RNase H-dependent DNA thresholder modulated by cancer marker concentration.

Threshold antisense oligonucleotide constructs were designed to cleave mRNA within different biomarker concentrations. The mRNA cleavage is activated by 2.6, 7.5 or 39.5 nM of biomarker depending on the construct design. The constructs can be used to differentiate cancer from normal cells by the level of oncogene expression followed by silencing of a targeted gene.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Ribonuclease A-polymer conjugates via in situ growth for cancer treatment.

Efficient delivery of therapeutic proteins is a critical aspect for protein-based cancer treatment. Herein, an in situ growth approach was employed to prepare ribonuclease A (RNase A)-polymer conjugates by incorporating a cationic polymer, poly(N,N'-dimethylamino-2-ethyl methacrylate) (P(DMAEMA)), and a hydrophobic polymer, poly(N-isopropylacrylamide) (P(NIPAM)), through atom transfer radical polymerization (ATRP). The synthesized RNase A-polymer conjugates (namely R-P(D-b-N)) could preserve the integrity of RNase A and exhibit a unique combination of cationic and hydrophobic properties, leading to enhanced intracellular delivery efficiency. The successful delivery of RNase A by R-P(D-b-N) conjugates effectively triggered the cell apoptosis through the mitochondria-dependent signaling pathway to achieve the anti-proliferative response. Additionally, the conjugates could inhibit cell migration and thus possess the potential for the suppression of tumor metastasis. Overall, our findings highlight that the introduction of cationic and hydrophobic moieties via ATRP provides a versatile platform for the intracellular delivery of therapeutic proteins, offering a new avenue for treating diverse diseases.

The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion.

RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favor and against this idea. RNase treatment of chromatin is commonly used to study RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (ChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase Polycomb repressive complex 2 (PRC2). This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here, we show that RNase A treatment during ChIP causes the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome-wide. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results emphasize substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.

Apparent RNA bridging between PRC2 and chromatin is an artifact of non-specific chromatin precipitation upon RNA degradation.

Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain repression of genes specific for other cell lineages. In vitro, RNA inhibits PRC2 activity, but the effect of RNA on PRC2 in cells is less clear, with studies concluding that RNA either antagonizes or promotes PRC2 chromatin association. The addition of RNase A to chromatin immunoprecipitation reactions has been reported to reduce detection of PRC2 target sites, suggesting the existence of RNA bridges connecting PRC2 to chromatin. Here, we show that the apparent loss of PRC2 chromatin association after RNase A treatment is due to non-specific chromatin precipitation. RNA degradation precipitates chromatin out of solution, thereby masking enrichment of specific DNA sequences in chromatin immunoprecipitation reactions. Maintaining chromatin solubility by the addition of poly-L-glutamic acid rescues detection of PRC2 chromatin occupancy upon RNA degradation. These findings undermine support for the model that RNA bridges PRC2 and chromatin in cells.

Association of plasma angiogenin with risk of major cardiovascular events in type 2 diabetes.

BACKGROUND: Angiogenin, an enzyme belonging to the ribonucleases A superfamily, plays an important role in vascular biology. Here, we sought to study the association of plasma angiogenin and major adverse cardiovascular events (MACEs) in patients with type 2 diabetes (T2D). METHODS: This prospective study included 1083 T2D individuals recruited from a secondary hospital and a primary care facility. The primary outcome was a composite of four-point MACE (nonfatal myocardial infarction, stroke, unstable angina pectoris leading to hospitalization and cardiovascular death). Circulating angiogenin was measured by a proximity extension assay. Cox regression models were used to evaluate the association of baseline plasma angiogenin with the risk of MACE. RESULTS: During a median follow-up of 9.3 years, 109 (10%) MACE were identified. Plasma angiogenin was significantly higher in participants with MACE than in those without MACE (P < 0.001). Doubling of plasma angiogenin concentration was associated with a 3.10-fold (95% CI 1.84-5.22) increased risk for MACE. The association was only moderately attenuated after adjustment for demographic and cardiometabolic risk factors (adjusted HR 2.38, 95% CI 1.34-4.23) and remained statistically significant after additional adjustment for estimated glomerular filtration rate (eGFR) and urinary albumin to creatinine ratio (uACR) (adjusted HR 1.90, 95% CI 1.02-3.53). A consistent outcome was obtained when plasma angiogenin was analysed as a categorical variable in tertiles. CONCLUSIONS: Plasma angiogenin was associated with the risk of future cardiovascular events in patients with T2D and may be a promising novel biomarker for identifying high-risk T2D patients for early management.

