Efficient Nucleic Acid Research Articles

Structural and functional characterization of a histidylated liposome for mRNA delivery.

The development of lipid-based mRNA delivery systems has significantly facilitated recent advances in mRNA-based therapeutics. Liposomes, as the pioneering class of mRNA vectors, continue to lead in clinical trials. We previously developed a histidylated liposome that demonstrated efficient nucleic acid delivery. In this study, the liposome preparation process was optimized by freeze-drying followed by extrusion to homogenize size distribution and improve storage stability. A comprehensive characterization of these LYX liposomes was performed, including evaluation in cellular and murine animal models. LYX liposomes can be stored for up to one year at 4°C, maintaining a stable size (150±10nm) and polydispersity index (0.10±0.02), while preserving their transfection efficacy. They exhibit high encapsulation efficacy (∼95%) and protect mRNA from RNase degradation. Lamellar organization was confirmed by Small Angle X-ray Scattering and CryoTEM, and intracellular trafficking was examined using confocal microscopy. LYX-mRNA lipoplexes can transfect both cell lines and primary cells, albeit with a lower transfection efficacy compared to the commercial Lipofectamine MessengerMAX™ vector. Our data suggest that this could be attributed to slower cell uptake and reduced endosomal escape of LYX. LYX liposomes effectively delivered mRNA encoding therapeutic BMP2 and BMP9 molecules, producing significant amounts of functional proteins that successfully induced BMP signaling. In addition, in vivo studies demonstrated the potential of LYX lipoplexes when incorporated into hydrogels and implanted subcutaneously in mice. These findings provided evidence that LYX liposomes are a promising platform for mRNA delivery, offering versatility for multiple applications.

Lab-on-a-Chip Devices for Nucleic Acid Analysis in Food Safety.

Lab-on-a-chip (LOC) devices have been developed for nucleic acid analysis by integrating complex laboratory functions onto a miniaturized chip, enabling rapid, cost-effective, and highly sensitive on-site testing. This review examines the application of LOC technology in food safety, specifically in the context of nucleic acid-based analyses for detecting pathogens and contaminants. We focus on microfluidic-based LOC devices that optimize nucleic acid extraction and purification on the chip or amplification and detection processes based on isothermal amplification and polymerase chain reaction. We also explore advancements in integrated LOC devices that combine nucleic acid extraction, amplification, and detection processes within a single chip to minimize sample preparation time and enhance testing accuracy. The review concludes with insights into future trends, particularly the development of portable LOC technologies for rapid and efficient nucleic acid testing in food safety.

Microfluidic Chip-based Enrichment and Nucleic Acid Extraction for Quantitative Detection of Mycobacterium Smegmatis in Aerosols.

Tuberculosis (TB) is ranked as the third most prevalent infectious disease globally. Early detection and treatment are crucial for effective management. Conventional diagnostic methods primarily rely on sputum samples, which present challenges in accessibility and have limited accuracy in certain populations such as children, individuals with HIV, and those with extrapulmonary TB. To address the need for point-of-care diagnostics, this study introduces a rapid diagnostic approach for TB using exhaled breath aerosol as a more easily obtainable specimen. Mycobacterium smegmatis, a non-pathogenic bacterium genetically similar to Mycobacterium tuberculosis, is used as a surrogate organism. The method involves the use of microfluidic chips for concentrating and electrolyzing mycobacteria in the aerosol, followed by extracting and quantifying nucleic acids using real-time fluorescence quantitative PCR. Notably, successful enrichment and quantification of bacterial content were achieved even at a minimal bacterial aerosol concentration of 104 CFU/mL. The developed chips are characterized by their cost-effectiveness, ease of use, high bacterial enrichment, efficient nucleic acid extraction, and low detection threshold (4.4 × 10-18 mol/L). This innovative approach offers a promising method for early TB screening and opens avenues for the rapid identification of other aerosol-transmitted diseases.

