Angewandte Chemie

Angiotensin-converting enzyme 2 (ACE2) is a key node in the protective axis of the renin-angiotensin-aldosterone system (RAAS) for blood pressure and hydroelectrolyte regulation, and the main protein receptor recognized by the spike glycoproteins of the severe acute respiratory syndrome (SARS) coronaviruses (CoV) SARS-CoV and SARS-CoV-2. We identified the macrocyclic peptide WJL-63, developed using mRNA display, with high ACE2-binding affinity. The peptide was characterized in vitro in terms of purity, stability, hydrophilicity and ACE2 binding. The crystal structure of the extracellular region of ACE2 in complex with the peptide at 2.2 [A] resolution was elucidated. The structure revealed a binding mode in which WJL-63 is accommodated towards one side of the wide catalytic cleft of the ACE2 peptidase domain, with no direct contact to the conserved zinc ion site. WJL-63 residues Q4, R7, R11 and R14 anchor the peptide deep inside the binding pocket. The opposite edges of the peptide were found to be in contact with subdomain 1 and subdomain 2 of the peptidase domain. This upright binding mode requires an open ACE2 conformation, in contrast to small molecule carboxypeptidase inhibitors, which typically bind to the closed conformation of the enzyme. As a consequence of the open conformation binding mode, the front edge of WJL-63 is accessible for modification such as the herein reported conjugation of a chelator for radiometal labeling. The radiolabeled DOTA-WJL-63 was evaluated on ACE2-transfected HEK cells on which it revealed relatively strong binding with a KD value of 90 {+/-} 28 nM. WJL-63 provides a strong basis for the development of new classes of compounds for the modulation of ACE2 conformation, and for the development of imaging agents for the visualization of ACE2, for example in fluorescence or electron microscopy, or positron emission tomography (PET) imaging.

With the increasing interest in RNA-based therapies, there is a pressing need to incorporate new chemistries into more complex RNA molecules. These modifications can protect RNA from degradation, improve its pharmacokinetics, and enhance its targeting properties. Here we describe the enzymatic synthesis of chemically modified RNA derivatives using a mutant T7 RNA polymerase to incorporate 23 different base modifications alongside stabilizing ribose modifications, such as 2'-fluoro and 2'-deoxy groups. To investigate the impact on transcription efficiency and fidelity, we employed a pool of 38 template sequences and analyzed the transcripts by next-generation sequencing of the cDNA. Results demonstrated that all modifications were successfully incorporated into RNA, with transcription efficiency influenced by three main factors: type of modification, base modified, and the sequence context. Misincorporation levels during transcription and reverse transcription into cDNA were generally low (<1%) but included noticeable exceptions for some nucleobase-modification combinations. As a robust proof-of-concept we demonstrated the selection of Histidine-U modified aptamer, relying on multiple rounds of transcription and amplification, binding Influenza hemagglutinin protein with low nanomolar KD. We anticipate that this work will significantly contribute to the design and production of chemically modified RNAs with novel functionalities, advancing applications in biomedicine and synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/720138v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@184d010org.highwire.dtl.DTLVardef@77fa67org.highwire.dtl.DTLVardef@d89e2eorg.highwire.dtl.DTLVardef@178ebc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Of the various conjugation strategies for cellular biomolecules, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the preferred click chemistry approach due to its fast reaction rate and the commercial availability of a wide range of conjugates. While extracellular labeling of biomolecules using CuAAC has been widely adopted, intracellular labeling in live cells has been challenging as the high copper concentrations required for CuAAC reaction is toxic to biological systems. As a critical first step towards CuAAC-mediated intracellular labeling, an ultrasensitive CuAAC ligand is needed to reduce cytosolic copper concentrations while maintaining fast reaction kinetics. Here, we developed BTT-DNA, a new DNA oligomer-conjugated CuAAC ligand for click reaction biomolecular labeling. The DNA oligo attachment serves several purposes, including: 1. Increased localization of copper atoms near the ligand, which enables ligation of azide tags with much lower copper concentrations than commercially available CuAAC ligands and without the addition of exogenous copper salt; 2. Allows nucleic acid template-driven proximity ligation by choosing the attached DNA sequence, 3. Enables the liposome encapsulation and delivery of the ligand into live cells, and 4. Facilitates intracellular labeling of nascent phospholipids in live cells. We demonstrate that BTT-DNA mediated labeling has little to no effect on the overall cell health.

