Electrochemical Detection of Nucleic Acids Using Three-Dimensional Graphene Screen-Printed Electrodes.

Electrochemical approaches, along with miniaturization of electrodes, are increasingly being employed to detect and quantify nucleic acid biomarkers. Miniaturization of the electrodes is achieved through the use of screen-printed electrodes (SPEs), which consist of one to a few dozen sets of electrodes, or by utilizing printed circuit boards. Electrode materials used in SPEs include glassy carbon (Chiang H-C, Wang Y, Zhang Q, Levon K, Biosensors (Basel) 9:2-11, 2019), platinum, carbon, and graphene (Cheng FF, He TT, Miao HT, Shi JJ, Jiang LP, Zhu JJ, ACS Appl Mater Interfaces 7:2979-2985, 2015). There are numerous modifications to the electrode surfaces as well (Cheng FF, He TT, Miao HT, Shi JJ, Jiang LP, Zhu JJ, ACS Appl Mater Interfaces 7:2979-2985, 2015). These approaches offer distinct advantages, primarily due to their demonstrated superior limit of detection without amplification. Using the SPEs and potentiostats, we can detect cells, proteins, DNA, and RNA concentrations in the nanomolar (nM) to attomolar (aM) range. The focus of this chapter is to describe the basic approach adopted for the use of SPEs for nucleic acid measurement.

Biophysical characterization of hydrogen sulfide: A fundamental exploration in understanding significance in cell signaling.

Hydrogen sulfide (H2S) has emerged as a significant signaling molecule involved in various physiological processes, including vasodilation, neurotransmission, and cytoprotection. Its interactions with biomolecules are critical to understand its roles in health and disease. Recent advances in biophysical characterization techniques have shed light on the complex interactions of H2S with proteins, nucleic acids, and lipids. Proteins are primary targets for H2S, which can modify cysteine residues through S-sulfhydration, impacting protein function and signaling pathways. Advanced spectroscopic techniques, such as mass spectrometry and NMR, have enabled the identification of specific sulfhydrated sites and provided insights into the structural and functional consequences of these modifications. Nucleic acids also interact with H2S, although this area is less explored compared to proteins. Recent studies have demonstrated that H2S can induce modifications in nucleic acids, affecting gene expression and stability. Techniques like gel electrophoresis and fluorescence spectroscopy have been utilized to investigate these interactions, revealing that H2S can protect DNA from oxidative damage and modulate RNA stability and function. Lipids, being integral components of cell membranes, interact with H2S, influencing membrane fluidity and signaling. Biophysical techniques such as electron paramagnetic resonance (EPR) and fluorescence microscopy have elucidated the effects of H2S on lipid membranes. These studies have shown that H2S can alter lipid packing and dynamics, which may impact membrane-associated signaling pathways and cellular responses to stress. In the current work we have integrated this with key scientific explainations to provide a comprehensive review.

Diffusiophoresis promotes phase separation and transport of biomolecular condensates.

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart directional motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced enhanced motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.

Delivery approaches of immunomodulatory nucleic acids for cancer therapy.

Messenger RNA (mRNA) vaccines have made remarkable public health contributions during the pandemic and initiated a new era for nucleic acid-based therapeutics. With the unique strength of nucleic acids, including not only mRNA but also DNA, microRNA, small interfering RNA (siRNA), and other nucleic acids, either in tuning off genes or introducing function, nucleic acid therapeutics have been regarded as potential candidates for the treatment of many different diseases, especially for the immunomodulation in cancer. However, the scope of the applications was limited by the challenges in delivery due to intrinsic properties of nucleic acids including low stability, immunogenicity, and toxicity. Bioengineering approaches toward efficient and targeted delivery of therapeutic nucleic acids have gained momentum in clinical applications in the past few decades. Recent advances in the biotechnological approaches for the delivery of mRNA, siRNA, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas for immunomodulatory are promising alternatives in designing future cancer immunotherapy.

Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution.

