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ACS Chemical Biology Nov 2023Therapeutic nucleic acids represent a powerful class of drug molecules to control gene expression and protein synthesis. A major challenge in this field is that soluble...
Therapeutic nucleic acids represent a powerful class of drug molecules to control gene expression and protein synthesis. A major challenge in this field is that soluble oligonucleotides have limited serum stability, and the majority of nucleic acids that enter the cells are trapped within endosomes. Delivery efficiency can be improved using lipid scaffolds. One such example is the nanodisc (ND), a self-assembled nanostructure composed of phospholipids and peptides and modeled after high density lipoproteins (HDLs). Herein, we describe the development of the nanodiscoidal nucleic acid (NNA) which is a ND covalently modified with nucleic acids on the top and bottom lipid faces as well as the lateral peptide belt. The 13 nm ND was doped with thiolated phospholipids and thiol-containing peptides and coupled in a one-pot reaction with oligonucleotides to achieve ∼30 DNA/NNA nucleic acid density. NNAs showed superior nuclease resistance and enhanced cellular uptake that was mediated through the scavenger receptor B1. Time-dependent Förster resonance energy transfer (FRET) analysis of internalized NNA confirmed that NNAs display increased stability. NNAs modified with clinically validated antisense oligonucleotides (ASOs) that target hypoxia inducible factor 1-α (HIF-1-α) mRNA showed enhanced activity compared with that of the soluble DNA across multiple cell lines as well as a 3D cancer spheroid model. Lastly, experiments show that ASO-modified NNAs are primarily localized into livers and kidneys, and NNAs were potent in downregulating HIF-1-α using 5-fold lower doses than previously reported. Collectively, our results highlight the therapeutic potential for NNAs.
Topics: Nucleic Acids; Oligonucleotides; DNA; Lipids; Peptides
PubMed: 37910400
DOI: 10.1021/acschembio.3c00038 -
Nucleic Acids Research Nov 2023G-quadruplexes (G4) are special nucleic acid structures with diverse conformational polymorphisms. Selective targeting of G-quadruplex conformations and regulating their...
G-quadruplexes (G4) are special nucleic acid structures with diverse conformational polymorphisms. Selective targeting of G-quadruplex conformations and regulating their biological functions provide promising therapeutic intervention. Despite the large repertoire of G4-binding tools, only a limited number of them can specifically target a particular G4 conformation. Here, we introduce a novel method, G4-SELEX-Seq and report the development of the first L-RNA aptamer, L-Apt12-6, with high binding selectivity to parallel G4 over other nucleic acid structures. Using parallel dG4 c-kit 1 as an example, we demonstrate the strong binding affinity between L-Apt12-6 and c-kit 1 dG4 in vitro and in cells, and notably report the applications of L-Apt12-6 in controlling DNA replication and gene expression. Our results suggest that L-Apt12-6 is a valuable tool for targeting parallel G-quadruplex conformation and regulating G4-mediated biological processes. Furthermore, G4-SELEX-Seq can be used as a general platform for G4-targeting L-RNA aptamers selection and should be applicable to other nucleic acid structures.
Topics: G-Quadruplexes; Aptamers, Nucleotide; Nucleic Acids
PubMed: 37870474
DOI: 10.1093/nar/gkad900 -
Current Protocols Jul 2023RNAstructure is a user-friendly program for the prediction and analysis of RNA secondary structure. It is available as a web server, a program with a graphical user...
RNAstructure is a user-friendly program for the prediction and analysis of RNA secondary structure. It is available as a web server, a program with a graphical user interface, or a set of command line tools. The programs are available for Microsoft Windows, macOS, or Linux. This article provides protocols for prediction of RNA secondary structure (using the web server, the graphical user interface, or the command line) and high-affinity oligonucleotide binding sites to a structured RNA target (using the graphical user interface). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Predicting RNA secondary structure using the RNAstructure web server Alternate Protocol 1: Predicting secondary structure and base pair probabilities using the RNAstructure graphical user interface Alternate Protocol 2: Predicting secondary structure and base pair probabilities using the RNAstructure command line interface Basic Protocol 2: Predicting binding affinities of oligonucleotides complementary to an RNA target using OligoWalk.