Nucleic acid‐templated chemical reactions for nucleic acid detection

AbstractNucleic acid‐templated reactions are chemical processes driven by the increased effective concentration of reactants on nucleic acids through the sequence‐specific hybridization of nucleic acids. Because these reactions translate the signals of target nucleic acids to detectable specific outputs, such as fluorescence, they can be applied for nucleic acid sensing and imaging. Owing to their advantageous features, such as signal amplification, isothermal nonenzymatic operation, and diverse reaction outputs and designs, the templated reactions have considerable potential for designing next‐generation nucleic acid sensors with high sensitivity, selectivity, rapidity, and user‐friendliness. Thus, over the past two decades, numerous templated reactions have been developed for more efficient nucleic acid detection. This review highlights recent advances in nucleic acid‐templated reactions since 2020, focusing on the newly developed reactions and strategies for designing highly sensitive, selective, and accurate nucleic acid sensing systems. We also summarize templated reaction research since 2015 and explore how integrating these reactions with other signal amplification systems and readout methods has led to the development of practical nucleic acid sensors with improved properties. According to the analysis of each type of templated reactions (ligation, releasing, and transformation), design trends are discussed that inform the outlook for the future development of nucleic acid sensors utilizing templated reactions.

Step Pulse-Mediated Low-Triggering Potential Electrochemiluminescence of Polyfluorene Nanoparticles for Bioassay.

When the electrochemiluminescence (ECL) reaction occurs at a triggering potential beyond ±1.0 V, the interference from the adverse oxidation-reduction reaction cannot be ignored. However, currently reported anode ECL usually occurs above +1.0 V. This study innovatively developed a convenient and simple step pulse (SP) method to modulate the low ECL triggering potential of poly [(9,9-dioctyl-fluorenyl-2,7-diacyl)-alt-co-(9-hexyl-3,6-carbazole)] (PFA) nanoparticles (NPs). Compared to cyclic voltammetry with a triggering potential exceeding +1.25 V for PFA NPs, SP scanning enabled PFA NPs to exhibit a strong and stable ECL emission with a triggering potential as low as +0.75 V and tripropylamine (TPrA) as a coreactant. PFA NPs coupled an efficient aptameric recognition-driven cascade nucleic acid amplification strategy to construct a sensitive biosensing platform for measuring phosphorylated Tau (p-Tau) protein as an Alzheimer's disease biomarker. p-Tau could release the secondary target (ST) chain through the aptameric recognition reaction with the aptamer, and the released ST could further trigger cascade catalytic hairpin assembly (CHA) and rolling circle amplification (RCA) at the PFA NP-modified electrode, producing a large number of long chains. The large amount of G-quadruplex/hemin formed by long chains and hemin will consume the ECL quencher H2O2 added in detection solution, thereby restoring the ECL signal and enabling the low potential quantitative analysis of p-Tau with a limit of detection of 4.15 fg/mL. SP technique provides a new way to reduce ECL triggering potential, and PFA NPs create a promising low-triggering potential ECL-sensing platform for bioanalysis.

B-216 Ethanol/isopropanol-free and enzyme-free universal nucleic acid extraction and purification system for fast molecular detection from swab samples