Colorectal Cancer-Associated Myofibroblasts Exhibit Enhanced Angiogenin Expression and Signaling via the PLXNB2 Receptor.

INTRODUCTION: Dynamic cell-cell interactions shape the tumor microenvironment to regulate tumor growth and invasiveness. Myofibroblasts are gastrointestinal stromal cells that are upregulated in the setting of colorectal cancer (CRC) and may play an important role in tumor-stromal cell communication. Angiogenin is a 14-kDa ribonuclease that regulates myofibroblast function and has been implicated in myofibroblast-CRC cell communication in mouse models. However, its role in human patients has not been well established. METHODS: Open access, annotated single-cell RNA sequencing data of paired normal human colon and CRC tissue were available in the National Center for Biotechnology Information Gene Expression Omnibus Database. We supplemented and verified these data by analyzing scRNA-seq data from an independent set of paired normal human colon and CRC tissue. CellChat was used to quantitatively infer biologically meaningful cell-cell communication networks from scRNA-seq data. PLXNB2 and α-2 actin (ACTA2) are cell surface angiogenin receptors that regulate angiogenin signaling. Ligand-receptor interactions involving angiogenin, PLXNB2, and ACTA2 were analyzed between cell populations in each sample. RESULTS: We found no difference in overall angiogenin expression comparing normal colon and CRC tissue. In normal colon tissue, myofibroblasts do not express angiogenin or the PLXNB2 receptor. In the presence of CRC, there was a striking increase in the number of myofibroblast cells within the surrounding stroma. CRC-associated myofibroblasts were characterized by a significant upregulation of both angiogenin and PLXNB2 receptor expression (P < 0.05), while no difference was seen in ACTA2. CRC cells not only use angiogenin for autocrine signaling but also communicate with myofibroblasts via the PLXNB2 receptor. CONCLUSIONS: Compared to normal human colon tissue, CRC tissue is associated with an enrichment of myofibroblasts that exhibit upregulated expression of angiogenin and the angiogenin receptor PLXNB2. CRC cells engage in autocrine signaling via angiogenin and paracrine signaling with myofibroblasts via PLXNB2. Angiogenin appears to be directly involved in tumor-stromal cell communication in human CRC tissue and may play an important role in disease progression.

Neural stem cell homeostasis is affected in cortical organoids carrying a mutation in Angiogenin.

Mutations in Angiogenin (ANG) and TARDBP encoding the 43 kDa transactive response DNA binding protein (TDP-43) are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). ANG is neuroprotective and plays a role in stem cell dynamics in the haematopoietic system. We obtained skin fibroblasts from members of an ALS-FTD family, one with mutation in ANG, one with mutation in both TARDBP and ANG, and one with neither mutation. We reprogrammed these fibroblasts to induced pluripotent stem cells (iPSCs) and generated cortical organoids as well as induced stage-wise differentiation of the iPSCs to neurons. Using these two approaches we investigated the effects of FTD-associated mutations in ANG and TARDBP on neural precursor cells, neural differentiation, and response to stress. We observed striking neurodevelopmental defects such as abnormal and persistent rosettes in the organoids accompanied by increased self-renewal of neural precursor cells. There was also a propensity for differentiation to later-born neurons. In addition, cortical neurons showed increased susceptibility to stress, which is exacerbated in neurons carrying mutations in both ANG and TARDBP. The cortical organoids and neurons generated from patient-derived iPSCs carrying ANG and TARDBP gene variants recapitulate dysfunctions characteristic of frontotemporal lobar degeneration observed in FTD patients. These dysfunctions were ameliorated upon treatment with wild type ANG. In addition to its well-established role during the stress response of mature neurons, ANG also appears to play a role in neural progenitor dynamics. This has implications for neurogenesis and may indicate that subtle developmental defects play a role in disease susceptibility or onset. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Structure and function of Campylobacter jejuni polynucleotide phosphorylase (PNPase): Insights into the role of this RNase in pathogenicity.