Self and nonself discrimination is fundamental to immunity. However, it remains largely enigmatic how the mechanisms of distinguishing nonself from self originated. As an intracellular nucleic acid sensor, protein kinase R (PKR) recognizes double-stranded RNA (dsRNA) and represents a crucial component of antiviral innate immunity. Here, we combine phylogenomic and functional analyses to show that PKR proteins probably originated from a preexisting kinase protein through acquiring dsRNA binding domains at least before the last common ancestor of jawed vertebrates during or before the Silurian period. The function of PKR appears to be conserved across jawed vertebrates. Moreover, we repurpose a protein closely related to PKR proteins into a putative dsRNA sensor, recapturing the making of PKR. Our study illustrates how a nucleic acid sensor might have originated via molecular tinkering with preexisting proteins and provides insights into the origins of innate immunity.

Nucleic acid-based nanotherapeutics for treating sepsis and associated organ injuries.

In recent years, gene therapy has been made possible with the success of nucleic acid drugs against sepsis and its related organ dysfunction. Therapeutics based on nucleic acids such as small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), and plasmid DNAs (pDNAs) guarantee to treat previously undruggable diseases. The advantage of nucleic acid-based therapy against sepsis lies in the development of nanocarriers, achieving targeted and controlled gene delivery for improved efficacy with minimal adverse effects. Entrapment into nanocarriers also ameliorates the poor cellular uptake of naked nucleic acids. In this study, we discuss the current state of the art in nanoparticles for nucleic acid delivery to treat hyperinflammation and apoptosis associated with sepsis. The optimized design of the nanoparticles through physicochemical property modification and ligand conjugation can target specific organs-such as lung, heart, kidney, and liver-to mitigate multiple sepsis-associated organ injuries. This review highlights the nanomaterials designed for fabricating the anti-sepsis nanosystems, their physicochemical characterization, the mechanisms of nucleic acid-based therapy in working against sepsis, and the potential for promoting the therapeutic efficiency of the nucleic acids. The current investigations associated with nanoparticulate nucleic acid application in sepsis management are summarized in this paper. Noteworthily, the potential application of nanotherapeutic nucleic acids allows for a novel strategy to treat sepsis. Further clinical studies are required to confirm the findings in cell- and animal-based experiments. The capability of large-scale production and reproducibility of nanoparticle products are also critical for commercialization. It is expected that numerous anti-sepsis possibilities will be investigated for nucleic acid-based nanotherapeutics in the future.

Strategies and mechanisms for endosomal escape of therapeutic nucleic acids.

Despite impressive recent establishment of therapeutic nucleic acids as drugs and vaccines, their broader medical use is impaired by modest performance in intracellular delivery. Inefficient endosomal escape presents a major limitation responsible for inadequate cytosolic cargo release. Depending on the carrier, this endosomal barrier can strongly limit or even abolish nucleic acid delivery. Different recent endosomal escape strategies and their hypothesized mechanisms are reviewed.

A Calibration Strategy for Silicon Nanowire Field-Effect Transistor Biosensors and Its Application in Ultra-Sensitive, Label-Free Biosensing.

The silicon nanowire field-effect transistor (SiNW FET) has been developed for over two decades as an ultrasensitive, label-free biosensor for biodetection. However, inconsistencies in manufacturing and surface functionalization at the nanoscale have led to poor sensor-to-sensor consistency in performance. Despite extensive efforts to address this issue through process improvements and calibration methods, the outcomes have not been satisfactory. Herein, based on the strong correlation between the saturation response of SiNW FET biosensors and both their feature size and surface functionalization, we propose a calibration strategy that combines the sensing principles of SiNW FET with the Langmuir-Freundlich model. By normalizing the response of the SiNW FET biosensors (ΔI/I0) with their saturation response (ΔI/I0)max, this strategy fundamentally overcomes the issues mentioned above. It has enabled label-free detection of nucleic acids, proteins, and exosomes within 5 min, achieving detection limits as low as attomoles and demonstrating a significant reduction in the coefficient of variation. Notably, the nucleic acid test results exhibit a strong correlation with the ultraviolet-visible (UV-vis) spectrophotometer measurements, with a correlation coefficient reaching 0.933. The proposed saturation response calibration strategy exhibits good universality and practicability in biological detection applications, providing theoretical and experimental support for the transition of mass-manufactured nanosensors from theoretical research to practical application.