Peptide nucleic acids (PNAs) are versatile tools for diagnostic and therapeutic applications, including gene regulation and miRNA targeting. However, their therapeutic potential is often limited by challenges such as biological efficacy. To address this, PNAs offer a key advantage over other nucleic acids--their ease of modification, which allows for enhanced properties. In this study, we introduced a novel{gamma} -amino carboxylic acid ({gamma}-ACA) modification to PNAs targeting miR-221-3p, a key miRNA implicated in various pathological processes. The modified PNAs showed significantly improved binding affinity to their targets and more efficient inhibition of miR-221-3p expression compared to unmodified PNAs in A549 cells, leading to effective regulation of downstream gene and protein expression. These results highlight the potential of{gamma} -modified PNAs as a platform for developing miRNA-targeted therapeutics.

Enzymes are instrumental to life and key actors of pathologies, making them relevant drug targets. Most enzyme inhibitors consist of small molecules. Although efficient, their development is long, costly and can come with unwanted off-targeting. Substantial gain in specificity and discovery efficiency is possible using biologicals. Best exemplified by antibodies, these drugs derived from living systems display high specificity and their development is eased by harnessing natural evolution. Aptamers are nucleic acids sharing functional similarities with antibodies while being deprived of many of their limitations. Yet, the success rate of inhibitory aptamer discovery remained hampered by the lack of an efficient discovery pipeline. In this work, we addressed this issue by introducing an ultrahigh-throughput strategy combining in vitro selection, microfluidic screening and bioinformatics. We demonstrate its efficiency by discovering a modified aptamer that specifically and strongly inhibits SPM-1, a beta-lactamase that remained recalcitrant to the development of potent inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/608213v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b15e8forg.highwire.dtl.DTLVardef@b7259forg.highwire.dtl.DTLVardef@6f24borg.highwire.dtl.DTLVardef@d4f48_HPS_FORMAT_FIGEXP M_FIG C_FIG

RNA therapeutics are powerful tools for gene modulation and targeted therapies, but their clinical application is hindered by nuclease degradation and immunogenicity. Incorporating chemical modifications, like locked nucleic acids (LNAs), can enhance nuclease resistance, targeting properties, and thermal stability. Traditionally, LNA incorporation has relied on solid-phase synthesis of short RNAs. Engineered polymerases capable of incorporating xenonucleic acids (XNAs), including LNA, into longer RNAs have been described. However, their XNA yield is limited by primer and template copy numbers, and the generated DNA-XNA duplexes can be difficult to purify. We present a novel approach for incorporating LNA-ATP and LNA-TTP alongside 2Fluoro (2F)-modified pyrimidines via in vitro transcription using a mutant T7 RNA polymerase. This method enables efficient, primer-independent synthesis and amplification of LNA-modified RNA with low error rates. To demonstrate its utility, we performed in vitro selection (SELEX) of LNA- and 2F-modified aptamers targeting Influenza hemagglutinin (HA) and human CD40 ligand (hCD40L), two therapeutically relevant proteins. Iterative SELEX cycles yielded aptamers with low-nanomolar affinities, high specificity, and high nuclease resistance. Overall, this approach provides a scalable and versatile platform for generating chemically stabilized RNAs, fully compatible with SELEX, and holds potential for developing next-generation RNA-based therapeutics with improved pharmacokinetics.

c-Myc is an oncogene that is dysregulated in ~70% of cancers. Its multifaceted function complicates effective drug targeting of the protein. i-Motif DNA structures in gene promotor regions have gained attention for their potential role in modulation of gene expression. These include the i-motif formed by the cytosine-rich sequence that lies upstream of the key P1 promotor of the c-Myc gene. Currently, selective ligands interacting with i-motif structures are limited. Here, peptide ligands for the i-motif from the promoter of c-Myc were identified via phage display. Hit peptides were filtered for selective binding to i-motif structures over other DNA structures using displacement assays and DNA melting experiments. Two lead peptides were found to produce dose-dependent changes on c-Myc gene expression after delivery into HEK293 cells expressing a c-Myc luciferase reporter construct. These leads may be used as chemical tools for the manipulation of c-Myc i-motif in vitro and have potential to be developed into cell-permeable peptidomimetics for delivery in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/656635v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18a4e9aorg.highwire.dtl.DTLVardef@12e72b0org.highwire.dtl.DTLVardef@6ba866org.highwire.dtl.DTLVardef@1fcd338_HPS_FORMAT_FIGEXP M_FIG C_FIG