Ribonucleases are in charge of the processing, degradation and quality control of all cellular transcripts, which makes them crucial factors in RNA regulation. This post-transcriptional regulation allows bacteria to promptly react to different stress conditions and growth phase transitions, and also to produce the required virulence factors in pathogenic bacteria. Campylobacter jejuni is the main responsible for human gastroenteritis in the world. In this foodborne pathogen, exoribonuclease PNPase (CjPNP) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization. Here we report the crystallographic structure of CjPNP, complemented with SAXS, which confirms the characteristic doughnut-shaped trimeric arrangement and evaluates domain arrangement and flexibility. Mutations in highly conserved residues were constructed to access their role in RNA degradation and polymerization. Surprisingly, we found two mutations that altered CjPNP into a protein that is only capable of degrading RNA even in conditions that favour polymerization. These findings will be important to develop new strategies to combat C. jejuni infections.

Escherichia coli strains with precise domain deletions in the ribonuclease RNase E can achieve greatly enhanced levels of membrane protein production.

Escherichia coli is one of the most widely utilized hosts for production of recombinant membrane proteins (MPs). Bacterial MP production, however, is usually accompanied by severe toxicity and low-level volumetric accumulation. In previous work, we had discovered that co-expression of RraA, an inhibitor of the RNA-degrading activity of RNase E, can efficiently suppress the cytotoxicity associated with the MP overexpression process and, simultaneously, enhance significantly the cellular accumulation of membrane-incorporated recombinant MPs in bacteria. Based on this, we constructed the specialized MP-producing E. coli strain SuptoxR, which can achieve dramatically enhanced volumetric yields of well-folded recombinant MPs. Ιn the present work, we have investigated whether domain deletions in the E. coli RNase E, which exhibit reduced ribonucleolytic activity, can result in suppressed MP-induced toxicity and enhanced recombinant MP production, in a manner resembling the conditions of rraA overexpression in E. coli SuptoxR. We have found that some strains encoding specific RNase E truncation variants can achieve significantly enhanced levels of recombinant MP production. Among these, we have found a single RNase E variant strain, which can efficiently suppress MP-induced toxicity and achieve greatly enhanced levels of recombinant MP production for proteins of both prokaryotic and eukaryotic origin. Based on its properties, and in analogy to the original SuptoxR strain, we have termed this strain SuptoxRNE22. E. coli SuptoxRNE22 can perform better than commercially available bacterial strains, which are frequently utilized for recombinant MP production. We anticipate that SuptoxRNE22 will become a widely utilized host for recombinant MP production in bacteria.

Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease.

In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.

Characterization of RNase J.

RNase J is involved in RNA maturation as well as degradation of RNA to the level of mononucleotides. This enzyme plays a vital role in maintaining intracellular RNA levels and governs different steps of the cellular metabolism in bacteria. RNase J is the first ribonuclease that was shown to have both endonuclease and 5'-3' exonuclease activity. RNase J enzymes can be identified by their characteristic sequence features and domain architecture. The quaternary structure of RNase J plays a role in regulating enzyme activity. The structure of RNase J has been characterized from several homologs. These reveal extensive overall structural similarity alongside a distinct active site topology that coordinates a metal cofactor. The metal cofactor is essential for catalytic activity. The catalytic activity of RNase J is influenced by oligomerization, the choice and stoichiometry of metal cofactors, and the 5' phosphorylation state of the RNA substrate. Here we describe the sequence and structural features of RNase J alongside phylogenetic analysis and reported functional roles in diverse organisms. We also provide a detailed purification strategy to obtain an RNase J enzyme sample with or without a metal cofactor. Different methods to identify the nature of the bound metal cofactor, the binding affinity and stoichiometry are presented. Finally, we describe enzyme assays to characterize RNase J using radioactive and fluorescence-based strategies with diverse RNA substrates.

