Cytohesins are cytoplasmic ErbB receptor activators.

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in vitro and in vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.

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.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

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.

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. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

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.

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. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

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.

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. The DNA nanojoints reversibly switch between the static and mobile state at different temperatures without a special annealing process. The temperature response range of the DNA nanojoint can be easily tuned by changing the length or the sequence of the hybridized region in its structure, and because of its interlocked structure the temperature response range of the DNA nanojoint is largely unaffected by its own concentration; this contrasts with systems that consist of separated components.

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.

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. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg2+ and a preQ1 ligand by continuous wave electron paramagnetic resonance spectroscopy at room temperature and pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) in the frozen state.

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.

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.

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. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof-of-concept that inhibitor-based spin labeling enables the convenient introduction of site-specific spin labels into kinases for which covalent or tight-binding small-molecule modulators are available.

Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease.

Mutant genes that underlie Mendelian forms of amyotrophic lateral sclerosis (ALS) and biochemical investigations of genetic disease models point to potential driver pathophysiological events involving endoplasmic reticulum (ER) stress and autophagy. Several steps in these cell biological processes are known to be controlled physiologically by small ADP-ribosylation factor (ARF) signaling. Here, we investigated the role of ARF guanine nucleotide exchange factors (GEFs), cytohesins, in models of ALS. Genetic or pharmacological inhibition of cytohesins protects motor neurons in vitro from proteotoxic insults and rescues locomotor defects in a Caenorhabditis elegans model of disease. Cytohesins form a complex with mutant superoxide dismutase 1 (SOD1), a known cause of familial ALS, but this is not associated with a change in GEF activity or ARF activation. ER stress evoked by mutant SOD1 expression is alleviated by antagonism of cytohesin activity. In the setting of mutant SOD1 toxicity, inhibition of cytohesin activity enhances autophagic flux and reduces the burden of misfolded SOD1. These observations suggest that targeting cytohesins may have potential benefits for the treatment of ALS.

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.

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 dU. 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 dU and Çm on a self-complementary RNA duplex. Our results demonstrate that the conformational diversity of dU is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation-selective PELDOR time traces of the dU-labeled RNA duplex.

Switchable Reconfiguration of a Seven-Ring Interlocked DNA Catenane Nanostructure.

The synthesis, purification, and structure characterization of a seven-ring interlocked DNA catenane is described. The design of the seven-ring catenane allows the dynamic reconfiguration of any of the four rings (R1, R3, R4, and R6) on the catenane scaffold, or the simultaneous switching of any combination of two, three, or all four rings to yield 16 different isomeric states of the catenane. The dynamic reconfiguration across the states is achieved by implementing the strand-displacement process in the presence of appropriate fuel/antifuel strands and is probed by fluorescence spectroscopy. Each of the 16 isomers of the catenane can be transformed into any of the other isomers, thus allowing for 240 dynamic transitions within the system.