the long-term utility and effectiveness of psychometric tests. This article reviews the psychometric properties of the Orthotics and Prosthetics Users Survey (OPUS). The purpose, in addition to its adaptation to the Spanish-speaking population, involves a review/update of content, statistical analyses, and validity studies, until a larger number of studies are conducted. Study design: a Systematic review. A systematic literature search was carried out in specialized search engines: Alcorze (University of Zaragoza), MEDLINE (PubMed), and EMBASE of original articles published since 2000. Eleven items belonging to the OPUS were obtained, according to the language of the country where they were validated, and promising psychometric properties were confirmed (reflecting reliability values between 0.62 and 0.95; Cronbach's α scores between 0.73 and 0.98) with sample sizes between 10 and 321. The study concluded by stating that the OPUS was validated in different languages, reporting good psychometric robustness so far. Further deployment, refinement, and validation of this survey by country is warranted in view of its promising use.

A rhythmically pulsing leaf-spring DNA-origami nanoengine that drives a passive follower.

Molecular engineering seeks to create functional entities for modular use in the bottom-up design of nanoassemblies that can perform complex tasks. Such systems require fuel-consuming nanomotors that can actively drive downstream passive followers. Most artificial molecular motors are driven by Brownian motion, in which, with few exceptions, the generated forces are non-directed and insufficient for efficient transfer to passive second-level components. Consequently, efficient chemical-fuel-driven nanoscale driver-follower systems have not yet been realized. Here we present a DNA nanomachine (70 nm × 70 nm × 12 nm) driven by the chemical energy of DNA-templated RNA-transcription-consuming nucleoside triphosphates as fuel to generate a rhythmic pulsating motion of two rigid DNA-origami arms. Furthermore, we demonstrate actuation control and the simple coupling of the active nanomachine with a passive follower, to which it then transmits its motion, forming a true driver-follower pair.

Reconfigurable Nanopolygons Made of DNA Catenanes.

The development of new types of bonds and linkages that can reversibly tune the geometry and structural features of molecules is an elusive goal in chemistry. Herein, we report the use of catenated DNA structures as nanolinkages that can reversibly switch their angle and form different kinds of polygonal nanostructures. We designed a reconfigurable catenane that can self-assemble into a triangular or hexagonal structure upon addition of programmable DNA strands that function via toehold strand-displacement. The nanomechanical and structural features of these catenated nanojoints can be applied for the construction of dynamic systems such as molecular motors with switchable functionalities.

A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

A Self-Regulating DNA Rotaxane Linear Actuator Driven by Chemical Energy.

Nature-inspired molecular machines can exert mechanical forces by controlling and varying the distance between two molecular subunits in response to different inputs. Here, we present an automated molecular linear actuator composed of T7 RNA polymerase (T7RNAP) and a DNA [2]rotaxane. A T7 promoter region and terminator sequences are introduced into the rotaxane axle to achieve automated and iterative binding and detachment of T7RNAP in a self-controlled fashion. Transcription by T7RNAP is exploited to control the release of the macrocycle from a single-stranded (ss) region in the T7 promoter to switch back and forth from a static state (hybridized macrocycle) to a dynamic state (movable macrocycle). During transcription, the T7RNAP keeps restricting the movement range on the axle available for the interlocked macrocycle and prevents its return to the promotor region. Since this range is continuously depleted as T7RNAP moves along, a directional and active movement of the macrocycle occurs. When it reaches the transcription terminator, the polymerase detaches, and the system can reset as the macrocycle moves back to hybridize again to the ss-promoter docking site. The hybridization is required for the initiation of a new transcription cycle. The rotaxane actuator runs autonomously and repeats these self-controlled cycles of transcription and movement as long as NTP-fuel is available.

Regeneration of Burnt Bridges on a DNA Catenane Walker.

DNA walkers are molecular machines that can move with high precision onthe nanoscale due to their structural and functional programmability. Despite recent advances in the field that allow exploring different energy sources, stimuli, and mechanisms of action for these nanomachines, the continuous operation and reusability of DNA walkers remains challenging because in most cases the steps, once taken by the walker, cannot be taken again. Herein we report the path regeneration of a burnt-bridges DNA catenane walker using RNase A. This walker uses a T7RNA polymerase that produces long RNA transcripts to hybridize to the path and move forward while the RNA remains hybridized to the path and blocks it for an additional walking cycle. We show that RNA degradation triggered by RNase A restores the path and returns the walker to the initial position. RNase inhibition restarts the function of the walker.

Mechanisms, Methods of Tracking and Applications of DNA Walkers: A Review.

In recent years, DNA nanotechnology expanded its scope from structural DNA nanoarchitecture towards designing dynamic and functional nanodevices. This progress has been evident in the development of an advanced class of DNA nanomachines, the so-called DNA walkers. They represent an evolution of basic switching between distinctly defined states into continuous motion. Inspired by the naturally occurring walkers such as kinesin, research on DNA walkers has focused on developing new ways of powering them and investigating their walking mechanisms and advantages. New techniques allowing the visualization of walkers as single molecules and in real time have provided a deeper insight into their behavior and performance. The construction of novel DNA walkers bears great potential for applications in therapeutics, nanorobotics or computation. This review will cover the various examples and breakthrough designs of recently reported DNA walkers that pushed the limits of their performance. It will also mention the techniques that have been used to investigate walker nanosystems, as well as discuss the applications that have been explored so far.