Advances in the Application of Protein Language Modeling for Nucleic Acid Protein Binding Site Prediction.

Protein and nucleic acid binding site prediction is a critical computational task that benefits a wide range of biological processes. Previous studies have shown that feature selection holds particular significance for this prediction task, making the generation of more discriminative features a key area of interest for many researchers. Recent progress has shown the power of protein language models in handling protein sequences, in leveraging the strengths of attention networks, and in successful applications to tasks such as protein structure prediction. This naturally raises the question of the applicability of protein language models in predicting protein and nucleic acid binding sites. Various approaches have explored this potential. This paper first describes the development of protein language models. Then, a systematic review of the latest methods for predicting protein and nucleic acid binding sites is conducted by covering benchmark sets, feature generation methods, performance comparisons, and feature ablation studies. These comparisons demonstrate the importance of protein language models for the prediction task. Finally, the paper discusses the challenges of protein and nucleic acid binding site prediction and proposes possible research directions and future trends. The purpose of this survey is to furnish researchers with actionable suggestions for comprehending the methodologies used in predicting protein-nucleic acid binding sites, fostering the creation of protein-centric language models, and tackling real-world obstacles encountered in this field.

Brain Nucleic Acid Delivery and Genome Editing via Focused Ultrasound-Mediated Blood-Brain Barrier Opening and Long-Circulating Nanoparticles.

We introduce a two-pronged strategy comprising focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening and long-circulating biodegradable nanoparticles (NPs) for systemic delivery of nucleic acids to the brain. Biodegradable poly(ß-amino ester) polymer-based NPs were engineered to stably package various types of nucleic acid payloads and enable prolonged systemic circulation while retaining excellent serum stability. FUS was applied to a predetermined coordinate within the brain to transiently open the BBB, thereby allowing the systemically administered long-circulating NPs to traverse the BBB and accumulate in the FUS-treated brain region, where plasmid DNA or mRNA payloads produced reporter proteins in astrocytes and neurons. In contrast, poorly circulating and/or serum-unstable NPs, including the lipid NP analogous to a platform used in clinic, were unable to provide efficient nucleic acid delivery to the brain regardless of the BBB-opening FUS. The marriage of FUS-mediated BBB opening and the long-circulating NPs engineered to copackage mRNA encoding CRISPR-associated protein 9 and single-guide RNA resulted in genome editing in astrocytes and neurons precisely in the FUS-treated brain region. The combined delivery strategy provides a versatile means to achieve efficient and site-specific therapeutic nucleic acid delivery to and genome editing in the brain via a systemic route.

Advances in nucleic acid therapeutics: structures, delivery systems, and future perspectives in cancer treatment.

Cancer has emerged as a significant threat to human health. Nucleic acid therapeutics regulate the gene expression process by introducing exogenous nucleic acid fragments, offering new possibilities for tumor remission and even cure. Their mechanism of action and high specificity demonstrate great potential in cancer treatment. However, nucleic acid drugs face challenges such as low stability and limited ability to cross physiological barriers in vivo. To address these issues, various nucleic acid delivery vectors have been developed to enhance the stability and facilitate precise targeted delivery of nucleic acid drugs within the body. In this review article, we primarily introduce the structures and principles of nucleic acid drugs commonly used in cancer therapy, as well as their cellular uptake and intracellular transportation processes. We focus on the various vectors commonly employed in nucleic acid drug delivery, highlighting their research progress and applications in recent years. Furthermore, we propose potential trends and prospects of nucleic acid drugs and their carriers in the future.

