Two for one: We have established an assay for nucleic acid detection by using the target ssRNA-activated protease from the type III-E CRISPR-Cas system. This assay would allow simultaneous detection of one or two target genes or viruses, which could be further expanded by incorporating more type III-E systems of various species. This assay provides an easily adaptable platform for biological and clinical diagnosis.

Abstract

RNA-guided protease activity was recently discovered in the type III-E CRISPR-Cas systems (Craspase), providing a novel platform for engineering a protein probe instead of the commonly used nucleic acid probe in nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity- and fluorescent protein dual-tagged Csx30 protein substrate, we have established an assay monitoring Csx30 cleavage by target ssRNA-activated Craspase. Four Craspase-based nucleic acid detection systems for genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, and the influenza virus (IFV) were reconstituted with demonstrated specificity. The assay could reliably detect target ssRNAs at concentrations down to 25 pM, which could be further improved approximately 15 000-fold (ca. 2 fM) by incorporating a recombinase polymerase isothermal preamplification step. Importantly, the species-specific substrate cleavage specificity of Craspase enabled multiplexed diagnosis, as demonstrated by the reconstituted composite systems for simultaneous detection of two genes from the same virus (SARS-CoV-2, spike and nsp12) or two types of viruses (SARS-CoV-2 and IFV). The assay could be further expanded by diversifying the fluorescent tags in the substrate and including Craspase systems from various species, thus potentially providing an easily adaptable platform for clinical diagnosis.

Male Heliconius butterflies, which occur locally in three mimicry groups, show an extremely high diversity of compounds in their clasper scent glands. The more than 1,000 different compounds identified in 17 species are used for species-specific mixtures of volatile and non-volatile compounds, presumably used for chemical communication. (Photos: C. Jiggins)

Abstract

Male Heliconius butterflies possess two pheromone emitting structures, wing androconia and abdominal clasper scent glands. The composition of the clasper scent gland of males of 17 Heliconius and Eueides species from an overlapping area in Ecuador, comprising three mimicry groups, was investigated by GC/MS. The chemical signal serves as an anti-aphrodisiac signal that is transferred from males to females during mating, indicating the mating status of the female to prevent them from harassment by other males. In addition, the odour may also serve in predator defence. There is potential for convergence driven by mimicry, although, such convergence might be detrimental for species recognition of the butterflies within the mimicry ring, making mating more difficult. More than 500 compounds were detected, consisting of volatile, semi-volatile or non-volatile compounds, including terpenes, fatty acid esters or aromatic compounds. Several novel esters were identified by GC/MS and GC/IR data, microderivatisation and synthesis, including butyl (Z)-3-dodecenoate and other (Z)-3-alkenoates, 3-oxohexyl citronellate and 5-methylhexa-3,5-dienyl (E)-2,3-dihydrofarnesoate. The secretions were found to be species specific, potentially allowing for species differentiation. Statistical analysis of the compounds showed differentiation by phylogenetic clade and species, but not by mimicry group.

The cell redox balance can be disrupted by the oxidation of biological peptides and eventually lead to the cell death, which provides opportunities to develop cytotoxic drugs. With the aim of developing compounds capable of specifically inducing fatal redox reactions upon irradiation, we have developed a library of copper compounds. This metal is abundant and considered essential for human health, making it particularly attractive for the development of new anticancer drugs. Copper(I) clusters with thiol ligands (including 5 novel ones) have been synthesized and characterized. Structures were elucidated by X-ray diffraction and show that the compounds are oligomeric clusters. The clusters display high photooxidation capacity towards cysteine – an essential amino acid – upon irradiation in the visible range (450 nm), while remaining completely inactive in the dark. This photoredox activity against a biological thiol was very encouraging for the development of an anticancer photoredox drug. Besides, the in vitro assay on murine colorectal cancer cells (CT26) did not show any toxicity – whether in the dark or when exposed to 450 nm light, likely because of the poor solubility of the complexes in biological medium.

Stapled peptides have rapidly established themselves as a powerful technique to mimic α-helical interactions with a short peptide sequence. There are many examples of stapled peptides that successfully disrupt α-helix-mediated protein-protein interactions, with an example currently in clinical trials. DNA-proteins interactions are also often mediated by α-helices and are involved in all transcriptional regulation processes. Unlike DNA-binding small molecules, which typically lack DNA sequence selectivity, DNA-binding proteins bind with high affinity and high selectivity. These are ideal candidates for the design DNA-binding stapled peptides. Despite the parallel to protein-protein interaction disrupting stapled peptides and the need for sequence specific DNA binders, there are very few DNA-binding stapled peptides. In this review we examine all the known DNA-binding stapled peptides. Their design concepts are compared to stapled peptides that disrupt protein-protein interactions and based on the few examples in the literature, DNA-binding stapled peptide trends are discussed.

The sterically controlled, nuclease enhanced DNA assembly technique successfully assembles DNA structures containing multiple capture probes. Short (60 bp) DNA stands, with probes attached, are assembled with larger (1 kb) strands, overcoming the limitations of Gibson assembly, and offering a multiplex diagnostic tool.