Abstract Background Efficient nucleic acid (NA) extraction has been made technology advancement in molecular science. However, there are several challenges yet to address. Firstly, ethanol or isopropanol are important components of the buffers for the most commercially available NA extraction kits. When put on cartridge for POC applications, they can react with plastic materials, undermining their mechanical/physiochemical properties. They can also be problematic due to their volatility, flammability and potential to leak. Secondly, turn-around time for most commercial kits is long and procedure is rather tedious. Thirdly, most of the extraction buffers can only be used to extract either DNA or RNA from either viruses or bacteria. A universal buffer system is lacking. Lastly, special components in some extraction buffers require -20°C or 4°C storage for desired performance, especially for those kits containing proteinase for cell lysis and nuclease denaturation and carrier NA for low quantity of template, causing inconvenience in shipping and storage. Delta developed buffers and associated protocol to address the challenges listed above. This study is to evaluate the performance of the Delta TNA buffer system in NA extraction and purification from various target pathogens in swab samples when compared to that of the leading commercial products. Methods The stock strains of staphylococcus aureus, pseudomonas aeruginosa and H-coronavirus OC43 were diluted in Delta Molecular Transport Media containing swab sample matrix to the testing concentrations before performing NA extraction. Vircell SARS-CoV-2 RNA was spiked directly into PCR reactions to generate a standard curve with 10x serial dilutions for the recovery rate test using Delta TNA kit. 300ul of samples were used for the NA extraction and purification with 80 ul eluate generated. The manufacturers’ instructions were followed. The extracted RNA or DNA was amplified on BioRAD CFX96. Results The extraction efficiency on RNA from H-coronavirus OC43, DNA from pseudomonas aeruginosa and staphylococcus aureus in swab samples, was compared with Katsura Respi and MN Pathogen kits, Qiagen DNA and viral RNA kits respectively. Delta TNA kit outperformed in either earlier Cq value or higher detection rate for different concentrations of analytes in <10 min comparing to up to 95 min for the commercial kits tested. The RNA recovery rate of Delta TNA kit was comparable to that of the Qiagen Viral Kit with 37.54% vs 29.84% at 10 copies/rxn, 146.84% vs 140.76% at 100 cpies/rxn and 118.29% vs 111.76% at 1000 copies/rxn. Meanwhile, Delta Swab TNA buffer was compatible with large spectrum of magnetic beads possessing various coating groups and particle sizes. Conclusions We provided a stable, user- and environment-friendly total nucleic acid extraction and purification system which is ethanol/isopropanol-free and enzyme-free. There is no pre-treatment or drying steps in the protocol and no centrifugation required. Delta’s protocol has the least total number of steps and least amount of total time taken amongst the kits compared. It serves as a universal recipe for both DNA and RNA from pathogens in swab samples including viruses and bacteria in less than 10 min, with competitive performance in both PCR and LAMP.

A rapid visualization method for detecting rotavirus A by combining nuclear acid sequence-based amplification with the CRISPR-Cas12a assay.

Introduction. Rotavirus A is the most common pathogen causing diarrhoea in children less than 5 years, leading to severe complications such as dehydration, electrolyte imbalances, acidosis, myocarditis, convulsions, pneumonia, and other life-threatening conditions.Gap statement. There is an urgent need for a rapid and efficient nucleic acid detection strategy to enable early diagnosis and treatment, preventing rotavirus transmission and associated complications.Aim. This article aimed to develop a nuclear acid sequence-based amplification (NASBA)-Cas12a system for detecting rotavirus A using fluorescence intensity or lateral flow strips.Methodology. The NASBA technology was combined with the clustered regularly interspaced short palindromic repeats-Cas12a system to establish a NASBA-Cas12a system for detecting rotavirus A.Results. The NASBA-Cas12a system could detect rotavirus A at 37 ℃ within 70 min and had no cross-reactivity with other viruses, achieving a limit of detection of 1.2 copies μl-1. This system demonstrated a sensitivity of 100%, specificity of 90%, positive predictive value of 97.22% and negative predictive value of 100%. The kappa value was 0.933, indicating that the NASBA-Cas12a system was highly consistent with reverse transcription-PCR.Conclusion. The NASBA-Cas12a system exhibited high sensitivity and specificity for detecting rotavirus A, showing great potential for clinical application.

FRAME: flap endonuclease 1-engineered PAM module for precise and sensitive modulation of CRISPR/Cas12a trans-cleavage activity.

