Angewandte Chemie

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

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

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.

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

Prostate cancer (PCa) is one of the principal contributors to health burden in the aging male population. PCa develops through dysregulation of androgen receptor (AR) signaling pathways. Despite improvements in diagnostic techniques and interventions, no pharmacological measures with long term efficacy have been established once PCa advances to castration resistant prostate cancer (CRPC). To circumvent this issue, tetra-aryl cyclobutanes (CBs) have been proposed as structurally distinct compounds with a mechanism of action differing from traditional androgen receptor signaling inhibitor (ARSIs). Here, we apply principles of crystal engineering and solid state synthesis to expand the class of CBs through strategic derivatization. The synthesis of the CB occurs quantitatively, producing no side products and eliminating the need for product purification. We demonstrate how head-to-tail stacking interactions of halo-pyrimidine rings can be exploited to stack and align unsymmetrical alkenes to undergo [2+2] photodimerization to generate the CB in the solid state. We examine the structure-function relationships of CBs in vitro by profiling AR mediated transcriptional activity, receptor translocation, and cell viability. Moreover, we explore and identify putative binding interactions within CB/AR complexes and establish an adaptive ligand-binding potential using molecular docking platforms. In total, our data suggests that CBs have unexploited therapeutic potential in CRPC and that green chemistry and crystal engineering principles offer a unique route to generating these drug candidates.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

DNA methylation is a critical epigenetic modification that regulates gene expression, maintains genome stability, and influences cellular function during development and disease. Accurate analysis of DNA methylation often requires amplification to generate sufficient material, yet preserving the original epigenetic information during this process is challenging because standard amplification methods can disrupt methylation patterns. To address this, we developed a one-pot strategy that combines helicase-dependent amplification (HDA) with DNA methyltransferase 1 (DNMT1)-mediated methylation, enabling simultaneous DNA amplification and preservation of native methylation marks. A key challenge is that HDA is optimized at 65 {degrees}C, whereas DNMT1 is unstable at elevated temperatures. We overcame this by establishing a unified buffer and isothermal reaction at 42 {degrees}C that supports both enzymatic activities. Under these conditions, HDA achieved robust amplification ([~]5 Ct), while DNMT1 faithfully methylated the newly synthesized DNA, as confirmed by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), with methylation levels proportional to the input template. This one-pot workflow demonstrates the feasibility of concurrent amplification and methylation, providing a foundation for scalable, accurate, and methylation-preserving DNA analyses for epigenetic and clinical applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/709983v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@fb8d3aorg.highwire.dtl.DTLVardef@f50c94org.highwire.dtl.DTLVardef@cda7aorg.highwire.dtl.DTLVardef@1db82b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Human immunodeficiency virus (HIV) infection remains a global health threat. Although antiretroviral therapy (ART) has significantly transformed HIV into a manageable chronic disease, emergence of drug resistance to current ART is a continuing concern. Broadly neutralizing antibodies, as well as reagents containing both a soluble CD4 mimetic and an HIV co-receptor inhibitor--such as CD4-CD4i antibodies--are promising strategies for the prevention and treatment of HIV infection. We previously developed a CD4 D1mimetic (mD1.22) with enhanced neutralization potency, surpassing that of the clinically validated sCD4-D1D2 mimetic. We also previously identified a novel CD4i antibody (X5) that targets the conserved coreceptor binding site on the gp120 core and recognizes an epitope partially overlapping with 17b monoclonal antibody binding site. X5 binding to gp120 was augmented by CD4 and modestly enhanced by CCR5. To leverage these favorable interactions, we designed and optimized sCD4-X5 bispecific antibodies by computational structure-aided modification of antibody size and fusion linkers between the two binding moieties. The bispecific D1X5, with a (G4S)7 long linker between D1 and X5 (IgG1-LL D1X5), exhibited broad neutralization against 11 diverse HIV subtypes across B, C, G clades and AC, BC recombinants. The TZM-bl cell neutralization assay showed IgG1-LL D1X5 neutralization geometric mean IC50 and IC80 are 0.6 g/mL and 3.4 g/mL respectively, which are within the range of potent bnAbs. This work has identified a novel single domain soluble CD4 based CD4-CD4i bispecific antibody with broad HIV-1 neutralization.