Mechanisms, Methods of Tracking and Applications of DNA Walkers: A Review.

The front cover artwork is provided by Julián Valero and Marko Skugor from Aarhus University and University of Bonn. The image shows an ad libitum representation of a new class of artificial molecular motors, coined as DNA walkers, on an athletic track testing their performance i.e. speed, directionality and progressivity. Read the full text of the Review at 10.1002/cphc.202000235.

Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures.

Mechanically interlocked DNA nanostructures are useful as flexible entities for operating DNA-based nanomachines. Interlocked structures made of double-stranded (ds) DNA components can be constructed by irreversibly threading them through one another to mechanically link them. The interlocked components thus remain bound to one another while still permitting large-amplitude motion about the mechanical bond. The construction of interlocked dsDNA architectures is challenging because it usually involves the synthesis and modification of small dsDNA nanocircles of various sizes, dependent on intrinsically curved DNA. Here we describe the design, generation, purification, and characterization of interlocked dsDNA structures such as catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs). Their construction requires precise control of threading and hybridization of the interlocking components at each step during the assembly process. The protocol details the characterization of these nanostructures with gel electrophoresis and atomic force microscopy (AFM), including acquisition of high-resolution AFM images obtained in intermittent contact mode in liquid. Additional functionality can be conferred on the DNA architectures by incorporating proteins, molecular switches such as photo-switchable azobenzene derivatives, or fluorophores for studying their mechanical behavior by fluorescence quenching or fluorescent resonance energy transfer experiments. These modified interlocked DNA architectures provide access to more complex mechanical devices and nanomachines that can perform a variety of desired functions and operations. The assembly of catenanes can be completed in 2 d, and that of rotaxanes in 3 d. Addition of azobenzene functionality, fluorophores, anchor groups, or the site-specific linkage of proteins to the nanostructure can extend the time line.

Orthogonally Photocontrolled Non-Autonomous DNA Walker.

There is considerable interest in developing progressively moving devices on the nanoscale, with the aim of using them as parts of programmable therapeutics, smart materials, and nanofactories. Present here is an entirely light-induced DNA walker based on orthogonal photocontrol. Implementing two azobenzene derivatives, S-DM-Azo and DM-Azo, enabled precise coordination of strand displacement reactions that powered a biped walker and guided it along a defined track in a non-autonomous way. This unprecedented type of molecular walker design offers high precision control over the movement in back-and-forth directions as desired, and is regulated solely by the sequence of the irradiation wavelengths. This concept may open new avenues for advancing non-autonomous progressive molecular motors, ultimately facilitating their application at the nanoscale.

Temporal and Reversible Control of a DNAzyme by Orthogonal Photoswitching.

The reversible switching of catalytic systems capable of performing complex DNA  computing operations using the temporal control of two orthogonal photoswitches is described. Two distinct photoresponsive molecules have been separately incorporated into a split horseradish peroxidase-mimicking DNAzyme. We show that its catalytic function can be turned on and off reversibly upon irradiation with specific wavelengths of light. The system responds orthogonally  to a  selection of irradiation wavelengths    and   durations of irradiation. Furthermore, the DNAzyme exhibits reversible switching and retains this ability throughout multiple switching cycles. We apply our system as a light-controlled 4:2 multiplexer. Orthogonally photoswitchable DNAzyme-based catalysts as introduced here have potential use for controlling complex logical operations and for future applications in DNA nanodevices.

A bio-hybrid DNA rotor-stator nanoengine that moves along predefined tracks.

Biological motors are highly complex protein assemblies that generate linear or rotary motion, powered by chemical energy. Synthetic motors based on DNA nanostructures, bio-hybrid designs or synthetic organic chemistry have been assembled. However, unidirectionally rotating biomimetic wheel motors with rotor-stator units that consume chemical energy are elusive. Here, we report a bio-hybrid nanoengine consisting of a catalytic stator that unidirectionally rotates an interlocked DNA wheel, powered by NTP hydrolysis. The engine consists of an engineered T7 RNA polymerase (T7RNAP-ZIF) attached to a dsDNA nanoring that is catenated to a rigid rotating dsDNA wheel. The wheel motor produces long, repetitive RNA transcripts that remain attached to the engine and are used to guide its movement along predefined ssDNA tracks arranged on a DNA nanotube. The simplicity of the design renders this walking nanoengine adaptable to other biological nanoarchitectures, facilitating the construction of complex bio-hybrid structures that achieve NTP-driven locomotion.

Expanding the Toolbox of Photoswitches for DNA Nanotechnology Using Arylazopyrazoles.