CRISPR/Cas12a system, renowned for its precise recognition and efficient nucleic acid cleavage capabilities, has demonstrated remarkable performance in molecular diagnostics and biosensing. However, the reported Cas12a activity regulation methods often involved intricate CRISPR RNA (crRNA) structural adjustments or costly chemical modifications, which limited their applications. Here, we demonstrated a unique enzyme activity engineering strategy using flap endonuclease 1 (FEN1) to regulate the accessibility of the protospaceradjacent motif (PAM) module in the double-stranded DNA activator (FRAME). By identifying the three-base overlapping structure between the target inputs and substrate, FEN1 selectively cleaved and released the 5'-flap containing the 'TTTN' sequence, which triggered the secondary cleavage of FEN1 while forming a nicked PAM, ultimately achieving the sensitive switching of Cas12a's activity. The FRAME strategy exemplified the 'two birds with one stone' principle, as it not only precisely programmed Cas12a's activity but also simultaneously triggered isothermal cyclic amplification. Moreover, the FRAME strategy was applied to construct a sensing platform for detecting myeloperoxidase and miR-155, which demonstrated high sensitivity and specificity. Importantly, it proved its versatility in detecting multiple targets using a single crRNA without redesign. Collectively, the FRAME strategy opens up a novel avenue for modulating Cas12a's activity, promising immense potential in the realm of medical diagnostics.

Highly efficient nucleic acid encapsulation method for targeted gene therapy using antibody conjugation system

Gene therapy has surfaced as a promising avenue for treating cancers, offering the advantage of deliberate adjustment of targeted genes. Nonetheless, the swift degradation of nucleic acids in the bloodstream necessitates an effective and secure delivery system. The widespread utilization of poly lactic-co-glycolic acid (PLGA) nanoparticles as drug delivery systems has highlighted challenges in controlling particle size and release properties. Moreover, the encapsulation of nucleic acids exacerbates these difficulties due to the negatively charged surface of PLGA nanoparticles. In this study, we aimed to improve the encapsulation efficiency of nucleic acids by employing negatively charged microbeads and optimizing the timing of the specific formulation steps. Furthermore, by conjugating PSMA-617, a ligand for the prostate-specific membrane antigen (PSMA), with PLGA nanoparticles, we assessed the antitumor effects and the efficacy of a nucleic acid delivery system on prostate cancer model. The employed technique within the nucleic acid encapsulation system represents a novel approach that could be adapted to encapsulate various kinds of nucleic acids. Moreover, it enables the attachment of targeting moieties to different cell membrane proteins, thereby unveiling new prospects for precise therapeutics in cancer therapy.

Development of polymerase chain reaction-lateral flow dipstick assay for detection of Mycoplasma bovis in cattle

Mycoplasma bovis (M. bovis) is capable of causing a range of diseases in cattle, encompassing calf pneumonia, arthritis, conjunctivitis, meningitis, and mastitis. It is widely recognized as one of the predominant pathogens posing a significant threat to the global cattle industry. Therefore, accurate and sensitive methods are urgently needed to detect M. bovis. This study aims to detect M. bovis by combining colloidal gold with biotin-labeled oligonucleotides to improve detection sensitivity and form a chromogenic detection probe based on signal amplification technology. Here, we developed a sensitive and specific polymerase chain reaction-lateral flow dipstick assay (PCR-LFD) strip for efficient nucleic acid detection of M. bovis. A pair of specific primers with 5’ ends labeled with biotin and digoxigenin probes was designed for PCR experiments. Colloidal gold particles-labeled anti-digoxigenin IgG coated gold-labeled test strip was prepared, streptavidin was used as the detection probe, and nitrocellulose membrane coated goat anti-mouse IgG was used as the control line. Our results showed that the detection limit of the PCR-LFD was 89 fg/µL for the M. bovis DNA. The results from the test strip were highly consistent with those from real-time qPCR. This assay were highly specific for M. bovis, as there were no cross-reactions with other microorganisms tested and the detection sensitivity of the test was also relatively high (97.67%). The novel strips present a promising tool for the cost-effective and sensitive diagnosis of M. bovis.

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(β-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.

Programmable Lanthanide Metal-Organic Framework for Ultra-Efficient Nucleic Acids Extraction and Interaction Analysis.