Profiling the in vivo RNA interactome associated with the endoribonuclease RNase III in Staphylococcus aureus.

Regulatory small RNA (sRNA) have been extensively studied in model Gram-negative bacteria, but the functional characterisation of these post-transcriptional gene regulators in Gram-positives remains a major challenge. Our previous work in enterohaemorrhagic E. coli utilised the proximity-dependant ligation technique termed CLASH (UV-crosslinking, ligation, and sequencing of hybrids) for direct high-throughput sequencing of the regulatory sRNA-RNA interactions within the cell. Recently, we adapted the CLASH technique and demonstrated that UV-crosslinking and RNA proximity-dependant ligation can be applied to Staphylococcus aureus, which uncovered the first RNA-RNA interaction network in a Gram-positive bacterium. In this chapter, we describe modifications to the CLASH technique that were developed to capture the RNA interactome associated with the double-stranded endoribonuclease RNase III in two clinical isolates of S. aureus. To briefly summarise our CLASH methodology, regulatory RNA-RNA interactions were first UV-crosslinked in vivo to the RNase III protein and protein-RNA complexes were affinity-purified using the His6-TEV-FLAG tags. Linkers were ligated to RNase III-bound RNA during library preparation and duplexed RNA-RNA species were ligated together to form a single contiguous RNA 'hybrid'. The RNase III-RNA binding sites and RNA-RNA interactions occurring on RNase III (RNA hybrids) were then identified by paired-end sequencing technology. RNase III-CLASH represents a step towards a systems-level understanding of regulatory RNA in Gram-positive bacteria.

Angiogenin as a Possible Mediator of Macrophage-Mediated Regulation of Fibroblast Functions.

We studied angiogenin production by human macrophages and evaluated the role of this factor in the macrophage-mediated regulation of fibroblasts. All macrophage subtypes, and especially the efferocytosis-polarized macrophages, M2(LS), actively produced angiogenin. Exogenous recombinant angiogenin dose-dependently enhanced the proliferation and differentiation of dermal fibroblasts. The addition of the angiogenin inhibitor to fibroblasts cultures suppressed the stimulating effect of exogenous angiogenin or M2(LS) conditioned media. These findings indicate the involvement of angiogenin in the macrophage-mediated paracrine regulation of skin fibroblasts.

Synergistic correlation between host angiogenin and dengue virus replication.

DENV infection poses a major health concern globally and the pathophysiology relies heavily on host-cellular machinery. Although virus replication relies heavily on the host, the mechanistic details of DENV-host interaction is not fully characterized yet. Here, we are focusing on characterizing the mechanistic basis of virus-induced stress on the host cell. Specifically, we aim to characterize the role of the stress modulator ribonuclease Angiogenin during DENV infection. Our results suggested that the levels of Angiogenin are up-regulated in DENV-infected cells and the levels increase proportionately with DENV replication. Our efforts to knockdown Angiogenin using siRNA were unsuccessful in DENV-infected cells but not in mock-infected control. To further investigate the modulation between DENV replication and Angiogenin, we treated Huh7 cells with Ivermectin prior to DENV infection. Our results suggest a significant reduction in DENV replication specifically at the later stages as a consequence of Ivermectin treatment. Interestingly, Angiogenin levels were also found to be decreased proportionately. Our results suggest that Angiogenin modulation during DENV infection is important for DENV replication and pathogenesis.

High-performance peptide and disulfide mapping by direct injection of intact proteins using on-line coupled UV-liquid chromatography microdroplet mass spectrometry (UVLC-MMS).

