Clustered regularly interspaced short palindromic repeats (CRISPR) have established itself as a frontier technology in genetic engineering. Researchers have successfully used the CRISPR/Cas system as precise gene editing tools and have further expanded their scope beyond both imaging and diagnostic applications. The most prominent utility of CRISPR is its capacity for gene therapy, serving as the contemporary, disease-modifying drug at the genetic level of human medical disorders. Correcting these diseases using CRISPR-based gene editing has developed to the extent of preclinical trials and possible patient treatments. A major impediment in actualizing this is the complications associated with in vivo delivery of the CRISPR/Cas complex. Currently, only the viral vectors (e.g., lentivirus) and non-viral encapsulation (e.g., lipid particles, polymer-based, and gold nanoparticles) techniques have been extensively reviewed, neglecting the efficiency of direct delivery. However, the direct delivery of CRISPR/Cas for in vivo gene editing therapies is an intricate process with numerous drawbacks. Hence, this paper discusses in detail both the need and the strategies that can potentially improve the direct delivery aspects of CRISPR/Cas biomolecules for gene therapy of human diseases. Here, we focus on enhancing the molecular and functional features of the CRISPR/Cas system for targeted in vivo delivery such as on-site localization, internalization, reduced immunogenicity, and better in vivo stability. We additionally emphasize the CRISPR/Cas complex as a multifaceted, biomolecular vehicle for co-delivery with therapeutic agents in targeted disease treatments. The delivery formats of efficient CRISPR/Cas systems for human gene editing are also briefly elaborated.
Aptamers are a class of single-stranded (ss) nucleic acid molecules generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that involves iterations of time-consuming and tedious selection, amplification, and enrichment steps. To compensate for the drawbacks of conventional SELEX, we have devised an in-silico methodology that facilitates a cost-effective and facile manner of aptamer selection. Here, we report the isolation of DNA aptamers against androgen receptors (ARs) using androgen response elements (ARE) that possess natural affinity toward AR. A virtual library of ARE sequences was prepared and subjected to a stringent selection criterion to generate a sequence pool having stable hairpin conformations and high GC content. The 3D-structures of the selected ss AREs were modeled and screened through rigid docking and molecular dynamic (MD) simulation to examine their potency as potential AR binders. The predicted sequences were further validated using direct enzyme-linked aptasorbent assay (ELASA), which includes the measurement of their binding affinity, specificity, and target discrimination properties under complex biological enviroments. A short, 15 nucleotides (nts), ssDNA aptamer, termed ARapt1 with the estimated Kd value of 5.5 ± 3 nm, was chosen as the most prominent aptamer against AR based on the coherence of both the in-silico and in-vitro evaluation results. The high target-binding affinity and selectivity of ARapt1 signify its potential use as a versatile tool in diagnostic applications relevant to prostate cancer and related diseases.
Aptamers are a class of folded nucleic acid strands capable of binding to different target molecules with high affinity and selectivity. Over the years, they have gained a substantial amount of interest as promising molecular tools for numerous medical applications, particularly in targeted therapeutics. However, only the different treatment approaches and current developments of aptamer-drug therapies have been discussed so far, ignoring the crucial technical and functional aspects of constructing a therapeutically effective aptamer-driven drug delivery system that translates to improved in-vivo performance. Hence, this paper provides a comprehensive review of the strategies used to improve the therapeutic performance of aptamer-guided delivery systems. We focus on the different functional features such as drug deployment, payload capacity, in-vivo stability and targeting efficiency to further our knowledge in enhancing the cell-specific delivery of aptamer-drug conjugates. Each reported strategy is critically discussed to emphasize both the benefits provided in comparison with other similar techniques and to outline their potential drawbacks with respect to the molecular properties of the aptamers, the drug and the system to be designed. The molecular architecture and design considerations for an efficient aptamer-based delivery system are also briefly elaborated.
