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Why are DNA bases stacked in a double helix structure? We combined three theoretical approaches to demonstrate how one core concept derived from quantum mechanics (Pauli repulsion) annihilates the contribution of dispersion to the π-π stacking. The helical architecture is governed by a combination of exchange and electrostatic forces, a result that is interpreted from both a computational and a biological perspective.

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Articular cartilage injury (ACI) remains one of the key challenges in regenerative medicine, as current treatment strategies do not result in ideal regeneration of hyaline-like cartilage. Enhancing endogenous repair via microRNAs (miRNAs) shows promise as a regenerative therapy. miRNA-140 and miRNA-455 are two key and promising candidates for regulating the chondrogenic differentiation of mesenchymal stem cells (MSCs). In this study, we innovatively synthesized a multifunctional tetrahedral framework in which a nucleic acid (tFNA)-based targeting miRNA codelivery system, named A-T-M, was used. With tFNAs as vehicles, miR-140 and miR-455 were connected to and modified on tFNAs, while Apt19S (a DNA aptamer targeting MSCs) was directly integrated into the nanocomplex. The relevant results showed that A-T-M efficiently delivered miR-140 and miR-455 into MSCs and subsequently regulated MSC chondrogenic differentiation through corresponding mechanisms. Interestingly, a synergistic effect between miR-140 and miR-455 was revealed. Furthermore, A-T-M successfully enhanced the endogenous repair capacity of articular cartilage in vivo and effectively inhibited hypertrophic chondrocyte formation. A-T-M provides a new perspective and strategy for the regeneration of articular cartilage, showing strong clinical application value in the future treatment of ACI.

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Psychiatric disorders are among the leading causes of disease burden worldwide. Despite their significant impact, their diagnosis remains challenging due to symptom heterogeneity, psychiatric comorbidity, and the lack of objective diagnostic tests and well-defined biomarkers. Leveraging genomic, epigenomic, and fragmentomic technologies, circulating cell-free DNA (ccfDNA)-based liquid biopsies have emerged as a potential non-invasive diagnosis and disease-monitoring tool. ccfDNA is a DNA species released into circulation from all types of cells through passive and active mechanisms and can serve as a biomarker for various diseases, namely, cancer. Despite their potential, the application of ccfDNA in neuropsychiatry remains underdeveloped. In this review, we provide an overview of liquid biopsies and their components, with a particular focus on ccfDNA. With a summary of pre-analytical practices and current ccfDNA technologies, we highlight the current state of research regarding the use of ccfDNA as a biomarker for neuropsychiatric disorders. Finally, we discuss future steps to unlock ccfDNA's potential in clinical practice.

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Using high-resolution 3D printing, a novel class of microneedle array patches (MAPs) is introduced, called latticed MAPs (L-MAPs). Unlike most MAPs which are composed of either solid structures or hollow needles, L-MAPs incorporate tapered struts that form hollow cells capable of trapping liquid droplets. The lattice structures can also be coated with traditional viscous coating formulations, enabling both liquid- and solid-state cargo delivery, on a single patch. Here, a library of 43 L-MAP designs is generated and in-silico modeling is used to down-select optimal geometries for further characterization. Compared to traditionally molded and solid-coated MAPs, L-MAPs can load more cargo with fewer needles per patch, enhancing cargo loading and drug delivery capabilities. Further, L-MAP cargo release kinetics into the skin can be tuned based on formulation and needle geometry. In this work, the utility of L-MAPs as a platform is demonstrated for the delivery of small molecules, mRNA lipid nanoparticles, and solid-state ovalbumin protein. In addition, the production of programmable L-MAPs is demonstrated with tunable cargo release profiles, enabled by combining needle geometries on a single patch.

