An Amphiphilic Cell-Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA and CRISPR Cas9.

The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that self-assembled and dynamic macrocycles can passively diffuse through the cell membrane and deliver a broad range of biologics including proteins, CRISPR Cas9 and ssDNA directly to the cytosol while retaining their bioactivity. Cell penetrating macrocycles (CPMs) can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPMs to their amphiphilic nature and chameleonic properties. They adopt conformations that partially bury polar groups and expose hydrophobic side chains thus self-assembling into micellar-like structures. Their superior fluorescence renders CPMs trackable inside cells where they follow the endomembrane system. CPMs outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this class of macrocycles will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.

SpyDirect: A Novel Biofunctionalization Method for High Stability and Longevity of Electronic Biosensors.

Electronic immunosensors are indispensable tools for diagnostics, particularly in scenarios demanding immediate results. Conventionally, these sensors rely on the chemical immobilization of antibodies onto electrodes. However, globular proteins tend to adsorb and unfold on these surfaces. Therefore, self-assembled monolayers (SAMs) of thiolated alkyl molecules are commonly used for indirect gold-antibody coupling. Here, a limitation associated with SAMs is revealed, wherein they curtail the longevity of protein sensors, particularly when integrated into the state-of-the-art transducer of organic bioelectronics-the organic electrochemical transistor. The SpyDirect method is introduced, generating an ultrahigh-density array of oriented nanobody receptors stably linked to the gold electrode without any SAMs. It is accomplished by directly coupling cysteine-terminated and orientation-optimized spyTag peptides, onto which nanobody-spyCatcher fusion proteins are autocatalytically attached, yielding a dense and uniform biorecognition layer. The structure-guided design optimizes the conformation and packing of flexibly tethered nanobodies. This biolayer enhances shelf-life and reduces background noise in various complex media. SpyDirect functionalization is faster and easier than SAM-based methods and does not necessitate organic solvents, rendering the sensors eco-friendly, accessible, and amenable to scalability. SpyDirect represents a broadly applicable biofunctionalization method for enhancing the cost-effectiveness, sustainability, and longevity of electronic biosensors, all without compromising sensitivity.

Convection Driven Ultrarapid Protein Detection via Nanobody-Functionalized Organic Electrochemical Transistors.

Conventional biosensors rely on the diffusion-dominated transport of the target analyte to the sensor surface. Consequently, they require an incubation step that may take several hours to allow for the capture of analyte molecules by sensor biorecognition sites. This incubation step is a primary cause of long sample-to-result times. Here, alternating current electrothermal flow (ACET) is integrated in an organic electrochemical transistor (OECT)-based sensor to accelerate the device operation. ACET is applied to the gate electrode functionalized with nanobody-SpyCatcher fusion proteins. Using the SARS-CoV-2 spike protein in human saliva as an example target, it is shown that ACET enables protein recognition within only 2 min of sample exposure, supporting its use in clinical practice. The ACET integrated sensor exhibits better selectivity, higher sensitivity, and lower limit of detection than the equivalent sensor with diffusion-dominated operation. The performance of ACET integrated sensors is compared with two types of organic semiconductors in the channel and grounds for device-to-device variations are investigated. The results provide guidelines for the channel material choice in OECT-based biochemical sensors, and demonstrate that ACET integration substantially decreases the detection speed while increasing the sensitivity and selectivity of transistor-based sensors.

How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application.

CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.

Rapid single-molecule detection of COVID-19 and MERS antigens via nanobody-functionalized organic electrochemical transistors.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for rapid and sensitive protein detection and quantification in simple and robust formats for widespread point-of-care applications. Here, we report on nanobody-functionalized organic electrochemical transistors with a modular architecture for the rapid quantification of single-molecule-to-nanomolar levels of specific antigens in complex bodily fluids. The sensors combine a solution-processable conjugated polymer in the transistor channel and high-density and orientation-controlled bioconjugation of nanobody-SpyCatcher fusion proteins on disposable gate electrodes. The devices provide results after 10 min of exposure to 5 µl of unprocessed samples, maintain high specificity and single-molecule sensitivity in human saliva and serum, and can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available. We used the sensors to detect green fluorescent protein, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) spike proteins, and for the COVID-19 screening of unprocessed clinical nasopharyngeal swab and saliva samples with a wide range of viral loads.

Endogenous Control Mechanisms of FAK and PYK2 and Their Relevance to Cancer Development.

Focal adhesion kinase (FAK) and its close paralogue, proline-rich tyrosine kinase 2 (PYK2), are key regulators of aggressive spreading and metastasis of cancer cells. While targeted small-molecule inhibitors of FAK and PYK2 have been found to have promising antitumor activity, their clinical long-term efficacy may be undermined by the strong capacity of cancer cells to evade anti-kinase drugs. In healthy cells, the expression and/or function of FAK and PYK2 is tightly controlled via modulation of gene expression, competing alternatively spliced forms, non-coding RNAs, and proteins that directly or indirectly affect kinase activation or protein stability. The molecular factors involved in this control are frequently deregulated in cancer cells. Here, we review the endogenous mechanisms controlling FAK and PYK2, and with particular focus on how these mechanisms could inspire or improve anticancer therapies.