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Scientific Reports Aug 2023Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading...
Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of [Formula: see text] w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of [Formula: see text] turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the [Formula: see text]-globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems.
Topics: Humans; Peptide Nucleic Acids; Zirconium; Nanoparticles; Nucleic Acids; Nanostructures
PubMed: 37648689
DOI: 10.1038/s41598-023-40916-w -
Science Advances Sep 2023Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The...
Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, β-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).
Topics: Humans; COVID-19; DNA; Nucleic Acids; Oligonucleotides; Electronics
PubMed: 37672583
DOI: 10.1126/sciadv.adi4997 -
Nucleic Acid Therapeutics Dec 2023One advantage of antisense oligonucleotides (ASOs) for drug development is their long-lasting gene knockdown after administration . In this study, we examine the effect...
One advantage of antisense oligonucleotides (ASOs) for drug development is their long-lasting gene knockdown after administration . In this study, we examine the effect on gene expression after intraocular injection in target tissues in the eye. We examined expression levels of the gene after intracameral or intravitreal (IV) injection of an anti- ASO in corneal epithelium/stroma, corneal endothelium, lens capsule epithelium, neurosensory retina, and retinal pigment epithelium/choroid of the mouse eye. We assessed potency of the compound at 7 days as well as duration of the gene knockdown at 14, 28, 60, 90, and 120 days. The ASO was more potent when delivered by IV injection relative to intracameral injection, regardless of whether the tissues analyzed were at the front or back of the eye. For corneal endothelium, inhibition was >50% after 120 days for ASO at 50 μg. At IV dosages of 6 μg, we observed >75% inhibition of gene expression in the retina and lens epithelium for up to 120 days. ASOs have potential as long-lasting gene knockdown agents in the mouse eye, but efficacy varies depending on the specific ocular target tissue and injection protocol.
Topics: Mice; Animals; Oligonucleotides, Antisense; Retina; Endothelium, Corneal; Gene Expression
PubMed: 37917066
DOI: 10.1089/nat.2023.0044 -
Mikrochimica Acta Mar 2024The three decades of experience with piezoelectric devices applied in the field of bioanalytical chemistry are shared. After introduction to principles and suitable... (Review)
Review
The three decades of experience with piezoelectric devices applied in the field of bioanalytical chemistry are shared. After introduction to principles and suitable measuring approaches, active and passive methods based on oscillators and impedance analysis, respectively, the focus is directed towards biosensing approaches. Immunosensing examples are provided, followed by other affinity sensing approaches based on hybridization of nucleic acids, aptamers, monitoring of enzyme activities, and detection of pathogenic microbes. The combination of piezosensors with cell lines and testing of drugs is highlighted, including mechanically active cells. The combination of piezosensors with other measuring techniques providing original hybrid devices is briefly discussed.
Topics: Cell Line; Electric Impedance; Nucleic Acids; Oligonucleotides
PubMed: 38451295
DOI: 10.1007/s00604-024-06257-9 -
Naunyn-Schmiedeberg's Archives of... Mar 2024Exposure to toxicants/stressors has been linked to the development of many human diseases. They could affect various cellular components, such as DNA, proteins, lipids,... (Review)
Review
Exposure to toxicants/stressors has been linked to the development of many human diseases. They could affect various cellular components, such as DNA, proteins, lipids, and non-coding RNAs (ncRNA), thereby triggering various cellular pathways, particularly oxidative stress, inflammatory responses, and apoptosis, which can contribute to pathophysiological states. Accordingly, modulation of these pathways has been the focus of numerous investigations for managing related diseases. The involvement of various ncRNAs, such as small interfering RNA (siRNA), microRNAs (miRNA), and long non-coding RNAs (lncRNA), as well as various proteins and peptides in mediating these pathways, provides many target sites for pharmaceutical intervention. In this regard, various oligonucleotide- and protein/peptide-based therapies have been developed to treat toxicity-induced diseases, which have shown promising results in vitro and in vivo. This comprehensive review provides information about various aspects of toxicity-related diseases including their causing factors, main underlying mechanisms and intermediates, and their roles in pathophysiological states. Particularly, it highlights the principles and mechanisms of oligonucleotide- and protein/peptide-based therapies in the treatment of toxicity-related diseases. Furthermore, various issues of oligonucleotides and proteins/peptides for clinical usage and potential solutions are discussed.
Topics: Humans; Oligonucleotides; MicroRNAs; RNA, Untranslated; RNA, Small Interfering; Peptides
PubMed: 37688622
DOI: 10.1007/s00210-023-02683-3 -
Computational Biology and Chemistry Jun 2024Protein variant libraries produced by site-directed mutagenesis are a useful tool utilized by protein engineers to explore variants with potentially improved properties,...