Deep learning-based structure prediction enables the design of peptide ligands without relying on naturally occurring scaffolds. However, most computationally generated peptides are not advanced beyond initial activity measurements, leaving the path to drug-like optimization and in vivo validation underexplored. Here we establish an end-to-end workflow for de novo peptide agonist discovery and maturation using the melanocortin-4 receptor (MC4R) as a model target. Using an AlphaFold2-based hallucination protocol implemented in ColabDesign, we generated more than 5,000 linear and head-to-tail cyclic candidate peptides directed towards the MC4R orthosteric pocket. Functional screening of a prioritized subset revealed measurable activity in 74% of linear peptides and 23% of cyclic peptides, from which we identified a cyclic agonist with an EC50 of 340 nM despite lacking the canonical melanocortin activation motif. We then performed systematic in vitro maturation by deep mutational scanning, half-life extender conjugation scanning, and a combinatorial optimization library, coupled with data-driven analysis to map sequence-activity relationships. These experiments identified an alternative activation motif centered on an APWR segment and yielded single-site variants with substantially improved potency. The most effective substitution, a proline at position 5, produced the E5P variant with an EC50 of 6.7 nM against the human melanocortin-4 receptor (hMC4R). Finally, central administration of E5P (10 nmol) reduced acute food intake in mice, providing in vivo proof of concept. Together, our results demonstrate a generalizable design-to-validation strategy for converting de novo peptide designs into optimized, pharmacologically active peptides, and expand the space of MC4R agonist chemotypes beyond endogenous melanocortins.

The ability to encode and reliably read nanoscale information is increasingly important for multiplexed biomolecular detection and super-resolution imaging. DNA origami provides a uniquely programmable platform for arranging structural and functional elements with nanometer precision, enabling the creation of identifiable nanoscale patterns. In this context, DNA origami-based barcodes that incorporate gold nanoparticles (AuNPs) to encode either origami geometry or the identity of specific biological targets within defined nanoparticle patterns have been paired with transmission electron microscopy imaging for decoding. However, surface-bond AuNPs may detach during handling, purification, or biological incubation, leading to misidentification or decoding errors in barcode analysis. Here we report a rational design for the controlled encapsulation of AuNPs within DNA origami tubes to enhance nanoparticle retention and structural integrity. We engineered curvature-inducing modifications in a flat rectangular DNA origami scaffold to promote inward folding and confinement of AuNPs. These barcodes can be further functionalized on the outer surface with bioactive aptamers and/or fluorescence dyes, enabling targeted interactions with cells and optical readout. Programable dimerization further expands multiplexing capacity. This design provides a robust framework for structurally stable origami barcodes and advances the development of high-resolution, multiplexed labeling and diagnostic platforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/725969v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@686c1aorg.highwire.dtl.DTLVardef@1914c4eorg.highwire.dtl.DTLVardef@28ad47org.highwire.dtl.DTLVardef@8847ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

I-motif (iM) DNA structures, formed by cytosine-rich sequences, are increasingly acknowledged for their involvement in gene regulation, maintenance of genomic stability, and their emerging potential as therapeutic targets, particularly in cancer. Despite their biological relevance, the discovery of selective small-molecule probes that can specifically recognize and interact with iM DNA remains an ongoing challenge. In this study, we have used TMPyP4 and screened for its ability to bind various iM DNA constructs, including HRAS1, HRAS2, VEGF, CMYC, CKIT and H-Telo. Structure-activity relationship analyses revealed that specific substitution patterns conferred selectivity towards HRAS2 iM target. Comprehensive spectroscopic investigations, including UV-Vis absorption, steady-state and time-resolved fluorescence, and fluorescence anisotropy, uncovered key photophysical signatures of binding, including significant hypochromic and bathochromic shifts, enhanced fluorescence emission, and prolonged fluorescence lifetimes. Circular dichroism (CD),thermal denaturation (UV-melting) and thermodynamic investigations confirmed that TMPyP4 effectively stabilized the HRAS2 iM structures without disrupting their native topologies. Meanwhile, FT-IR spectroscopy revealed local structural rearrangements upon TMPyP4 binding, offering further evidence of molecular interaction. Collectively, these findings provide valuable insights into the molecular recognition of iM DNA by TMPyP4 and highlight its promise as both selective HRAS2 iM-binding agent and responsive fluorescent probe. This work lays a strong foundation for the development of novel tools for studying iM structures in biological systems and for designing future therapeutics targeting iM DNA in cancer and related diseases.