Functional DNA molecules such as aptamers, catalytic DNAzymes, and dynamic nanostructures have many promising applications in diagnostics and therapeutics yet suffer from limited utility due to immunogenicity and unwanted interactions with other biomolecules, leading to nuclease degradation and unintended sequence driven binding to native sample DNA. Bio-orthogonal, (L)-form or mirror image DNA overcome these challenges yet suffers from the inability to sequence these molecules in a high-throughput manner, a necessary step in the rapid creation of functional (L)-DNA. Here, we report for the first time, design, synthesis, and functional demonstration of (L)-DNA sequencing by synthesis (SBS) using a fluorescent nucleotide reversible terminator, (L)-3'-O-azidomethyl-dGTP-N3-fluorophore paired with a (D)-9{degrees}N mutant DNA polymerase, both enantiomers (i.e., mirror image) of their natural biologically active forms. TABLE OF CONTENTS (GRAPHICAL ABSTRACT) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/692211v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@160650forg.highwire.dtl.DTLVardef@18add51org.highwire.dtl.DTLVardef@1c80104org.highwire.dtl.DTLVardef@1bad99b_HPS_FORMAT_FIGEXP M_FIG C_FIG

The continual emergence and re-emergence of infectious diseases has led to a pressing need for the development of swift and targeted therapeutic interventions. SARS-CoV-2, the causative agent of COVID-19, is a prime example of such a rapidly spreading virus[1]. The global crisis caused by this virus has propelled researchers to explore and adopt novel techniques in the hopes of effectively combating its spread[2]. One such innovative approach is the use of aptamers--single-stranded RNA molecules that can bind targets with high specificity.

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@287a47org.highwire.dtl.DTLVardef@17407eborg.highwire.dtl.DTLVardef@b40435org.highwire.dtl.DTLVardef@804352_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi (D1R) = 8.34, pKi (D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.

Summary paragraphThe elucidation of gene-silencing mechanisms by RNA interference (RNAi) and antisense oligomers has drawn increasing attention to nucleic acid medicine. However, several challenges remain to be overcome, such as in vivo stability1, target selectivity 2,3, drug delivery4,5, and induced innate immunity6. Here, we report a new, versatile, and highly-selective method to hack RNA by controlling RNA structure using short oligonucleotides (RNA hacking: RNAh) in living cells. The oligonucleotide, named Staple oligomer, hybridizes specifically to a target mRNA and artificially induces an RNA higher-order structure, RNA G-quadruplex (RGq)7, on the mRNA. As a result, the RGq allows effective suppression of the target protein translation. This technology does not require cooperation with bioprocesses including enzymatic reactions as in RNAi or antisense technologies, permitting the introduction of artificial nucleic acids into Staple oligomers to increase their in vivo stability without compromising their effectiveness. The method was validated by translational regulation of the mRNAs of TPM3, MYD88, and TRPC6, in a cell-free system and in living mammalian cells. In vivo application of the technology to TRPC6 mRNA allowed us to prevent cardiac hypertrophy in transverse aortic constriction (TAC)-treated mice with no detectable off-target effects. This technology provides new insights into gene therapy after RNAi and antisense technologies.

Aptamers are oligonucleotides with antibody-like binding function, selected from large combinatorial libraries. In this study, we modified a DNA aptamer library with N-hydroxysuccinimide esters, enabling covalent reactivity with cognate proteins. We selected for the ability to bind to mouse monoclonal antibodies, resulting in the isolation of two distinct covalent binding motifs. The covalent aptamers are specific for the Fc region of mouse monoclonal IgG1 and are cross-reactive with mouse IgG2a and other IgGs. Investigation into the covalent reactivity of the aptamers revealed a dependence on micromolar concentrations of Cu2+ ions which can be explained by residual catalyst remaining after modification of the aptamer library. The aptamers were successfully used as adapters in the formation of antibody-oligonucleotide conjugates (AOCs) for use in detection of HIV protein p24 and super-resolution imaging of actin. This work introduces a new method for the site-specific modification of native monoclonal antibodies and may be useful in applications requiring AOCs or other antibody conjugates.

Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntingtons disease: MSH3 and HTT; Alzheimers disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.

