[Application of the CRISPR/Cas system in gene editing and nucleic acid detection of parasitic diseases: a review].

CRISPR/Cas system, an adaptive immune system with clustered regularly interspaced short palindromic repeats, may interfere with exogenous nucleic acids and protect prokaryotes from external damages, is an effective gene editing and nucleic acid detection tools. The CRISPR/Cas system has been widely applied in virology and bacteriology; however, there is relatively less knowledge about the application of the CRISPR/Cas system in parasitic diseases. The review summarizes the mechanisms of action of the CRISPR/Cas system and provides a comprehensive overview of their application in gene editing and nucleic acid detection of parasitic diseases, so as to provide insights into future studies on parasitic diseases.

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.

Force Field X: A computational microscope to study genetic variation and organic crystals using theory and experiment.

Force Field X (FFX) is an open-source software package for atomic resolution modeling of genetic variants and organic crystals that leverages advanced potential energy functions and experimental data. FFX currently consists of nine modular packages with novel algorithms that include global optimization via a many-body expansion, acid-base chemistry using polarizable constant-pH molecular dynamics, estimation of free energy differences, generalized Kirkwood implicit solvent models, and many more. Applications of FFX focus on the use and development of a crystal structure prediction pipeline, biomolecular structure refinement against experimental datasets, and estimation of the thermodynamic effects of genetic variants on both proteins and nucleic acids. The use of Parallel Java and OpenMM combines to offer shared memory, message passing, and graphics processing unit parallelization for high performance simulations. Overall, the FFX platform serves as a computational microscope to study systems ranging from organic crystals to solvated biomolecular systems.

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.

Nucleic Acid Target Sensing Using a Vibrating Sharp-Tip Capillary and Digital Droplet Loop-Mediated Isothermal Amplification (ddLAMP).

Nucleic acid tests are key tools for the detection and diagnosis of many diseases. In many cases, the amplification of the nucleic acids is required to reach a detectable level. To make nucleic acid amplification tests more accessible to a point-of-care (POC) setting, isothermal amplification can be performed with a simple heating source. Although these tests are being performed in bulk reactions, the quantification is not as accurate as it would be with digital amplification. Here, we introduce the use of the vibrating sharp-tip capillary for a simple and portable system for tunable on-demand droplet generation. Because of the large range of droplet sizes possible and the tunability of the vibrating sharp-tip capillary, a high dynamic range (~2 to 6000 copies/µL) digital droplet loop-mediated isothermal amplification (ddLAMP) system has been developed. It was also noted that by changing the type of capillary on the vibrating sharp-tip capillary, the same mechanism can be used for simple and portable DNA fragmentation. With the incorporation of these elements, the present work paves the way for achieving digital nucleic acid tests in a POC setting with limited resources.

Self-Assembly of Ultrasmall 3D Architectures of (l)-Acyclic Threoninol Nucleic Acids with High Thermal and Serum Stability.

The primary challenge of implementing DNA nanostructures in biomedical applications lies in their vulnerability to nuclease degradation and variations in ionic strength. Furthermore, the size minimization of DNA and RNA nanostructures is limited by the stability of the DNA and RNA duplexes. This study presents a solution to these problems through the use of acyclic (l)-threoninol nucleic acid (aTNA), an artificial acyclic nucleic acid, which offers enhanced resilience under physiological conditions. The high stability of homo aTNA duplexes enables the design of durable nanostructures with dimensions below 5 nm, previously unattainable due to the inherent instability of DNA structures. The assembly of a stable aTNA-based 3D cube and pyramid that involves an i-motif formation is demonstrated. In particular, the cube outperforms its DNA-based counterparts in terms of stability. We furthermore demonstrate the successful attachment of a nanobody to the aTNA cube using the favorable triplex formation of aTNA with ssDNA. The selective in vitro binding capability to human epidermal growth factor receptor 2 is demonstrated. The presented research presents the use of aTNA for the creation of smaller durable nanostructures for future medical applications. It also introduces a new method for attaching payloads to these structures, enhancing their utility in targeted therapies.

A Visual Compendium of Principal Modifications within the Nucleic Acid Sugar Phosphate Backbone.