Abstract

Traditional methods for the assembly of functionalised DNA structures, involving enzyme restriction and modification, present difficulties when working with small DNA fragments (<100 bp), in part due to a lack of control over enzymatic action during the DNA modification process. This limits the design flexibility and range of accessible DNA structures. Here, we show that these limitations can be overcome by introducing chemical modifications into the DNA that spatially restrict enzymatic activity. This approach, sterically controlled nuclease enhanced (SCoNE) DNA assembly, thereby circumvents the size limitations of conventional Gibson assembly (GA) and allows the preparation of well-defined, functionalised DNA structures with multiple probes for specific analytes, such as IL-6, procalcitonin (PCT), and a biotin reporter group. Notably, when using the same starting materials, conventional GA under typical conditions fails. We demonstrate successful analyte capture based on standard and modified sandwich ELISA and also show how the inclusion of biotin probes provides additional functionality for product isolation.

3’,5’ cyclic nucleotides play a fundamental role in modern biochemical processes and have been suggested to play a central role at the origin of terrestrial life. In the current work we suggest that a formamide-based systems chemistry may account for their availability on the early Earth. In particular, we demonstrate that in a liquid formamide environment at elevated temperatures 3’,5’ cyclic nucleotides are obtained in good yield and selectivity upon intramolecular cyclization of 5’ phosphorylated nucleosides in the presence of carbodiimides.

Trifunctional chemical probes derived from the potent shellfish poison, (+)-saxitoxin (STX), irreversibly inhibit wild-type voltage-gated sodium channels (Na_Vs). Saxitoxin derivatives decorated with a maleimide electrophile and either biotin, a fluorescent dye, or biorthogonal-reactive group were synthesized and evaluated using whole-cell, voltage-clamp electrophysiology.

Abstract

Voltage-gated sodium ion channels (Na_Vs) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of Na_Vs hold potential value for understanding how channel regulation and post-translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)-saxitoxin (STX) – a potent and reversible inhibitor of multiple Na_V isoforms – and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or ‘click’ functional group for labeling wild-type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX-maleimide-coumarin derivative is most effective at irreversibly blocking Na⁺ conductance when applied to recombinant Na_Vs and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high-affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next-generation tool compounds for selective modification of Na_Vs expressed in the plasma membranes of cells.

Telomere elongation by telomerase consists of two types of translocation: duplex translocation during each repeat synthesis and template translocation at the end of repeat synthesis. Our replica exchange molecular dynamics simulations show that in addition to the Watson-Crick interactions in the active site, templating RNA can also form base pairs with the upstream regions of DNA, mostly with the second upstream DNA repeat with respect to the 3' end. At the end of the repeat synthesis, dG10-P442 and dG11-N446 hydrogen bonds form. Then, active site base pairs dissociate one by one and the RNA bases reanneal with the complementary base on the upstream DNA repeat. For each dissociating base pair a new one forms, conserving the number of base pairs during template translocation.

Peptoids were designed to mimic receptors for advanced glycation end-products (RAGE) ligand amyloid-β (Aβ) and curtail RAGE inflammatory activation. Here, we reveal the nanomolar binding capability of the peptoid-based mimics to RAGE and demonstrate their ability to attenuate lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production as well as reduce upregulation of cell surface RAGE at non-toxic concentrations.

Abstract

While the primary pathology of Alzheimer's disease (AD) is defined by brain deposition of amyloid-β (Aβ) plaques and tau neurofibrillary tangles, chronic inflammation has emerged as an important factor in AD etiology. Upregulated cell surface expression of the receptor for advanced glycation end-products (RAGE), a key receptor of innate immune response, is reported in AD. In parallel, RAGE ligands, including Aβ aggregates, HMGB1, and S100B, are elevated in AD brain. Activation of RAGE by these ligands triggers release of inflammatory cytokines and upregulates cell surface RAGE. Despite such observation, there are currently no therapeutics that target RAGE for treatment of AD-associated neuroinflammation. Peptoids, a novel class of potential AD therapeutics, display low toxicity, facile blood-brain barrier permeability, and resistance to proteolytic degradation. In the current study, peptoids were designed to mimic Aβ, a ligand that binds the V-domain of RAGE, and curtail RAGE inflammatory activation. We reveal the nanomolar binding capability of peptoids JPT1 and JPT1a to RAGE and demonstrate their ability to attenuate lipopolysaccharide-induced pro-inflammatory cytokine production as well as upregulation of RAGE cell surface expression. These results support RAGE antagonist peptoid-based mimics as a prospective therapeutic strategy to counter neuroinflammation in AD and other neurodegenerative diseases.

Fast but not furious. A rapid on-resin N-formylation protocol for peptides was developed using formic acid, acetic anhydride, pyridine, and DMF. This method is simple in execution and provides near quantitative yield independent of peptide length and sequence.

Abstract

N-formylation is a common pre- and post-translational modification of the N-terminus or the lysine side chain of peptides and proteins that plays a role in the initiation of immune responses, gene expression, or epigenetics. Despite its high biological relevance, protocols for the chemical N-formylation of synthetic peptides are scarce. The few available methods are elaborate in their execution and the yields are highly sequence-dependent. We present a rapid, easy-to-use one-pot procedure that runs at room temperature and can be used to formylate protected peptides at both the N-terminus and the lysine side chain on the resin in near-quantitative yields. Only insensitive, storage-stable standard chemicals – formic acid, acetic anhydride, pyridine and DMF – are used. Formylation works for both short and long peptides of up to 34 amino acids and over the spectrum of canonical amino acids.