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With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

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RNA G-quadruplex structures (rG4s) play important roles in the regulation of biological processes. So far, all the l-RNA aptamers developed to target rG4 of interest contain G4 motif itself, raising the question of whether non-G4-containing l-RNA aptamer can be developed to target rG4. Furthermore, it is unclear whether an l-Aptamer-based tool can be generated for G4 detection in vitro and imaging in cells. Herein, a new strategy is designed using a low GC content template library to develop a novel non-G4-containing l-RNA aptamer with strong binding affinity and improved binding specificity to rG4 of interest. The first non-G4-containing l-Aptamer, l-Apt.1-1, is identified with nanomolar binding affinity to amyloid precursor protein (APP) D-rG4. l-Apt.1-1 is applied to control APP gene expression in cells via targeting APP D-rG4 structure. Moreover, the first l-RNA-based fluorogenic bi-functional aptamer (FLAP) system is developed, and l-Apt.1-1_Pepper is engineered for in vitro detection and cellular imaging of APP D-rG4. This work provides an original approach for developing non-G4-containing l-RNA aptamer for rG4 targeting, and the novel l-Apt.1-1 developed for APP gene regulation, as well as the l-Apt.1-1_Pepper generated for imaging of APP rG4 structure can be further used in other applications in vitro and in cells.

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Chronic wound management is affected by three primary challenges: bacterial infection, oxidative stress and inflammation, and impaired regenerative capacity. Conventional treatment methods typically fail to deliver optimal outcomes, thus highlighting the urgency to develop innovative materials that can address these issues and improve efficacy. Recent advances in DNA nanotechnology have garnered significant interest, particularly in the field of functional nucleic acid (FNA) nanomaterials, owing to their exceptional biocompatibility, programmability, and therapeutic potential. Among them, FNAs with unique nanostructures have garnered considerable attention. First, they inherit the biological properties of FNAs, including biocompatibility, reactive oxygen species (ROS)-scavenging capabilities, and modulation of cellular functions. Second, based on a precise design, these nanostructures exhibit superior physical properties, stability, and cellular uptake. Third, by leveraging the programmability of DNA strands, FNA nanostructures can be customized to accommodate therapeutic nucleic acids, peptides, and small-molecule drugs, thereby enabling a stable and controlled drug delivery system. These unique characteristics enable the use of FNA nanostructures to effectively address the major challenges in chronic wound management. This review focuses on various FNA nanostructures, including tetrahedral framework nucleic acids (tFNAs), DNA hydrogels, DNA origami, and rolling-circle amplification (RCA) DNA assembly. Additionally, a summary of recent advancements in their design and application for chronic wound management as well as insights for future research in this field are provided.

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Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible lung disease, and developing an effective treatment remains a challenge. The limited therapeutic options are primarily delivered by the oral route, among which pirfenidone (PFD) improves pulmonary dysfunction and patient quality of life. However, its high dose and severe side effects (dyspepsia and systemic photosensitivity) limit its clinical value. Intratracheal aerosolization is an excellent alternative method for treating lung diseases because it increases the concentration of the drug needed to reach the focal site. Tetrahedral framework nucleic acid (tFNA) is a drug delivery system with exceptional delivery capabilities. Therefore, we synthesized a PFD-tFNA (Pt) complex using tFNA as the delivery vehicle and achieved quantitative nebulized drug delivery to the lungs via micronebulizer for lung fibrosis treatment. In vivo, Pt exhibited excellent immunomodulatory capacity and antioxidant effects. Furthermore, Pt reduced mortality, gradually restored body weight and improved lung tissue structure. Similarly, Pt also exhibited superior fibrosis inhibition in an in vitro fibrosis model, as shown by the suppression of excessive fibroblast activation and epithelial-mesenchymal transition (EMT) in epithelial cells exposed to TGF-ß1. Conclusively, Pt, a complex with tFNA as a transport system, could enrich the therapeutic regimen for IPF via intratracheal aerosolization inhalation.

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Oral cavity cancer is the most common type of head and neck cancer. There is no definitive standard diagnosis, prognosis, or treatment response biomarker panel based on simple, specific, non-invasive, and reliable methods for head and neck squamous cell carcinoma (HNSCC) patients. On the other hand, the frequent post-treatment biopsies make it challenging to discriminate residual disease or recurrent tumors following postoperative reparative and post-radiation changes. Saliva, blood plasma, and serum samples were commonly used to monitor HNSCC through liquid biopsies. Based on the evidence, the most prominent molecular-based fluid biomarker, such as circulating tumor DNA (ctDNA), has potential applications for early cancer diagnosis, screening, patient management, and surveillance. ctDNA showed genomic and epigenomic changes and the status of human papillomavirus (HPV) with the real-time monitoring of tumor status through cancer therapy. Due to the intra and inter-tumor heterogeneity of tumor cells like cancer stem cells (CSCs) and tumor microenvironment (TME) in HNSCC, the tiny tissue biopsy cannot reflect all genomic and transcriptomic abnormality. Most liquid biopsies are applied to detect circulating molecular biomarkers consisting of cell-free DNA (cfDNA), ctDNA, microRNA, mRNA, and exosome for monitoring tumor progression. Based on the results of previous studies, liquid biopsy can be applied for comprehensive multi-omic discovery by assessing the predictive value of ctDNA in both early and advanced cancers. Liquid biopsy can be used to evaluate molecular signature profiles in HNSCC patients, with great potential to help in early diagnosis, prognosis, surveillance, and treatment monitoring of tumors. These happen by designing longitudinal extensive cohort studies and the utility of organoid technology that promotes the context of personalized and precision cancer medicine.