Nucleic acid chemistry is a huge research area that has received new impetus due to the recent explosive success of oligonucleotide therapy. In order for an oligonucleotide to become clinically effective, its monomeric parts are subjected to modifications. Although a large number of redesigned natural nucleic acids have been proposed in recent years, the vast majority of them are combinations of simple modifications proposed over the past 50 years. This review is devoted to the main modifications of the sugar phosphate backbone of natural nucleic acids known to date. Here, we propose a systematization of existing knowledge about modifications of nucleic acid monomers and an acceptable classification from the point of view of chemical logic. The visual representation is intended to inspire researchers to create a new type of modification or an original combination of known modifications that will produce unique oligonucleotides with valuable characteristics.

A Repurposed Drug Interferes with Nucleic Acid to Inhibit the Dual Activities of Coronavirus Nsp13.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.

Suppression of Ehrlich ascites tumor cell proliferation via G1 arrest induced by dietary nucleic acid-derived nucleosides.

The nucleic acids found in food play a crucial role in maintaining various bodily functions. This study investigated the potential anticancer effects of dietary nucleic acids, an area that is still not fully understood. By utilizing an in vivo mouse model and an in vitro cell model, we discovered an anti-proliferative impact of RNA in both systems. DNA exhibited anti-proliferative effects in the mouse model, while this phenomenon wasn't observed in the in vitro cell model using Ehrlich ascites tumor (EAT) cells. Conversely, DNA hydrolysate demonstrated distinct anti-proliferative effects in EAT cells, suggesting that nucleotides or nucleosides generated during nucleic acid digestion act as active constituents. Furthermore, we examined various nucleosides and two sodium-independent equilibrative nucleoside transporter inhibitors (ENTs), identifying guanosine and 2'-deoxyguanosine as pivotal in the anti-proliferative effect. We also found that the anti-proliferation activity with both nucleosides was suppressed by the treatment of dipyridamole, a non-selective inhibitor for ENT1 and ENT2, but not nitrobenzylthioinosine, a low inhibitor for ENT2. The uptake of these compounds into cells is likely facilitated by ENT2. These nucleotides impeded the progression of cancer cells from the G1 phase to the S phase in the cell cycle. Another significant finding is the increased expression of CCAAT/enhancer-binding protein (C/EBPß) induced by guanosine and 2'-deoxyguanosine. Furthermore, immunostaining revealed that C/EBPß diffuses into the nucleus, indicating its presence. This suggests that guanosine or 2-deoxyguanosine induces G1 arrest in cancer cells via the activation of C/EBPß. Encouraged by these promising results, guanosine and 2'-deoxyguanosine show potential applications in cancer prevention.

Wonderful World of Nucleic Acids.

Wonderful World of Nucleic Acids.

Measuring Interactions Between Proteins and Small Molecules or Nucleic Acids.

Interactions between proteins and small molecules or nucleic acids play a pivotal role in numerous biological processes critical for human health and are fundamental for advancing our understanding of biological systems. Proteins are the workhorses of the cell, executing various functions ranging from catalyzing biochemical reactions to transmitting signals within the body. Small molecules, including drugs and metabolites, can modulate protein activity, thereby impacting cellular processes and disease pathways. Similarly, nucleic acids, such as DNA and RNA, regulate protein synthesis and function through intricate interactions. Understanding these interactions is crucial for drug discovery and development and can shed light on gene regulation, transcriptional control, and RNA processing, providing insights into genetic diseases and developmental disorders. Moreover, studying protein-small molecule and protein-nucleic acid interactions enhances our comprehension of fundamental biological mechanisms. A wide array of methods to study these interactions range in cost, sensitivity, materials usage, throughput, and complexity. Notably in the last decade, new techniques have been developed that enhance our understanding of these interactions. In this review, we aim to summarize the new state-of-the-art methods for detecting interactions between proteins and small molecules or nucleic acids, as well as discuss older methods that still hold value today. © 2024 Wiley Periodicals LLC.

Heterogeneous Integration of Acoustic Microextraction with an Optoelectronic Sensor on Glass for Nucleic Acid Testing.