Therapeutic peptides combine high target specificity with potent biological activity.1 However, treatment success is often limited by rapid clearance and the need for frequent injections.2, 3 This challenge is particularly acute for therapeutic peptides used in obesity, where clinical benefit must be balanced against dose-dependent adverse effects. In nature, these constraints are overcome by storing hormones as reversible fibrils,4 but pharmacokinetic control is essential for widespread adoption of bio-inspired self-assembled depots for therapeutic peptides. Here, we show that tuneable pharmacokinetics can be achieved and modelled by mapping the fundamental chemical parameters of reversibly self-assembly in vitro. We demonstrate this approach for the amylin analogue pramlintide. Amylin analogues are under development for the next generation of diabetes and obesity treatments, with improved mechanism of action e.g. preserving lean body mass.5-8 Pramlintide is an approved drug with a well-established safety profile, however, it has a comparable half-life to native amylin.8-12 In a pilot study, we achieve in vitro-in vivo correlation, increasing the half-life of pramlintide 20-82-fold in rats, while controlling burst release. These findings demonstrate that the optimisation of pharmacokinetics can be decoupled from peptide engineering, establishing a generalisable framework for generating long-acting peptide formulations by emulating native storage mechanisms.

High-risk Human Papillomaviruses (HR-HPVs) are responsible for 5% of global cancers. While vaccines against HR-HPVs exist, there are no treatments available for individuals already infected. Cell-penetrating peptides (CPPs) have demonstrated antiviral properties against viruses by blocking viral entry and delivering antivirals into infected cells. Developing CPP-based therapies faces challenges including inefficient delivery of macromolecules and endosomal entrapment, which must be overcome for effective clinical application. This study identifies an HPV16 major capsid protein L1 derived cationic peptide as a potent CPP. Peptide uptake depended on both a cluster of cationic residues and the specific peptide sequence. Mechanistic studies showed peptide entry occurred via cell surface heparan sulfate-mediated, lipid-raft dependent endocytosis. The peptide efficiently delivered GFP into HaCaT keratinocytes, and associated with the Golgi apparatus, demonstrating endosomal escape. GFP fusion protein endocytosis relied on binding of the cationic peptide to cell surface heparan sulfates. Cell-penetrating ability was conserved among homologous regions of various HPV types. The peptide showed potent antiviral activity by inhibiting infection of HaCaT cells by several HR-HPV types collectively responsible for nearly all HPV-associated cancers. Excitingly, HPV18 L1-derived peptide from the homologous region exhibited potent antiviral activity against HPV16 by preventing viral internalization. Our findings characterize HPV-derived peptides as highly efficient CPPs with potential to deliver therapeutic agents into cells and assist in development of treatments for high-risk HPVs.

There has been a surge in antibiotic resistance in recent years, making traditional antibiotics less effective against key pathogens. RNA has recently emerged as a potential target for antibiotics due to its involvement in crucial microbial functions. It is possible to expand the range of therapeutic targets by using RNA-based therapies, but it remains necessary to improve the molecular-level understanding of interactions between RNA and known and potential binders. The SAM-I riboswitch, which controls the transcriptional termination of gene expression involved in sulfur metabolism in most bacteria, is an excellent ligand target. Thus, understanding its behavior with and without ligand complexes would be very helpful for drug design applications. In this manuscript, we studied the interactions between the SAM-I riboswitch and its natural ligand, SAM, which controls riboswitch function, and compared those interactions to its interactions with the very similar small molecular SAH, which does not control riboswitch function, and to its interactions with a potential binder JS4, identified via virtual screening. From our simulations, we gain a deeper understanding of small molecule interactions with the SAM-I riboswitch. The results reveal how differently the small molecules (SAM, SAH and JS4) bind to and potentially induce conformational changes in the riboswitch. Our findings offer valuable insight into the molecular mechanisms underlying riboswitch RNA-ligand interactions for the design of more effective RNA-targeting therapeutics.