Photoregulation is among the most promising tools for development of dynamic DNA nanosystems, due to its high spatiotemporal precision, biocompatibility, and ease of use. So far, azobenzene and its derivatives have shown high potential in photocontrolling DNA duplex hybridization by light-dependent photoisomerization. Despite many recent advances, obtaining sufficiently high photoswitching efficiency under conditions more suitable for work with DNA nanostructures are challenging. Here we introduce a pair of arylazopyrazoles as new photoswitches for efficient and reversible control of DNA hybridization achieved even at room temperature with a low number of required modifications. Their photophysical properties in the native state and in DNA strands result in near-quantitative isomerization rates by irradiation with UV and orange light. To demonstrate the applicability of these photoswitches, we have successfully applied one of them to open and close a DNA hairpin by light at room temperature.

Allosteric Control of Oxidative Catalysis by a DNA Rotaxane Nanostructure.

DNA is a versatile construction material for the bottom-up assembly of structures and functional devices in the nanoscale. Additionally, there are specific sequences called DNAzymes that can fold into tertiary structures that display catalytic activity. Here we report the design of an interlocked DNA nanostructure that is able to fine-tune the oxidative catalytic activity of a split DNAzyme in a highly controllable manner. As scaffold, we employed a double-stranded DNA rotaxane for its ability to undergo programmable and predictable conformational changes. Precise regulation of the DNAzyme's oxidative catalysis can be achieved by external stimuli (i.e., addition of release oligos) that modify the spatial arrangement within the system, without interfering with the catalytic core, similar to structural rearrangements that occur in allosterically controlled enzymes. We show that multiple switching steps between the active and inactive conformations can be performed consistent with efficient regulation and robust control of the DNA nanostructure.

Interlocked DNA topologies for nanotechnology.

Interlocked molecular architectures are well known in supramolecular chemistry and are widely used for various applications like sensors, molecular machines and logic gates. The use of DNA for constructing these interlocked structures has increased significantly within the current decade. Because of Watson-Crick base pairing rules, DNA is an excellent material for the self-assembly of well-defined interlocked nanoarchitectures. These DNA nanostructures exhibit sufficient stability, good solubility in aqueous media, biocompatibility, and can be easily combined with other biomolecules in bio-hybrid nano-assemblies. Therefore, the study of novel DNA-based interlocked systems is of interest for nanotechnology, synthetic biology, supramolecular chemistry, biotechnology, and for sensing purposes. Here we summarize recent developments and applications of interlocked supramolecular architectures made of DNA. Examples illustrating that these systems can be precisely controlled by switching on and off the molecular motion of its mechanically trapped components are discussed. Introducing different triggers into such systems creates molecular assemblies capable of performing logic gate operations and/or catalytic activity control. Interlocked DNA-based nanostructures thus represent promising frameworks for building increasingly complex and dynamic nanomachines with highly controllable functionality.

Single-Stranded Tile Stoppers for Interlocked DNA Architectures.

Interlocked DNA architectures are useful for DNA nanotechnology because of their mechanically bonded components, which can move relative to one another without disassembling. We describe the design, synthesis, and characterization of novel single-stranded tile (SST) stoppers for the assembly of interlocked DNA architectures. SST stoppers are shown to self-assemble into a square-shaped rigid structure upon mixing 97 oligodeoxynucleotide (ODN) strands. The structures are equipped with a sticky end that is designed for hybridization with the sticky ends of a dsDNA axle of a DNA rotaxane. Because the diameter of the macrocycle threaded onto the axle is 14 nm, the dimension of the square-shaped stopper was designed to be bulky enough to prevent the dethreading of the macrocycle. An asymmetric rotaxane with a SST- and a ring-shaped stopper featuring two stations for hybridization of the macrocycle to the axle was assembled. The macrocycle can be directed towards one or the other station upon triggering with fuel ODNs.

Cellular Antisense Activity of PNA-Oligo(bicycloguanidinium) Conjugates Forming Self-Assembled Nanoaggregates.

A series of peptide nucleic acid-oligo(bicycloguanidinium) (PNA-BGn ) conjugates were synthesized and characterized in terms of cellular antisense activity by using the pLuc750HeLa cell splice correction assay. PNA-BG4 conjugates exhibited low micromolar antisense activity, and their cellular activity required the presence of a hydrophobic silyl terminal protecting group on the oligo(BG) ligand and a minimum of four guanidinium units. Surprisingly, a nonlinear dose-response with an activity threshold around 3-4 µM, indicative of large cooperativity, was observed. Supported by light scattering and electron microscopy analyses, we propose that the activity, and thus cellular delivery, of these lipo-PNA-BG4 conjugates is dependent on self-assembled nanoaggregates. Finally, cellular activity was enhanced by the presence of serum. Therefore we conclude that the lipo-BG-PNA conjugates exhibit an unexpected mechanism for cell delivery and are of interest for further in vivo studies.

Sequence-selective DNA binding with cell-permeable oligoguanidinium-peptide conjugates.

Conjugation of a short peptide fragment from a bZIP protein to an oligoguanidinium tail results in a DNA-binding miniprotein that selectively interacts with composite sequences containing the peptide-binding site next to an A/T-rich tract. In addition to stabilizing the complex with the target DNA, the oligoguanidinium unit also endows the conjugate with cell internalization properties.