Microdroplet mass spectrometry (MMS), achieving ultra-fast enzyme digestion in the ionization source, holds great promises for innovating protein analysis. Here, in-depth protein characterization is demonstrated by direct injection of intact protein mixtures via on-line coupling MMS with capillary C4 liquid chromatography (LC) containing UV windows (UVLC-MMS) through an enzyme introduction tee. We showed complete sets of peptides of individual proteins (hemoglobin, bovine serum albumin, and ribonuclease A) in a mixture could be obtained in one injection. Such full (100%) sequence coverage, however, could not be achieved by conventional nanoLC-MS method using bottom-up approach with single enzyme. Moreover, direct injection of a chaperone α-crystalline (α-Cry) complex yielded identification of post-translational modifications including novel sites and semi-quantitative characterization including 3:1 stoichiometry ratio of αA- and αB-Cry sub-units and ∼1.4 phosphorylation/subunit on S45 (novel site) and S122 (main site) of αA-Cry, ∼0.7 phosphorylation/subunit on S19 (main site) and S45 of αB-Cry, as well as 100% acetylation on both N-termini of each subunits by matching the mass and retention time of the intact and its digested peptides. Furthermore, trifluoroacetic acid was able to be used in the mobile phase with UVLC-MMS to improve the separation of differentially reduced intact species and detectability of the droplet-digested products. This allowed us to completely map four disulfide linkages of ribonuclease A based on collision-induced dissociation of disulfide clusters, some of which would otherwise not be detected, preventing scrambling or shuffling errors arising from lengthy bulk solution digestion by the bottom-up approach. Integration of UVLC and MMS greatly improves droplet digestion efficiency and MS detection, enabling highly efficient workflow for in-depth and accurate protein characterization.

Sugar osmolyte inhibits and attenuates the fibrillogenesis in RNase A: An in vitro and in silico characterizations.

Mechanisms of protein aggregation are of immense interest in therapeutic biology and neurodegenerative medicine. Biochemical processes within the living cell occur in a highly crowded environment. The phenomenon of macromolecular crowding affects the diffusional and conformational dynamics of proteins and modulates their folding. Macromolecular crowding is reported to cause protein aggregation in some cases, so it is a cause of concern as it leads to a plethora of neurodegenerative disorders and systemic amyloidosis. To divulge the mechanism of aggregation, it is imperative to study aggregation in well-characterized model proteins in the presence of macromolecular crowder. One such protein is ribonuclease A (RNase A), which deciphers neurotoxic function in humans; therefore we decided to explore the amyloid fibrillogenesis of this thermodynamically stable protein. To elucidate the impact of crowder, dextran-70 and its monomer glucose on the aggregation profile of RNase-A various techniques such as Absorbance, Fluorescence, Fourier Transforms Infrared, Dynamic Light Scattering and circular Dichroism spectroscopies along with imaging techniques like Atomic Force Microscopy and Transmission Electron Microscopy were employed. Thermal aggregation and fibrillation were further promoted by dextran-70 while glucose counteracted the effect of the crowding agent in a concentration-dependent manner. This study shows that glucose provides stability to the protein and prevents fibrillation. Intending to combat aggregation, which is the hallmark of numerous late-onset neurological disorders and systemic amyloidosis, this investigation unveils that naturally occurring osmolytes or other co-solutes can be further exploited in novel drug design strategies.

Novel XBP1s-independent function of IRE1 RNase in HIF-1α-mediated glycolysis upregulation in human macrophages upon stimulation with LPS or saturated fatty acid.