Charge Detection Mass Spectrometry Enables Molecular Characterization of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) are increasingly used in preclinical investigations as delivery vectors. Tools that can characterize assembly and assess quality will accelerate their development and clinical translation. Standard techniques used to characterize NANPs, like gel electrophoresis, lack the resolution for precise characterization. Here, we introduce the use of charge detection mass spectrometry (CD-MS) to characterize these materials. Using this technique, we determined the mass of NANPs varying in size, shape, and molecular mass, NANPs varying in production quality due to formulations lacking component oligonucleotides, and NANPs functionalized with protein and nucleic acid-based secondary molecules. Based on these demonstrations, CD-MS is a promising tool to precisely characterize NANPs, enabling more precise assessments of the manufacturing and processing of these materials.

Defective endomembrane dynamics in Rab27a deficiency impairs nucleic acid sensing and cytokine secretion in immune cells.

Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and ß2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo, CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination.

Construction of charge-reversible coordination-crosslinked spherical nucleic acids to deliver dual anti-cancer genes and ferroptosis payloads.

Spherical nucleic acids (SNAs) are nanostructures with the DNA arranged radially on the surface, thus allowing specific binding with cancer cells expressing high levels of scavenger receptor-A to enhance cellular uptake. However, conventional carriers for SNAs are cytotoxic, not degradable and difficult to deliver multiple payloads. In this study, we developed charge-reversible coordination-crosslinked SNAs to deliver dual anti-cancer genes and ferroptosis payload for anti-cancer purposes. To this end, we modified poly(lactic acid) (PLA) with functionalized side chains to allow its binding with antisense oligonucleotides (ASOs) and siRNA, annealed two single-stranded RNAs to obtain double-stranded RNA, and introduced a polyethylene glycol (PEG) shell to enhance the circulation time. Additionally, the ferroptosis payload imidazole was coordinated with iron ions as a core-crosslinked group to enhance the stability of SNAs and efficiency to kill cancer cells. We demonstrated that this novel nanocomplex efficiently internalized and killed CT-26 cells in vitro. In vivo data confirmed that the dual gene delivery system successfully targeted CT-26 tumors in tumor-bearing BALB/c mice, and exhibited strong tumor suppression ability, without inducing adverse toxic effects. Taken together, our dual gene therapy system offered an enhanced anti-tumor solution by simultaneously delivering dual anti-cancer genes and ferroptosis payload in tumor microenvironment.

Programmable Framework Nucleic Acid-Modified Nanomagnetic Beads for Efficient Isolation of Exosomes and Exosomal Proteomics Analysis.

Exosomes are increasingly being regarded as emerging and promising biomarkers for cancer screening, diagnosis, and therapy. The downstream molecular analyses of exosomes were greatly affected by the isolation efficiency from biosamples. Among the current exosome isolation strategies, affinity nanomaterials performed comparably better with selectivity and specificity. However, these techniques did not take the structure and size of exosomes into account, which may lead to a loss of isolation efficiency. In this article, a framework nucleic acid was employed to prepare a well-designed nanosized bead Fe3O4@pGMA@DNA TET@Ti4+ for enrichment of exosomes. The abundant phosphate groups in the framework nucleic acid provide binding sites to immobilize Ti4+, and its rigid three-dimensional skeleton makes them act as roadblocks to barricade exosomes and provide affinity interactions on a three-dimensional scale, resulting in the improvement of isolation efficiency. The model exosomes can be effectively isolated with 92% recovery in 5 min. From 100 µL of HeLa cell culture supernatant, 34 proteins out of the top 100 commonly identified exosomal proteins were identified from the isolated exosomes by the novel beads, which is obviously more than that by TiO2 (19 proteins), indicating higher isolation efficiency and exosome purity by Fe3O4@pGMA@DNA TET@Ti4+ beads. The nanobeads were finally applied for comparing exosomal proteomics analysis from real clinical serum samples. Twenty-five upregulated and 10 downregulated proteins were identified in the lung cancer patients group compared to the health donors group, indicating that the novel nanobeads have great potential in isolation of exosomes for exosomal proteomics analysis in cancer screening and diagnosis.

Nucleic Acid Armor: Fortifying RNA Therapeutics through Delivery and Targeting Innovations for Immunotherapy.