Acinetobacter baumannii is a top-priority, ESKAPE pathogen that poses a major challenge to human health. The pathogen is difficult to combat due to its extensive arsenal of antibiotic resistance and its protective polysaccharide capsule. In addition, A. baumannii isolates are highly heterogeneous, which complicates the development of rapid detection methods or novel targeted therapeutic approaches. Here, we discovered and characterized a new biotechnological tool, the nanobody H7 (NbH7), along with its conserved target, the surface-exposed Omp25 protein of A. baumannii, and elucidated their interaction at the molecular level. Moreover, we demonstrate that NbH7-functionalized magnetic beads enable selective and efficient capture of A. baumannii from bacterial mixtures, including non-pathogenic intestinal bacteria. This provides proof of concept for a new targeting system that remains effective across diverse A. baumannii clinical isolates and capsule types and holds potential for use in diagnostic cell enrichment and targeted therapies.

Nitazenes are emerging synthetic opioids that exhibit exceptionally high potency at the {micro}-opioid receptor (MOR) and contribute to rising overdose fatalities worldwide. Despite extensive in vitro profiling, the structural determinants underlying their structure-activity relationships (SARs) remain unresolved. Here, we combine functional profiling with quantum mechanical calculations and molecular dynamics (MD) simulations to establish a MOR structure-based SAR for nitazenes. Across functional assays, systematic variation at the R1, R2, and R3 positions revealed non-additive effects on potency and identified optimal R1 chain length, R2 N-desethylation, and retention of the 5-nitro group as key determinants of high MOR potency. Consistent with this framework, N-desethyl isotonitazene emerged as the most potent analogue. Structural analysis of the cryo-EM MOR-Gi-fluornitrazene complex, together with MD simulations of multiple nitazene analogues, revealed a conserved trivalent binding architecture in which each substituent engages distinct subpockets. N-desethylation at R2 increases the positive electrostatic surface at the protonated amine, reduces steric constraints near transmembrane helix (TM) 7, strengthens R3 interactions, and allosterically modulates R1 engagement in a substituent-dependent manner. Additionally, optimal R1 chain length and shape stabilize the TM5-TM6 interface and influence activation-relevant TM6 dynamics, defining a unified SAR at R1 across nitazene and fentanyl scaffolds. Together, these findings indicate that nitazene potency reflects substituent-dependent coupling among R1, R2, and R3 within the MOR binding pocket, with R3 engagement distinguishing nitazenes from fentanyl. This framework establishes a coherent structural model of nitazene-MOR recognition that accounts for their unusually high potency and efficacy.

Hyperuricemia, a major risk factor for gout and kidney disease, arises from the evolutionary loss of human uricase and remains a significant medical challenge due to its high prevalence. However, limited therapeutic options are available for refractory hyperuricemia that typically require long-term treatment. Here we developed a circRNA-based uricase replacement strategy and evaluated its efficacy in uricase-knockout mice as a model for severe hyperuricemia. Lipid nanoparticle-mediated delivery of circRNA enabled efficient in vivo expression of an engineered human-like uricase, which rapidly reduced serum urate levels after a single injection and maintained the urate-lowering effect for up to 10 days. Repeated administration led to sustained urate reduction for 10 weeks, mitigated renal injury, and exhibited favorable biosafety. These findings highlight the therapeutic potential of circRNA-based uricase replacement for the long-term treatment of hyperuricemia and its associated complications.

Conformationally responsive DNA nanoswitches have previously been developed and validated for a variety of biosensing applications including detection of DNA, microRNA, and viral RNA/DNA. Here we develop new methodology for enhancing the sensitivity of DNA-based sensing by recycling a fixed number of targets for repeated reuse. We achieved target-dependent enzymatic ligation of looped nanoswitches and showed that subsequent removal of target does not affect the ligated loop. Through cyclic annealing, ligation, and target removal, we can linearly control signal amplification up to hundreds of cycles. This method adds an important new capability for low abundance targets without the need for target amplification.