The creation of nanostructure is profound for the generation of nanobiosensors in several medical diagnosis. Here, we employed an aqueous hydrothermal route using Zinc-oxide (ZnO) and Gold (Au), which under optimal conditions formed an ultra-crystalline rose-like nanostructure textured with nanowires on the surface, coined as "spiked nanorosette." The spiked nanorosette structures was further characterized to possess crystallites of ZnO and Au grains with average sizes of 27.60 and 32.33 nm, respectively. The intensity for both ZnO (002) and Au (111) planes of the nanocomposite was inferred to be controlled by fine-tuning the percentage of Au nanoparticles doped in the ZnO/Au matrix, as referred by X-ray diffraction analysis. The formation of ZnO/Au-hybrid nanorosettes were additionally verified by the distinct corresponding peaks from photoluminescence and X-ray photoelectron spectroscopy, supported by electrical validations. The biorecognition properties of the spiked nanorosettes were also examined using custom targeted and non-target DNA sequences. The DNA targeting capabilities of the nanostructures were analyzed by Fourier Transform Infrared and electrochemical impedance spectroscopy. The fabricated nanowire-embedded nanorosette exhibited a detection limit at the lower picomolar range of 1 × 10-12 M, with high selectivity, stability and reproducibility and good linearity, under optimal conditions. Impedance-based techniques are more sensitive to the detection of nucleic acid molecule whereas this novel spiked nanorosette demonstrate promising attributes as excellent nanostructures for nanobiosensor developments and their potential future application for nucleic-acids or disease diagnostics.
The sudden global crisis of COVID-19, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demands swift containment measures due to its rapid spread and numerous problematic mutations, which complicate the establishment of herd immunity. With escalating fatalities across various nations no foreseeable end in sight, there is a pressing need to create swiftly deployable, rapid, cost-effective detection, and treatment methods. While various steps are taken to mitigate the transmission and severity of the disease, vaccination is proven throughout mankind history as the best method to acquire immunity and circumvent the spread of infectious diseases. Nonetheless, relying solely on vaccination might not be adequate to match the relentless viral mutations observed in emerging variants of SARS-CoV-2, including alterations to their RBD domain, acquisition of escape mutations, and potential resistance to antibody binding. Beyond the immune system activation achieved through vaccination, it is crucial to develop new medications or treatment methods to either impede the infection or enhance existing treatment modalities. This review emphasizes innovative treatment strategies that aim to directly disrupt the virus's ability to replicate and spread, which could play a role in ending the SARS-CoV-2 pandemic.
Aptamers are single-stranded DNA or RNA oligonucleotides generated by SELEX that exhibit binding affinity and specificity against a wide variety of target molecules. Compared to RNA aptamers, DNA aptamers are much more stable and therefore are widely adopted in a number of applications especially in diagnostics. The tediousness and rigor associated with certain steps of the SELEX intensify the efforts to adopt in silico molecular docking approaches together with in vitro SELEX procedures in developing DNA aptamers. Inspired by these endeavors, we carry out an overview of the in silico molecular docking approaches in DNA aptamer generation, by detailing the stepwise procedures as well as shedding some light on the various softwares used. The in silico maturation strategy and the limitations of the in silico approaches are also underscored.
The performance of aptamers as versatile tools in numerous analytical applications is critically dependent on their high target binding specificity and selectivity. However, only the technical or methodological aspects of measuring aptamer-target binding affinities are focused, ignoring the equally important mathematical components that play pivotal roles in affinity measurements. In this study, we aim to provide a comprehensive review regarding the utilization of different mathematical models and equations, along with a detailed description of the computational steps involved in mathematically deriving the binding affinity of aptamers against their specific target molecules. Mathematical models ranging from one-site binding to multiple aptameric binding site-based models are explained in detail. Models applied in several different approaches of affinity measurements such as thermodynamics and kinetic analysis, including cooperativity and competitive-assay based mathematical models have been elaborately discussed. Mathematical models incorporating factors that could potentially affect affinity measurements are also further scrutinized.