Efficient, mild, and reversible adsorption of nucleic acids onto nanomaterials represents a promising analytical approach for medical diagnosis. However, there is a scarcity of efficient and reversible nucleic acid adsorption nanomaterials. Additionally, the lack of comprehension of the molecular mechanisms governing their interactions poses significant challenges. These issues hinder the rational design and analytical applications of the nanomaterials. Herein, we propose an ultra-efficient nucleic acid affinity nanomaterial based on programmable lanthanide metal-organic frameworks (Ln-MOFs). Through experiments and density functional theory calculations, a rational design guideline for nucleic acid affinity of Ln-MOF was proposed, and a modular and flexible preparation scheme was provided. Then, Er-TPA (terephthalic acid) MOF emerged as the optimal candidate due to its pore size-independent adsorption and desorption capabilities for nucleic acids, enabling ultra-efficient adsorption (about 150% mass ratio) within 1 min. Furthermore, we elucidate the molecular-level mechanisms underlying the Ln-MOF adsorption of single- and double-stranded DNA and G4 structures. The affinity nanomaterial based on Ln-MOF exhibits robust nucleic acid extraction capability (4-fold higher than commercial reagent kits) and enables mild and reversible CRISPR/Cas9 functional regulation. This method holds significant promise for broad application in DNA/RNA liquid biopsy and gene editing, facilitating breakthroughs in analytical chemistry, pharmacy, and medical research.

Membrane Fusion-Mediated Loading of Therapeutic siRNA into Exosome for Tissue-Specific Application.

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Module-combinatorial design and screening of multifunctional polymers based on polyaspartic acid for DNA delivery

It is crucial to develop non-viral gene vectors that can efficiently and safely transfect plasmid DNA into cells. Low transfection efficiency and high cytotoxicity of cationic polymers hinder their application as gene carriers. Modification of cationic polymers has emerged as an attractive strategy for efficient and safe nucleic acids delivery. In this study, a simple and rapid method is developed to synthesize a series of multifunctional polymers by utilizing biodegradable polyaspartic acid as the backbone and modifying it with three modules. This one-component polymer possesses capabilities for nucleic acid condensation, cellular uptake, and endosomal escape. Polymers containing imidazole, triazole, or pyridine group exhibited promising transfection activity. Substituted with dodecylamine or 2-hexyldecan-1-amine enhance cellular uptake and subsequent transfection. Furthermore, the influence of ionizable amine side chains on gene delivery is investigated. Two optimal polymers, combined with the avian encephalomyelitis virus (AEV) plasmid vaccine, induced robust specific antibody responses and cellular immune responses in mice and chickens. Through module-combination design and screening of polyaspartamide polymers, this study presents a paradigm for the development of gene delivery vectors.

Rapid fluorescent nucleic acid sensing with ultra-thin gold nanosheets

Fluorescently labeled DNA oligonucleotides and gold nanospheres have been frequently utilized in biosensors, providing efficient nucleic acid detection. Nevertheless, the restricted loading capacity of gold nanospheres undermines overall sensitivity. In this study, we employed four-atom-thick ultrathin gold nanosheets (AuNSs), utilizing a “pre-mix model” for rapid target nucleic acid detection. In this approach, fluorescently labeled DNA probes were pre-incubated with the target nucleic acid, followed by the addition of AuNSs for probe adsorption and fluorescence quenching. With the developed method, we efficiently and rapidly detected the SARS-CoV-2 N gene sequence within 30 min, involving a brief 15-min target pre-incubation and a subsequent 15-min adsorption of free probes and fluorescence quenching by AuNSs. This method exhibited heightened sensitivity compared to gold nanospheres, boasting a limit of detection (LOD) of 0.808 nM. Furthermore, exceptional recovery was achieved in simulated biological samples. The study introduces an effective strategy for nucleic acid sensing characterized by rapidity, heightened sensitivity, ease of operation, and robustness. These findings encourage further development of rapid biomarker sensing methods employing 2D nanomaterials.