Lab-on-a-chip systems (LOCs), characterized by their high sensitivity, low sample consumption, and portability, have significantly advanced the field of on-site testing. Despite the evolution of integrated LOCs from qualitative to quantitative analyses, on-chip full integration of sample preparation, purification, and multiplexed detection remains a challenge. Here, we propose a strategy for the heterogeneous integration of a set of complementary metal oxide semiconductor-compatible devices including acoustic resonator, thin-film resistors, and temperature/photosensors as a new type of LOC for nucleic acid testing (NAT). Programmed acoustic streaming-based particles and fluid manipulations largely simplify the nucleic acid extraction process including cell lysis, nucleic acid capture, and elution. The design of the acoustic microextraction module and extraction process was thoroughly studied. Benefitted by the microelectromechanical system approach, the conventional mechanical actions and complex flow control are avoided, which enables a compact hand-held NAT instrument without complicated peripherals. Validation experiments conducted on plasma-harboring mutations in the epidermal growth factor receptor (EGFR) gene confirmed the robustness of the system, achieving an impressive nucleic acid (NA) extraction efficiency of approximately 90% within 5 min and a limit of detection of the target NA in the plasma of 1 copy/µL.

Evaluating Cationic Nanoparticles as Carriers of Antiviral Nucleic Acids.

Nanoparticle carriers enable the multivalent delivery of nucleic acids to cells and protect them from degradation. In this chapter, we present a comprehensive overview of four methodologies: electrophoretic mobility shift assay (EMSA), alamarBlue/CFDA-AM cell viability dyes, fluorescence microscopy, and antiviral assays, which collectively are tools to explore interactions between nucleic acids and nanoparticles, and their biological efficacy. These assays provide insights into binding potential, cytotoxicity, and antiviral efficacy of nucleic acid-based nanoparticle treatments furthering the development of effective antiviral therapeutics.

Nucleic Acid Sensor-Mediated PANoptosis in Viral Infection.

Innate immunity, the first line of host defense against viral infections, recognizes viral components through different pattern-recognition receptors. Nucleic acids derived from viruses are mainly recognized by Toll-like receptors, nucleotide-binding domain leucine-rich repeat-containing receptors, absent in melanoma 2-like receptors, and cytosolic DNA sensors (e.g., Z-DNA-binding protein 1 and cyclic GMP-AMP synthase). Different types of nucleic acid sensors can recognize specific viruses due to their unique structures. PANoptosis is a unique form of inflammatory cell death pathway that is triggered by innate immune sensors and driven by caspases and receptor-interacting serine/threonine kinases through PANoptosome complexes. Nucleic acid sensors (e.g., Z-DNA-binding protein 1 and absent in melanoma 2) not only detect viruses, but also mediate PANoptosis through providing scaffold for the assembly of PANoptosomes. This review summarizes the structures of different nucleic acid sensors, discusses their roles in viral infections by driving PANoptosis, and highlights the crosstalk between different nucleic acid sensors. It also underscores the promising prospect of manipulating nucleic acid sensors as a therapeutic approach for viral infections.

Design and synthesis of nucleic acid nano-environment interactome-targeting small molecule PROTACs and their anticancer activity.

Targeted protein degradation through PROteolysis TArgeting Chimeras (PROTACs) is a relatively new modality in cellular interventions. The minimum requirement for PROTACs to function is forming a tertiary complex of the protein of interest (POI), E3 ligase, and the molecular glue PROTAC. Here, we propose a new approach to modulate the nano-environment interactome of a non-protein target through a plausible quaternary complex of interactome-biomolecule of interest (BOI)-PROTAC and E3 ligase. We report nucleic acid-targeting PROTAC (NA-TAC) molecules by conjugating DNA-binding and E3 ligase ligands. We demonstrate that NA-TACs can target the G-quadruplex DNA and induce elevated DNA damage and cytotoxicity compared to the conventional G-quadruplex binding ligands. Our new class of NA-TACs lays the foundation for small molecule-based non-protein targeting PROTACs for interactome and nanoenvironment mapping and nucleic acid-targeted precision medicines.

Recent Advances in and Application of Fluorescent Microspheres for Multiple Nucleic Acid Detection.