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Most chemotherapeutic agents are poorly soluble in water, have low selectivity, and cannot reach the tumor in the desired therapeutic concentration. On the other hand, sensitive hydrophilic therapeutics like nucleic acids and proteins suffer from poor bioavailability and cell internalization. To solve this problem, new types of controlled release systems based on nano-sized self-assemblies of cyclodextrins able to control the speed, timing, and location of therapeutic release are being developed. Cyclodextrins are macrocyclic oligosaccharides characterized by a high synthetic plasticity and potential for derivatization. Introduction of new hydrophobic and/or hydrophilic domains and/or formation of nano-assemblies with therapeutic load extends the use of CDs beyond the tried-and-tested CD-drug host-guest inclusion complexes. The recent advances in nano drug delivery have indicated the benefits of the hybrid amphiphilic CD nanosystems over individual CD and polymer components. This review provides a comprehensive overview of the most recent advances in the design of CDs self-assemblies and their use for delivery of a wide range of therapeutic molecules. It aims to offer a valuable insight into the many roles of CDs within this class of drug nanocarriers as well as current challenges and future perspectives.

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Aim: Oligonucleotide therapeutics can be quantified using various bioanalytical methods, and these methods have been compared extensively. However, few comparisons exist where the same analyte is evaluated by multiple assay platforms.Materials & methods: Hybrid LC-MS, SPE-LC-MS, HELISA and SL-RT-qPCR methods were developed for an siRNA analyte, and samples from a pharmacokinetic study were analyzed by all four methods.Results: All assay platforms provided comparable data, though higher concentrations were observed using the non-LC-MS assays. Hybrid LC-MS and SL-RT-qPCR were the most sensitive methodologies, and SL-RT-qPCR and HELISA demonstrated the highest throughput.Conclusion: Each assay platform is suitable for oligonucleotide bioanalysis, and the ultimate choice of methodology will depend on the prioritization of needs such as sensitivity, specificity and throughput.

[Box: see text].

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Retinal neovascular disease is a leading cause of vision loss and blindness globally. It occurs when abnormal new blood vessels form in the retina. In this study, we utilized tetrahedral framework nucleic acids (tFNAs) as vehicles to load quercetin (QUE), a small-molecule flavonoid, forming a deoxyribonucleic acid (DNA) nanocomplex, tFNAs-QUE. Our data show this nanocomplex inhibits pathological neovascularization, reduces the area of retinal nonperfusion area, protects retinal neurons, and preserves the visual function. Further, we discovered that tFNAs-QUE selectively upregulates the AKT/Nrf2/HO-1 signaling pathway, which can suppress pathological vascular growth and exert antioxidative effects. Therefore, this study presents a promising small-molecule-loading mechanism for the treatment of ischemic retinal diseases.

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Carbon dots (CDs) represent an emerging class of nanomaterials that combine outstanding photoluminescent properties with low toxicity and excellent biocompatibility. These unique features have garnered significant interest for potential applications in sensing as well as nanovectors for bioactive compounds. Within this context, the possibility of synthesizing chiral carbon dots (CCDs) has paved the way for a plethora of bioapplications in their interaction with chiral biomolecules. In this study we report the synthesis and characterization of CCDs with opposite chiralities and their selective interaction with nucleic acids. A systematic study on their interaction with different oligonucleotides (ODNs) using UV-vis, photoluminescence, and circular dichroism analyses highlighted how the chiral surface of the CCDs induces distinct spectroscopic responses in CCDs-ODN conjugates. These findings establish the foundation for innovative applications of CCDs as nanosensors and nanocarriers for nucleic acids. Additionally, the antioxidant properties of CCDs were investigated, highlighting their dual potential as both sensing and preservative nanomaterials for genetic material. Our results suggest significant implications for the development of chiral-specific diagnostic tools, drug delivery systems, and therapeutic agents. Furthermore, these properties open new avenues for the use of CCDs in antibiotic residue detection, fluorescence imaging, and photodynamic therapy.