Development of the 12-Base Short Dimeric Myogenetic Oligodeoxynucleotide That Induces Myogenic Differentiation.

A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5'-AGA TTA GGG TGA GGG TGA-3'), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and induces myogenic differentiation, which is expected to be a nucleic acid drug for the prevention of disease-associated muscle wasting. To improve the drug efficacy and synthesis cost of myoDN, shortening the sequence while maintaining its structure-based function is a major challenge. Here, we report the novel 12-base non-telomeric myoDN, iMyo01 (5'-TTG GGT GGG GAA-3'), which has comparable myogenic activity to iSN04. iMyo01 as well as iSN04 promoted myotube formation of primary-cultured human myoblasts with upregulation of myogenic gene expression. Both iMyo01 and iSN04 interacted with nucleolin, but iMyo01 did not bind to berberine, the isoquinoline alkaloid that stabilizes iSN04. Nuclear magnetic resonance revealed that iMyo01 forms a G-quadruplex structure despite its short sequence. Native polyacrylamide gel electrophoresis and a computational molecular dynamics simulation indicated that iMyo01 forms a homodimer to generate a G-quadruplex. These results provide new insights into the aptamer truncation technology that preserves aptamer conformation and bioactivity for the development of efficient nucleic acid drugs.

A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 Tcells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Kinetics of RNA-LNP delivery and protein expression

Lipid nanoparticles (LNPs) employing ionizable lipids are the most advanced technology for delivery of RNA, most notably mRNA, to cells. LNPs represent well-defined core–shell particles with efficient nucleic acid encapsulation, low immunogenicity and enhanced efficacy. While much is known about the structure and activity of LNPs, less attention is given to the timing of LNP uptake, cytosolic transfer and protein expression. However, LNP kinetics is a key factor determining delivery efficiency. Hence quantitative insight into the multi-cascaded pathway of LNPs is of interest to elucidate the mechanism of delivery. Here, we review experiments as well as theoretical modeling of the timing of LNP uptake, mRNA-release and protein expression. We describe LNP delivery as a sequence of stochastic transfer processes and review a mathematical model of subsequent protein translation from mRNA. We compile probabilities and numbers obtained from time resolved microscopy. Specifically, live-cell imaging on single cell arrays (LISCA) allows for high-throughput acquisition of thousands of individual GFP reporter expression time courses. The traces yield the distribution of mRNA life-times, expression rates and expression onset. Correlation analysis reveals an inverse dependence of gene expression efficiency and transfection onset-times. Finally, we discuss why timing of mRNA release is critical in the context of codelivery of multiple nucleic acid species as in the case of mRNA co-expression or CRISPR/Cas gene editing.

Biporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection

Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.

Synthesis of Amino Acid-Based Cationic Lipids and Study of the Role of the Cationic Head Group for Enhanced Drug and Nucleic Acid Delivery.

Leveraging liposomes for drug and nucleic acid delivery, though promising due to reduced toxicity and ease of preparation, faces challenges in stability and efficiency. To address this, we synthesized cationic amphiphiles from amino acids (arginine, lysine, and histidine). Histidine emerged as the superior candidate, leading to the development of three histidine-rich cationic amphiphiles for liposomes. Using the hydration method, we have prepared the liposomes and determined the optimal N/P ratios for lipoplex formation via gel electrophoresis. In vitro transfection assays compared the efficacy of our lipids to Fugene, while MTT assays gauged biocompatibility across cancer cell lines (MDA-MB 231 and MCF-7). The histidine-based lipid demonstrated marked potential in enhancing drug and nucleic acid delivery. This improvement stemmed from increased zeta potential, enhancing electrostatic interactions with nucleic acids and cellular uptake. Our findings underscore histidine's crucial role over lysine and arginine for effective delivery, revealing a significant correlation between histidine abundance and optimal performance. This study paves the way for histidine-enriched lipids as promising candidates for efficient drug and nucleic acid delivery, addressing key challenges in the field.