Traditional single nucleic acid assays can only detect one target while multiple nucleic acid assays can detect multiple targets simultaneously, providing comprehensive and accurate information. Fluorescent microspheres in multiplexed nucleic acid detection offer high sensitivity, specificity, multiplexing, flexibility, and scalability advantages, enabling precise, real-time results and supporting clinical diagnosis and research. However, multiplexed assays face challenges like complexity, costs, and sample handling issues. The review explores the recent advancements and applications of fluorescent microspheres in multiple nucleic acid detection. It discusses the versatility of fluorescent microspheres in various fields, such as disease diagnosis, drug screening, and personalized medicine. The review highlights the possibility of adjusting the performance of fluorescent microspheres by modifying concentrations and carrier forms, allowing for tailored applications. It emphasizes the potential of fluorescent microsphere technology in revolutionizing nucleic acid detection and advancing health, disease treatment, and medical research.

High-content tailoring strategy to improve the multifunctionality of functional nucleic acids.

Functional nucleic acids (FNAs) have attracted increasing attention in recent years due to their diverse physiological functions. The understanding of their conformational recognition mechanisms has advanced through nucleic acid tailoring strategies and sequence optimization. With the development of the FNA tailoring techniques, they have become a methodological guide for nucleic acid repurposing. Therefore, it is necessary to systematize the relationship between FNA tailoring strategies and the development of nucleic acid multifunctionality. This review systematically categorizes eight types of FNA multifunctionality, and introduces the traditional FNA tailoring strategy from five aspects, including deletion, substitution, splitting, fusion and elongation. Based on the current state of FNA modification, a new generation of FNA tailoring strategy, called the high-content tailoring strategy, was unprecedentedly proposed to improve FNA multifunctionality. In addition, the multiple applications of rational tailoring-driven FNA performance enhancement in various fields were comprehensively summarized. The limitations and potential of FNA tailoring and repurposing in the future are also explored in this review. In summary, this review introduces a novel tailoring theory, systematically summarizes eight FNA performance enhancements, and provides a systematic overview of tailoring applications across all categories of FNAs. The high-content tailoring strategy is expected to expand the application scenarios of FNAs in biosensing, biomedicine and materials science, thus promoting the synergistic development of various fields.

Split T7 switch-mediated cell-free protein synthesis system for detecting target nucleic acids.

Cell-free protein synthesis (CFPS) reactions can be used to detect nucleic acids. However, most CFPS systems rely on a toehold switch and exhibit the following critical limitations: (i) off-target signals due to leaky translation in the absence of target nucleic acids, (ii) a suboptimal detection limit of approximately 30 nM without pre-amplification, and (iii) labor-intensive screening processes due to sequence constraints for the target nucleic acids. To overcome these shortcomings, we developed a new split T7 switch-mediated CFPS system in which the split T7 promoter was applied to a three-way junction structure to selectively initiate transcription-translation only in the presence of target nucleic acids. Both fluorescence and colorimetric detection systems were constructed by employing different reporter proteins. Notably, we introduced the self-complementation of split fluorescent proteins to streamline preparation of the proposed system, enabling versatile applications. Operation of this one-pot approach under isothermal conditions enabled the detection of target nucleic acids at concentrations as low as 10 pM, representing more than a thousand times improvement over previous toehold switch-based approaches. Furthermore, the proposed system demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins encoded in the expression region. By eliminating issues associated with the previous toehold switch system, our split T7 switch-mediated CFPS system could become a core platform for detecting various target nucleic acids.

Multiplexed Detection Strategies for Biosensors Based on the CRISPR-Cas System.

A growing number of applications require simultaneous detection of multiplexed nucleic acid targets in a single reaction, which enables higher information density in combination with reduced assay time and cost. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-Cas system have broad applications for the detection of nucleic acids due to their strong specificity, high sensitivity, and excellent programmability. However, realizing multiplexed detection is still challenging for the CRISPR-Cas system due to the nonspecific collateral cleavage activity, limited signal reporting strategies, and possible cross-reactions. In this review, we summarize the principles, strategies, and features of multiplexed detection based on the CRISPR-Cas system and further discuss the challenges and perspective.

Tetranuclear Polypyridylruthenium(II) Complexes as Selective Nucleic Acid Stains for Flow Cytometric Analysis of Monocytic and Epithelial Lung Carcinoma Large Extracellular Vesicles.

Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.