Topics: Binding Sites; Oligonucleotides; Probability; Protein Structure, Secondary; RNA
PubMed: 37487054
DOI: 10.1002/cpz1.846 -
Progress in Molecular Biology and... 2024RNA therapy is one of the new treatments using small RNA molecules to target and regulate gene expression. It involves the application of synthetic or modified RNA...
RNA therapy is one of the new treatments using small RNA molecules to target and regulate gene expression. It involves the application of synthetic or modified RNA molecules to inhibit the expression of disease-causing genes specifically. In other words, it silences genes and suppresses the transcription process. The main theory behind RNA therapy is that RNA molecules can prevent the translation into proteins by binding to specific messenger RNA (mRNA) molecules. By targeting disease-related mRNA molecules, RNA therapy can effectively silence or reduce the development of harmful proteins. There are different types of RNA molecules used in therapy, including small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamer, ribozyme, and antisense oligonucleotides (ASOs). These molecules are designed to complement specific mRNA sequences, allowing them to bind and degrade the targeted mRNA or prevent its translation into protein. Nanotechnology is also highlighted to increase the efficacy of RNA-based drugs. In this chapter, while examining various methods of RNA therapy, we discuss the advantages and challenges of each.
Topics: Humans; MicroRNAs; RNA, Small Interfering; Oligonucleotides; Oligonucleotides, Antisense; RNA, Messenger
PubMed: 38360005
DOI: 10.1016/bs.pmbts.2023.12.022 -
Angewandte Chemie (International Ed. in... Nov 2023Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have...
Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have rationally re-engineered tile-based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof-of-principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti-Dig, Anti-DNP and Anti-MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA-based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity.
Topics: DNA; Biosensing Techniques; Oligonucleotides; Proteins; Antibodies, Bispecific; Aptamers, Nucleotide
PubMed: 37804080
DOI: 10.1002/anie.202313243 -
Cells Oct 2023Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising... (Review)
Review
Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising biological and pharmacological properties for antisense applications. Despite their great potential, the efficient delivery of these therapeutic agents to target cells remains a major obstacle to their widespread use. Cellular uptake of naked PMO is poor. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular uptake and intracellular delivery of oligonucleotide-based drugs. Among these, the DG9 peptide has been identified as a versatile CPP with remarkable potential for enhancing the delivery of ASO-based therapeutics due to its unique structural features. Notably, in the context of phosphorodiamidate morpholino oligomers (PMOs), DG9 has shown promise in enhancing delivery while maintaining a favorable toxicity profile. A few studies have highlighted the potential of DG9-conjugated PMOs in DMD (Duchenne Muscular Dystrophy) and SMA (Spinal Muscular Atrophy), displaying significant exon skipping/inclusion and functional improvements in animal models. The article provides an overview of a detailed understanding of the challenges that ASOs face prior to reaching their targets and continued advances in methods to improve their delivery to target sites and cellular uptake, focusing on DG9, which aims to harness ASOs' full potential in precision medicine.
Topics: Animals; Humans; Oligonucleotides, Antisense; Cell-Penetrating Peptides; Oligonucleotides; Morpholinos; Muscular Dystrophy, Duchenne; Muscular Atrophy, Spinal
PubMed: 37830609
DOI: 10.3390/cells12192395 -
Cell Chemical Biology Jan 2024Synthetic antisense oligonucleotides (ASOs) and duplex RNAs (dsRNAs) are an increasingly successful strategy for drug development. After a slow start, the pace of... (Review)
Review
Synthetic antisense oligonucleotides (ASOs) and duplex RNAs (dsRNAs) are an increasingly successful strategy for drug development. After a slow start, the pace of success has accelerated since the approval of Spinraza (nusinersen) in 2016 with several drug approvals. These accomplishments have been achieved even though oligonucleotides are large, negatively charged, and have little resemblance to traditional small-molecule drugs-a remarkable achievement of basic and applied science. The goal of this review is to summarize the foundation underlying recent progress and describe ongoing research programs that may increase the scope and impact of oligonucleotide therapeutics.