In obesity, adipose tissue infiltrating macrophages acquire a unique pro-inflammatory polarization, thereby playing a key role in the development of chronic inflammation and Type 2 diabetes. Increased saturated fatty acids (SFAs) levels have been proposed to drive this specific polarization. Accordingly, we investigated the immunometabolic reprogramming in SFA-treated human macrophages. As expected, RNA sequencing highlighted a pro-inflammatory profile but also metabolic signatures including glycolysis and hypoxia as well as a strong unfolded protein response. Glycolysis upregulation was confirmed in SFA-treated macrophages by measuring glycolytic gene expression, glucose uptake, lactate production and extracellular acidification rate. Like in LPS-stimulated macrophages, glycolysis activation in SFA-treated macrophages was dependent on HIF-1α activation and fueled the production of pro-inflammatory cytokines. SFAs and LPS both induced IRE1α endoribonuclease activity, as demonstrated by XBP1 mRNA splicing, but with different kinetics matching HIF-1α activation and the glycolytic gene expression. Interestingly, the knockdown of IRE1α and/or the pharmacological inhibition of its RNase activity prevented HIF-1α activation and significantly decreased glycolysis upregulation. Surprisingly, XBP1s appeared to be dispensable, as demonstrated by the lack of inhibiting effect of XBP1s knockdown on glycolytic genes expression, glucose uptake, lactate production and HIF-1α activation. These experiments demonstrate for the first time a key role of IRE1α in HIF-1α-mediated glycolysis upregulation in macrophages stimulated with pro-inflammatory triggers like LPS or SFAs through XBP1s-independent mechanism. IRE1 could mediate this novel function by targeting other transcripts (mRNA or pre-miRNA) through a mechanism called regulated IRE1-dependent decay or RIDD. Deciphering the underlying mechanisms of this novel IRE1 function might lead to novel therapeutic targets to curtail sterile obesity- or infection-linked inflammation.

Delivery of Small-Molecule Drugs and Protein Drugs by Injectable Acid-Responsive Self-Assembled COF Hydrogels for Combinatorial Lung Cancer Treatment.

Covalent organic frameworks (COFs) have revealed enormous application prospects for cancer therapeutics recently, but their assembly systems face considerable challenges, such as the codelivery of hydrophobic and hydrophilic protein drugs with different physicochemical properties for in vivo delivery and release, as well as endosomal/lysosomal escape of protein drugs. To address these issues, we leveraged the high specific surface area, lipotropism, and structural tunability of boronate ester-linked COFs (COF-1) for the construction of advanced drug delivery systems. We first encapsulated the small-molecule drug doxorubicin (DOX) into a lipophilic COF (COF-1@DOX) and immobilized the functional protein drug ribonuclease A (RNase A) on the surface of the COF (RNase A-COF-1@DOX). We then created a novel composite delivery system (RNase A-COF-1@DOX gel) by cross-linking an albumin-oxygenated hydrogel (gel) network into the pores of COFs, allowing targeted codelivery of protein and small-molecule drugs in vivo. Using in-living body and multichannel fluorescence imaging, we analyzed the in vivo codelivery of protein and small-molecule drugs in a Lewis lung carcinoma (LLC) model. Finally, we applied the RNase A-COF-1@DOX gel to treat lung cancer in mice. This study paves an avenue for constructing COF-based drug delivery systems for lung cancer treatment and holds the potential to be extended to other types of cancer for more effective and targeted therapeutic treatments.

Human RNase 1 can extensively oligomerize through 3D domain swapping thanks to the crucial contribution of its C-terminus.

Human ribonuclease (RNase) 1 and bovine RNase A are the proto-types of the secretory "pancreatic-type" (pt)-RNase super-family. RNase A can oligomerize through the 3D domain swapping (DS) mechanism upon acetic acid (HAc) lyophilisation, producing enzymatically active oligomeric conformers by swapping both N- and C-termini. Also some RNase 1 mutants were found to self-associate through 3D-DS, however forming only N-swapped dimers. Notably, enzymatically active dimers and larger oligomers of wt-RNase 1 were collected here, in higher amount than RNase A, from HAc lyophilisation. In particular, RNase 1 self-associates through the 3D-DS of its N-terminus and, at a higher extent, of the C-terminus. Since RNase 1 is four-residues longer than RNase A, we further analyzed its oligomerization tendency in a mutant lacking the last four residues. The C-terminus role has been investigated also in amphibian onconase (ONC®), a pt-RNase that can form only a N-swapped dimer, since its C-terminus, that is three-residues longer than RNase A, is locked by a disulfide bond. While ONC mutants designed to unlock or cut this constraint were almost unable to dimerize, the RNase 1 mutant self-associated at a higher extent than the wt, suggesting a specific role of the C-terminus in the oligomerization of different RNases. Overall, RNase 1 reaches here the highest ability, among pt-RNases, to extensively self-associate through 3D-DS, paving the way for new investigations on the structural and biological properties of its oligomers.