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.

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.

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.

Spin Labeling of Long RNAs Via Click Reaction and Enzymatic Ligation.

Electron paramagnetic resonance (EPR) is a spectroscopic method for investigating structures, conformational changes, and dynamics of biomacromolecules, for example, oligonucleotides. In order to be applicable, the oligonucleotide has to be labeled site-specifically with paramagnetic tags, the so-called spin labels. Here, we provide a protocol for spin labeling of long oligonucleotides with nitroxides.

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.

A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2.

A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism*.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2.

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.

Interlocked DNA Nanojoints for Reversible Thermal Sensing.

The ability to precisely measure and monitor temperature at high resolution at the nanoscale is an important task for better understanding the thermodynamic properties of functional entities at the nanoscale in complex systems, or at the level of a single cell. However, the development of high-resolution and robust thermal nanosensors is challenging. The design, assembly, and characterization of a group of thermal-responsive deoxyribonucleic acid (DNA) joints, consisting of two interlocked double-stranded DNA (dsDNA) rings, is described.

ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes.

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells.

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.

Translocation of a Cell Surface Spliceosomal Complex Induces Alternative Splicing Events and Lymphoma Cell Necrosis.

Spliceosomal dysregulation dramatically affects many cellular processes, notably signal transduction, metabolism, and proliferation, and has led to the concept of targeting intracellular spliceosomal proteins to combat cancer. Here we show that a subset of lymphoma cells displays a spliceosomal complex on their surface, which we term surface spliceosomal complex (SSC). The SSC consists of at least 13 core components and was discovered as the binding target of the non-Hodgkin's lymphoma-specific aptamer C10.

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.

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.

High-Yield Spin Labeling of Long RNAs for Electron Paramagnetic Resonance Spectroscopy.

Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach, a 34-nucleotide aptamer domain of the preQ1 riboswitch and an 81-nucleotide TPP riboswitch aptamer could be labeled with two labels in several positions.

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.

Poly-ligand profiling differentiates trastuzumab-treated breast cancer patients according to their outcomes.

Assessing the phenotypic diversity underlying tumour progression requires the identification of variations in the respective molecular interaction networks. Here we report proof-of-concept for a platform called poly-ligand profiling (PLP) that surveys these system states and distinguishes breast cancer patients who did or did not derive benefit from trastuzumab. We perform tissue-SELEX on breast cancer specimens to enrich single-stranded DNA (ssDNA) libraries that preferentially interact with molecular components associated with the two clinical phenotypes.

Supramolecular aptamer nano-constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells.

Platforms for targeted drug-delivery must simultaneously exhibit serum stability, efficient directed cell internalization, and triggered drug release. Here, using lipid-mediated self-assembly of aptamers, we combine multiple structural motifs into a single nanoconstruct that targets hepatocyte growth factor receptor (cMet). The nanocarrier consists of lipidated versions of a cMet-binding aptamer and a separate lipidated GC-rich DNA hairpin motif loaded with intercalated doxorubicin.

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.

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.

Studying the Conformation of a Receptor Tyrosine Kinase in Solution by Inhibitor-Based Spin Labeling.

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer.

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.

Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach.

Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules.

Post-synthetic Spin-Labeling of RNA through Click Chemistry for PELDOR Measurements.

Site-directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron-electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here dŲ. We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW-EPR as well as PELDOR we analyzed and compared the influence of dŲ and Çm on a self-complementary RNA duplex.