G-quadruplex (G4) DNA structures are increasingly recognized for their roles in key cellular processes, including transcriptional regulation and genome stability, making them attractive therapeutic targets. Selective recognition of individual G4s remains challenging due to the high structural similarity among human G4 motifs. The G4 Ligand-conjugated Oligonucleotide strategy addresses this need by combining the G4-binding capabilities of small-molecule G4-ligands with the sequence specificity of an oligonucleotide complementary to the flanking region of the target G4. Here, we systematically explore how the oligonucleotide component governs G4 binding and stabilization by varying its length, backbone composition, and sequence complementarity. This revealed that efficient G4 recognition depends on a strong interdependence between hybridization and G4-ligand binding, such that both elements cooperatively reinforce complex stability and site specificity. Central mismatches disrupt this dual recognition and reduce selectivity. While longer oligonucleotides hybridize more slowly, they form more stable complexes and show stronger G4 stabilization in thermal melting and polymerase stop assays. Replacing the DNA oligonucleotide with peptide nucleic acid enhances binding strength, thermal stability, and metabolic stability, but selective G4 stabilization is achieved only upon ligand conjugation. Together, these results show how rational oligonucleotide design enables selective and potent recognition of G4 structures using GL-Os.

Functional imaging using fluorescent indicators has revolutionized biology but additional sensor scaffolds are needed to access properties such as bright, far-red emission. We introduce a new platform for chemigenetic fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. This approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in neurons.

In recent years, the CRISPR/Cas9 technology has emerged as a highly efficient tool for cell gene editing. However, the delivery of the CRISPR/Cas9 system into cells remains a significant challenge, drastically limiting in vivo gene therapy applications. In this study, we present a transfection/transduction-free tool for intracellular delivery of the Cas9:gRNA ribonucleoprotein. The Cas9 enzyme is conjugated to a 12 nm gold nanoparticle through affinity binding between the 6x His-tag of the protein and the NTA-Ni{superscript 2}LJ groups on the nanoparticles. This link chemistry allows a fine control of the density of the enzymes decorating the particle surface, the orientation of the bonding and the stability of the interaction. Importantly, the surface chemistry of this nanoformulation has been precisely engineered to modulate the cellular internalization and localization. Thanks to this approach of precision chemistry, this nanoformulation demonstrated the ability to spontaneously enter human melanoma cells as monodispersed particles that localize in cell cytoplasm, endosomes, and nucleus. It also shows effective gene editing efficiency similarly to conventional transfection tools. This gold-based formulation of Cas9 represents a ready-to-use biotech editing tool, and a promising solution for direct in vivo gene editing applications.

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI

G protein-coupled receptors (GPCRs) exist within a landscape of interconvertible conformational states and in dynamic equilibrium between monomers and higher-order oligomers, both influenced by ligand binding. Here, we have shown that a homobivalent ligand formed by equal chromenopyrazole moieties as pharmacophores, connected by 14 methylene units, can modulate the dynamics of the cannabinoid CB2 receptor (CB2R) homodimerization by simultaneously binding both protomers of the CB2R-CB2R homodimer. Computational and pharmacological experimentals showed that one of the ligand pharmacophores binds to the orthosteric site of one protomer, and the other pharmacophore to a membrane-oriented pocket between transmembranes 1 and 7 of the partner protomer. This provides unique pharmacological properties, such as increased potency in Gi binding and increased recruitment of {beta}-arrestin. Thus, by modulating dimerization dynamics, it may be possible to fine-tune CB2R activity with potentially improved therapeutic outcomes. HIGHLIGHTSO_LIA homobivalent ligand of CB2R (PM369) modulates the dynamics of receptor homodimerization C_LIO_LIPM369 binds to the orthosteric site of one protomer and to a complementary, membrane-facing, site of the other protomer C_LIO_LIPM369 triggers CB2R homodimerization via the TM 1/7 interface that provides unique pharmacological properties C_LIO_LIPM369 potentiates signaling, increased potency in Gi binding and increased recruitment of {beta}-arrestin C_LIO_LIThese results highlight new approaches to control GPCR signaling C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/593612v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1736aeborg.highwire.dtl.DTLVardef@707654org.highwire.dtl.DTLVardef@168925borg.highwire.dtl.DTLVardef@60bc11_HPS_FORMAT_FIGEXP M_FIG C_FIG

Currently the world is dealing with the third outbreak of the human-infecting coronavirus with potential lethal outcome, cause by a member of the Nidovirus family, the SARS-CoV-2. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused the last worldwide pandemic. Successful development of vaccines highly contributed to reduce the severeness of the COVID-19 disease. To establish a control over the current and newly emerging coronaviruses of epidemic concern requires development of substances able to cure severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA using antisense oligonucleotides (ASOs) and identify locked nucleic acid gapmers (LNA gapmers) potent to reduce by up to 96% the intracellular viral load in vitro. Our results strongly suggest promise of our preselected ASOs for further development as therapeutic or prophylactic anti-viral agents. One sentence summaryASOs (LNA gapmers) targeting the SARS-CoV-2 RNA genome have been effective in viral RNA (load) reduction in vitro.