Topics: RNA; Oligonucleotides; Oligonucleotides, Antisense; Drug Development
PubMed: 37804835
DOI: 10.1016/j.chembiol.2023.09.005 -
Chemical Communications (Cambridge,... Mar 2024To improve their properties or to introduce entirely new functionalities, the intriguing scaffolds of nucleic acids have been decorated with various modifications, most... (Review)
Review
To improve their properties or to introduce entirely new functionalities, the intriguing scaffolds of nucleic acids have been decorated with various modifications, most recently also organometallic ones. While challenging to introduce, organometallic modifications offer the potential of expanding the field of application of metal-dependent functionalities to metal-deficient conditions, notably those of biological media. So far, organometallic moieties have been utilized as probes, labels and catalysts. This Feature Article summarizes recent efforts and predicts likely future developments in each of these lines of research.
Topics: Oligonucleotides; Nucleic Acids; Metals
PubMed: 38385213
DOI: 10.1039/d4cc00305e -
Biomaterials Science Apr 2024Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), which were therapeutic DNA with high immunostimulatory activity, have been applied in... (Review)
Review
Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), which were therapeutic DNA with high immunostimulatory activity, have been applied in widespread applications from basic research to clinics as therapeutic agents for cancer immunotherapy, viral infection, allergic diseases and asthma since their discovery in 1995. The major factors to consider for clinical translation using CpG motifs are the protection of CpG ODNs from DNase degradation and the delivery of CpG ODNs to the Toll-like receptor-9 expressed human B-cells and plasmacytoid dendritic cells. Therefore, great efforts have been devoted to the advances of efficient delivery systems for CpG ODNs. In this review, we outline new horizons and recent developments in this field, providing a comprehensive summary of the nanoparticle-based CpG delivery systems developed to improve the efficacy of CpG-mediated immune responses, including DNA nanostructures, inorganic nanoparticles, polymer nanoparticles, metal-organic-frameworks, lipid-based nanosystems, proteins and peptides, as well as exosomes and cell membrane nanoparticles. Moreover, future challenges in the establishment of CpG delivery systems for immunotherapeutic applications are discussed. We expect that the continuously growing interest in the development of CpG-based immunotherapy will certainly fuel the excitement and stimulation in medicine research.
Topics: Humans; Oligodeoxyribonucleotides; Nanoparticles; Animals; Immunotherapy; Toll-Like Receptor 9; Drug Delivery Systems
PubMed: 38293828
DOI: 10.1039/d3bm01970e -
Analytical Sciences : the International... Oct 2023Aptamers have received extensive attention in recent years because of their advantages of high specificity, high sensitivity and low immunogenicity. Aptamers can perform... (Review)
Review
Aptamers have received extensive attention in recent years because of their advantages of high specificity, high sensitivity and low immunogenicity. Aptamers can perform almost all functions of antibodies through the combination of spatial structure and target, which are called "chemical antibodies". At present, aptamers have been widely used in cell imaging, new drug development, disease treatment, microbial detection and other fields. Due to the diversity of modifications, aptamers can be combined with different detection technologies to construct aptasensors. This review focuses on the diversity of aptamers in the field of detection and the development of aptamer-based detection technology and proposes new challenges for aptamers in this field.
Topics: Antibodies; Cell Differentiation; Oligonucleotides; Technology
PubMed: 37700097
DOI: 10.1007/s44211-023-00409-2