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Infected wounds are a complex disease involving bacterial infections and dysregulated inflammation. However, current research has mostly focused on bacterial inhibition rather than on inflammation. Thus, combined therapeutic strategies with anti-bacterial and anti-inflammation efficacies are urgently needed. Antibiotics are the main treatment strategy for infections. However, the excessive use of antibiotics throughout the body can cause serious side effects. In addition, miRNA-based therapeutics are superior for the treatment of wounds, but their rapid degradation and poor cellular uptake limit their clinical application. Tetrahedral framework DNA (tFNA) is an ideal drug delivery system owing to its excellent stability and remarkable transport ability. Herein, a novel multi-functional miRNA and antibiotic co-delivery system based on tFNA is presented for the first time, called B/L. B/L has heightened resistance to serum and excellent codelivery ability. After transdermal administration, B/L can specifically target TNF receptor-associated factor 6(TRAF6) and IL-1receptor-associated kinase 1(IRAK1), thereby regulating nuclear factor kappa-B (NF-𝜿B) and thus effectively reducing inflammation and promoting the healing of infected wounds. This novel multi-functional co-delivery system provides a versatile, simple, biocompatible, and powerful platform for the personalized and combined treatment of multiple diseases.

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Osteoarthritis (OA) is one of the most prevalent joint diseases and severely affects the quality of life in the elderly population. However, there are currently no effective prevention or treatment options for OA. Oxidative stress and pyroptosis play significant roles in the development and progression of OA. To address this issue, we have developed a novel therapeutic approach for OA that targets oxidative stress and pyroptosis. We synthesized tetrahedral framework nucleic acid (tFNAs) to form framework nucleic acid complexes (TNCs), which facilitate the delivery of the naturally occurring polymethoxyflavonoid nobiletin (Nob) to chondrocytes. TNC has demonstrated favorable bioavailability, stability, and biosafety for delivering Nob. Both in vitro and in vivo experiments have shown that TNC can alleviate OA and protect articular cartilage from damage by eliminating oxidative stress, inhibiting pyroptosis, and restoring the extracellular matrix anabolic metabolism of chondrocytes. These findings suggest that TNC has significant potential in the treatment of OA and cartilage injury.

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Inferring the 3D structure and conformation of disordered biomolecules, e.g., single stranded nucleic acids (ssNAs), remains challenging due to their conformational heterogeneity in solution. Here, we use escape-time electrometry (ETe) to measure with sub elementary-charge precision the effective electrical charge in solution of short to medium chain length ssNAs in the range of 5-60 bases. We compare measurements of molecular effective charge with theoretically calculated values for simulated molecular conformations obtained from Molecular Dynamics simulations using a variety of forcefield descriptions. We demonstrate that the measured effective charge captures subtle differences in molecular structure in various nucleic acid homopolymers of identical length, and also that the experimental measurements can find agreement with computed values derived from coarse-grained molecular structure descriptions such as oxDNA, as well next generation ssNA force fields. We further show that comparing the measured effective charge with calculations for a rigid, charged rod-the simplest model of a nucleic acid-yields estimates of molecular structural dimensions such as linear charge spacings that capture molecular structural trends observed using high resolution structural analysis methods such as X-ray scattering. By sensitively probing the effective charge of a molecule, electrometry provides a powerful dimension supporting inferences of molecular structural and conformational properties, as well as the validation of biomolecular structural models. The overall approach holds promise for a high throughput, microscopy-based biomolecular analytical approach offering rapid screening and inference of molecular 3D conformation, and operating at the single molecule level in solution.