Protein variant libraries produced by site-directed mutagenesis are a useful tool utilized by protein engineers to explore variants with potentially improved properties, such as activity and stability. These libraries are commonly built by selecting residue positions and alternative beneficial mutations for each position. All possible combinations are then constructed and screened, by incorporating degenerate codons at mutation sites. These degenerate codons often encode additional unwanted amino acids or even STOP codons. Our study aims to take advantage of annealing based recombination of oligonucleotides during synthesis and utilize multiple degenerate codons per mutation site to produce targeted protein libraries devoid of unwanted variants. Toward this goal we created an algorithm to calculate the minimum number of degenerate codons necessary to specify any given amino acid set, and a dynamic programming method that uses this algorithm to optimally partition a DNA target sequence with degeneracies into overlapping oligonucleotides, such that the total cost of synthesis of the target mutant protein library is minimized. Computational experiments show that, for a modest increase in DNA synthesis costs, beneficial variant yields in produced mutant libraries are increased by orders of magnitude, an effect particularly pronounced in large combinatorial libraries.
Topics: Mutation; Algorithms; Proteins; Mutagenesis, Site-Directed; Peptide Library; DNA; Oligonucleotides
PubMed: 38669847
DOI: 10.1016/j.compbiolchem.2024.108068 -
International Journal of Biological... Feb 2024Animal diseases often have significant consequences due to the unclear and time-consuming diagnosis process. Furthermore, the emergence of new viral infections and... (Review)
Review
Animal diseases often have significant consequences due to the unclear and time-consuming diagnosis process. Furthermore, the emergence of new viral infections and drug-resistant pathogens has further complicated the diagnosis and treatment of viral diseases. Aptamers, which are obtained through systematic evolution of ligands by exponential enrichment (SELEX) technology, provide a promising solution as they enable specific identification and binding to targets, facilitating pathogen detection and the development of novel therapeutics. This review presented an overview of aptasensors for animal virus detection, discussed the antiviral activity and mechanisms of aptamers, and highlighted advancements in aptamer-based antiviral research following the COVID-19 pandemic. Additionally, the challenges and prospects of aptamer-based virus diagnosis and treatment research were explored. Although this review was not exhaustive, it offered valuable insights into the progress of aptamer-based antiviral drug research, target mechanisms, as well as the development of novel antiviral drugs and biosensors.
Topics: Animals; Humans; Aptamers, Nucleotide; Pandemics; SELEX Aptamer Technique; Viruses; Antiviral Agents
PubMed: 38072350
DOI: 10.1016/j.ijbiomac.2023.128677 -
Analytical Chemistry Aug 2023Here, a novel rapid and ultrasensitive aptamer biosensor was designed for target-induced activation of AIE effect and followed by the activation of Crispr Cas12a...
Here, a novel rapid and ultrasensitive aptamer biosensor was designed for target-induced activation of AIE effect and followed by the activation of Crispr Cas12a (LbCpf1)-mediated cleavage to achieve dual-signal detection. The prepared DNA building blocks contain the target aptamer, ssDNA-Fc, and Activator1. In this system, the activation mode was divided into two steps. First, when the target interacts with the aptamers, the DNA building blocks would be disintegrated rapidly, releasing a mass of Ac1, generating ETTC-dsDNA aggregated to produce a fluorescence signal by the AIE effect. Second, with the release of Ac2, LbCpf1-crRNA was activated, which greatly improves the ssDNA-Fc cleavage efficiency to render signal amplification and ultrasensitive detection of the target. Satisfactorily, using this approach to detect gliotoxin, optimal conditions for detection was achieved for reducing the detection time to 55 min, achieving a low detection limit of 2.4 fM and a satisfactory linear in the range of 50 fM to 1 nM, which addressed the shortcoming of a weak electrochemical signal in previous sensors. The water-insoluble AIE material was coupled with DNA to obtain water-soluble ETTC-dsDNA and successfully introduced into the sensor system, with a low detection limit of 5.6 fM. Subsequently, the biosensor combined with handheld electrochemical workstation was successfully applied in the detection of gliotoxin in five actual samples, with a detection range of 32.0 to 2.09 × 10 pM. This strategy not only provides a novel and effective detection platform for mycotoxins in complex food matrices but also opens a promising avenue for various molecules detection in imaging and disease diagnosis.
Topics: Gliotoxin; CRISPR-Cas Systems; Mycotoxins; Oligonucleotides; DNA, Single-Stranded; Biosensing Techniques
PubMed: 37493946
DOI: 10.1021/acs.analchem.3c01760 -
Chemistry (Weinheim An Der Bergstrasse,... Oct 2023Fluorine labeling of ribonucleic acids (RNA) in conjunction with F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and...
Fluorine labeling of ribonucleic acids (RNA) in conjunction with F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA-ligand interactions. This study presents the first syntheses of 2'-OCF guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid-phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2'-OCF modification is associated with preferential C2'-endo conformation of the U and G ribose in single-stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2'-OCF label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2'-OCF modification has potential for applications in therapeutic oligonucleotides. To this end, three 2'-OCF modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2'-OCF nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2'-OCF group without any restrictions in placing the modification into the RNA target sequence.
Topics: RNA; RNA, Small Interfering; Oligonucleotides; Molecular Conformation; Magnetic Resonance Spectroscopy; Oligoribonucleotides; Nucleic Acid Conformation
PubMed: 37534701
DOI: 10.1002/chem.202302220 -
Expert Review of Proteomics 2024Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom... (Review)
Review
INTRODUCTION
Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described.
AREAS COVERED
Over the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results.
EXPERT OPINION
Understanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution.
Topics: Humans; Oligonucleotides; Antibodies
PubMed: 38363709
DOI: 10.1080/14789450.2024.2318565