RNA is a promising nucleic acid-based biomolecule for various treatments because of its high efficacy, low toxicity, and the tremendous availability of targeting sequences. Nevertheless, RNA shows instability and has a short half-life in physiological environments such as the bloodstream in the presence of RNAase. Therefore, developing reliable delivery strategies is important for targeting disease sites and maximizing the therapeutic effect of RNA drugs, particularly in the field of immunotherapy. In this mini-review, we highlight two major approaches: (1) delivery vehicles and (2) chemical modifications. Recent advances in delivery vehicles employ nanotechnologies such as lipid-based nanoparticles, viral vectors, and inorganic nanocarriers to precisely target specific cell types to facilitate RNA cellular entry. On the other hand, chemical modification utilizes the alteration of RNA structures via the addition of covalent bonds such as N-acetylgalactosamine or antibodies (antibody-oligonucleotide conjugates) to target specific receptors of cells. The pros and cons of these technologies are enlisted in this review. We aim to review nucleic acid drugs, their delivery systems, targeting strategies, and related chemical modifications. Finally, we express our perspective on the potential combination of RNA-based click chemistry with adoptive cell therapy (e.g., B cells or T cells) to address the issues of short duration and short half-life associated with antibody-oligonucleotide conjugate drugs.

The Application of Microsampling Disks in Circular Dichroism Spectroscopy for Peptide and Nucleic Acid Drugs.

In recent years, there has been a growing focus on the development of medium-sized drugs based on peptides or nucleic acids owing to their potential therapeutic benefits. As some of these medium-sized drugs exert their therapeutic effects by adopting specific secondary structures, evaluating their conformational states is crucial to ensure the efficacy, quality, and safety of the drug products. It is important to assess the structural integrity of biomolecular therapeutics to guarantee their intended pharmacological activity and maintain the required standards for drug development and manufacturing. One widely utilized technique for quality evaluation is secondary structural analysis using circular dichroism (CD) spectroscopy. Given the higher production and quality control costs associated with medium-sized drugs compared with small-molecule drugs, developing analytical techniques that enable CD analysis with reduced sample volumes is highly desirable. Herein, we focused on a microsampling disk-type cell as a potential solution for reducing the required sample volume. We investigated whether CD spectral analysis using a microsampling disk could provide equivalent spectra compared with the standard cell (sample volume: approx. 300 µL). Our findings demonstrated that the microsampling disk (sample volume: 2-10 µL) could be successfully applied to CD spectral analysis of peptide and nucleic acid drugs, paving the way for more efficient and cost-effective quality evaluation processes.

Recent advances in gene delivery nanoplatforms based on spherical nucleic acids.

Gene therapy is a therapeutic option for mitigating diseases that do not respond well to pharmacological therapy. This type of therapy allows for correcting altered and defective genes by transferring nucleic acids to target cells. Notably, achieving a desirable outcome is possible by successfully delivering genetic materials into the cell. In-vivo gene transfer strategies use two major classes of vectors, namely viral and nonviral. Both of these systems have distinct pros and cons, and the choice of a delivery system depends on therapeutic objectives and other considerations. Safe and efficient gene transfer is the main feature of any delivery system. Spherical nucleic acids (SNAs) are nanotechnology-based gene delivery systems (i.e., non-viral vectors). They are three-dimensional structures consisting of a hollow or solid spherical core nanoparticle that is functionalized with a dense and highly organized layer of oligonucleotides. The unique structural features of SNAs confer them a high potency in internalization into various types of tissue and cells, a high stability against nucleases, and efficay in penetrating through various biological barriers (such as the skin, blood-brain barrier, and blood-tumor barrier). SNAs also show negligible toxicity and trigger minimal immune response reactions. During the last two decades, all these favorable physicochemical and biological attributes have made them attractive vehicles for drug and nucleic acid delivery. This article discusses the unique structural properties, types of SNAs, and also optimization mechanisms of SNAs. We also focus on recent advances in the synthesis of gene delivery nanoplatforms based on the SNAs.