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The Hammerhead Ribozyme (HHR) is a ubiquitous RNA enzyme that catalyzes site-specific intramolecular cleavage. While mutations to its catalytic core have traditionally been viewed as detrimental to its activity, several discoveries of naturally occurring variants of the full-length ribozyme challenge this notion, suggesting a deeper understanding of HHR evolution and functionality. By systematically introducing mutations at key nucleotide positions within the catalytic core, we generated single-, double-, and triple-mutation libraries to explore the sequence requirements and evolution of a full-length HHR. In vitro selection revealed many novel hammerhead variants, some of which possess mutations at nucleotides previously considered to be essential. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids.

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Genetic basis underlying the biodiversity and phenotypic plasticity are fascinating questions in evolutionary biology. Such molecular diversity can be achieved at multi-omics levels. Here, we sequenced the first chromosome-level genome of assassin bug Rhynocoris fuscipes, a polyphagous generalist predator for biological control of agroecosystems. Compared to non-predatory true bugs Apolygus lucorum and Riptortus pedestris, the R. fuscipes-specific genes were enriched in diet-related genes (e.g., serine proteinase, cytochrome P450) which had higher expression level and more exons than non-diet genes. Extensive A-to-I RNA editing was identified in all three species and showed enrichment in genes associated with diet in R. fuscipes, diversifying the transcriptome. An extended analysis between five predaceous and 27 phytophagous hemipteran species revealed an expansion of diet-related genes in R. fuscipes. Our findings bridge the gap between genotype and phenotype, and also advance our understanding on genetic and epigenetic bases governing the diet shifts in ture bugs.

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Circulating cell-free nucleic acids are considered a promising source of biomarkers for diseases and cancer. Liquid biopsy biomarkers for brain tumours represent a major, still unmet, clinical need. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Here we report an easy and reproducible method to analyse cell-free nucleic acids in plasma and EVs by conventional flow cytometry easy to translate into the clinics. Nucleic acids associated with the EVs or present in plasma samples are stained by Pyronin Y, which is a fluorescent dye that is preferably binding double-stranded nucleic acids. Fluorescent staining of EVs isolated from cell-conditioned media is suitable for DNA and RNA detection by flow cytometry. The nucleic acids are partially protected from degradation by the EVs' membrane. Additionally, DNA and RNA can be stained in plasma samples and plasma-derived EVs. Remarkably, analysis of plasma from patients and healthy individuals reveals a difference in their nucleic acid profiles. Taken together, our results indicate that the proposed methodology, which is based on conventional direct flow cytometry, is a promising easy tool for plasma nucleic acid analysis.

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Introduction: In response to continuous advancements in synthetic biotechnologies and in the availability of synthetic nucleic acids to the biological research community since the publication of the 2010 HHS synthetic double-stranded DNA (dsDNA) screening framework, the U.S. government undertook a comprehensive and stakeholder-driven review and revision process. This culminated in the publication of a new screening framework for synthetic nucleic acids in October 2023, followed by an Executive Order directing departments and agencies of the U.S. government to take certain measures in support of implementing the screening framework. This review provides an overview of the process by which stakeholder comments were considered and by which the 2023 screening framework was drafted. A summary of expected impacts on the life sciences research community is also provided. Methods: Comments were solicited from synthetic biology stakeholders through the publication of two Federal Register Notices, in 2020 and 2022. The 2020 Notice elicited 15 unique responses totaling 220 pages, and the 2022 Notice elicited 26 unique responses totaling 79 pages. These were considered by a deliberative interagency group, resulting in a revised screening framework in 2023. Discussion and Conclusion: The adoption of the 2023 screening framework, and related provisions in the Executive Order that followed, will impact researchers and biosafety officers across the U.S. bioeconomy. For instance, this screening framework is no longer limited in its recommendations to providers of synthetic dsDNA containing sequences unique to regulated pathogens or toxins, but now includes recommendations to all entities involved in the sale, use, and transfer of all forms of synthetic nucleic acids encoding genetic sequences that contribute to pathogenicity or toxicity-whether from regulated agents or not. Biosafety professionals are emerging as a critical resource for establishing and fostering a culture of biosecurity surrounding synthetic nucleic acids containing these high consequence genetic sequences. Significance: The work presented is significant because the scope of the 2010 screening framework has been expanded to include roles and responsibilities for new entities across the life sciences research landscape. This will likely impact biosafety professionals, who may be well positioned in their institutions to coordinate these new responsibilities.