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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 -
Blood Advances Nov 2023Chimeric antigen receptor T-cell (CAR-T) therapy represents a major advance in cancer immunotherapy; however, it can be associated with life-threatening neurotoxicity...
Chimeric antigen receptor T-cell (CAR-T) therapy represents a major advance in cancer immunotherapy; however, it can be associated with life-threatening neurotoxicity linked to blood-brain barrier disruption and endothelial activation. Defibrotide was shown to reduce endothelial cell activation in vitro and is approved in the United States for treatment of veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) in patients with renal or pulmonary dysfunction after hematopoietic cell transplantation (HCT), and in the European Union for severe VOD/SOS after HCT in patients aged >1 month. Defibrotide may stabilize the endothelium during CAR-T therapy and reduce the rate of CAR-T-associated neurotoxicity. This phase 2 study evaluated the safety and efficacy of defibrotide for prevention of CAR-T-associated neurotoxicity in patients with relapsed/refractory large B-cell lymphoma receiving axicabtagene ciloleucel. Part 1 established the recommended phase 2 dose (RP2D; 6.25 mg/kg); 20 patients (from parts 1 and 2) receiving the RP2D were evaluable for efficacy. Rate of CAR-T-associated neurotoxicity by day 30 (primary end point) was ∼50%, lower than reported in the ZUMA-1 trial (64%). Median event duration of grade ≥3 neurotoxicity was 7 days. No unexpected defibrotide-related safety findings and defibrotide-related treatment-emergent adverse events or deaths were reported. Results showed modest reduction in rate of CAR-T-associated neurotoxicity and high-grade neurotoxicity event duration relative to historical data; however, reduction was unlikely to meet the primary end point, so the study was terminated early. Nevertheless, results contribute valuable data for potential therapeutic insight on the management of CAR-T-associated neurotoxicity. This trial was registered at www.clinicaltrials.gov as #NCT03954106.
Topics: Humans; United States; Receptors, Chimeric Antigen; Polydeoxyribonucleotides; Hepatic Veno-Occlusive Disease; T-Lymphocytes
PubMed: 37399456
DOI: 10.1182/bloodadvances.2023009961 -
Nucleic Acids Research Aug 2023Cell-free protein synthesis assays have become a valuable tool to understand transcriptional and translational processes. Here, we established a fluorescence-based...
Cell-free protein synthesis assays have become a valuable tool to understand transcriptional and translational processes. Here, we established a fluorescence-based coupled in vitro transcription-translation assay as a read-out system to simultaneously quantify mRNA and protein levels. We utilized the well-established quantification of the expression of shifted green fluorescent protein (sGFP) as a read-out of protein levels. In addition, we determined mRNA quantities using a fluorogenic Mango-(IV) RNA aptamer that becomes fluorescent upon binding to the fluorophore thiazole orange (TO). We utilized a Mango-(IV) RNA aptamer system comprising four subsequent Mango-(IV) RNA aptamer elements with improved sensitivity by building Mango arrays. The design of this reporter assay resulted in a sensitive read-out with a high signal-to-noise ratio, allowing us to monitor transcription and translation time courses in cell-free assays with continuous monitoring of fluorescence changes as well as snapshots of the reaction. Furthermore, we applied this dual read-out assay to investigate the function of thiamine-sensing riboswitches thiM and thiC from Escherichia coli and the adenine-sensing riboswitch ASW from Vibrio vulnificus and pbuE from Bacillus subtilis, which represent transcriptional and translational on- and off-riboswitches, respectively. This approach enabled a microplate-based application, a valuable addition to the toolbox for high-throughput screening of riboswitch function.
Topics: Adenine; Aptamers, Nucleotide; Fluorescence; Green Fluorescent Proteins; Nucleic Acid Conformation; Riboswitch; Cell-Free System
PubMed: 37409574
DOI: 10.1093/nar/gkad574 -
Nucleic Acids Research May 2024RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in...
RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.
Topics: Animals; Humans; Male; Mice; Acetylgalactosamine; Antagomirs; Apolipoproteins E; Central Nervous System; Gene Silencing; Liver; Mice, Inbred C57BL; MicroRNAs; RNA Interference; RNA, Small Interfering
PubMed: 38348876
DOI: 10.1093/nar/gkae100 -
Biosensors Dec 2023Food contaminants represent possible threats to humans and animals as severe food safety hazards. Prolonged exposure to contaminated food often leads to chronic diseases... (Review)
Review
Food contaminants represent possible threats to humans and animals as severe food safety hazards. Prolonged exposure to contaminated food often leads to chronic diseases such as cancer, kidney or liver failure, immunosuppression, or genotoxicity. Aflatoxins are naturally produced by strains of the fungi species , which is one of the most critical and poisonous food contaminants worldwide. Given the high percentage of contaminated food products, traditional detection methods often prove inadequate. Thus, it becomes imperative to develop fast, accurate, and easy-to-use analytical methods to enable safe food products and good practices policies. Focusing on the recent progress (2018-2023) of electrochemical aptasensors for aflatoxin B1 (AFB1) detection in food and beverage samples, without pretending to be exhaustive, we present an overview of the most important label-free and labeled sensing strategies. Simultaneous and competitive aptamer-based strategies are also discussed. The aptasensors are summarized in tabular format according to the detection mode. Sample treatments performed prior analysis are discussed. Emphasis was placed on the nanomaterials used in the aptasensors' design for aptamer-tailored immobilization and/or signal amplification. The advantages and limitations of AFB1 electrochemical aptasensors for field detection are presented.
Topics: Animals; Humans; Aflatoxin B1; Food; Food Safety; Kidney; Nanostructures; Oligonucleotides
PubMed: 38248384
DOI: 10.3390/bios14010007 -
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 -
Nucleic Acids Research Sep 2023Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions...
Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers.
Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.
Topics: Humans; DNA; DNA Replication; Friedreich Ataxia; Iron-Binding Proteins; Oligonucleotides; Peptide Nucleic Acids; Saccharomyces cerevisiae; Trinucleotide Repeat Expansion
PubMed: 37216608
DOI: 10.1093/nar/gkad441 -
Journal of Experimental & Clinical... Sep 2023Most of the endometrial cancer (EC) patients are diagnosis in early stage with a good prognosis while the patients with locally advanced recurrent or metastatic result...
A patient-derived organoid-based study identified an ASO targeting SNORD14E for endometrial cancer through reducing aberrant FOXM1 Expression and β-catenin nuclear accumulation.
BACKGROUND
Most of the endometrial cancer (EC) patients are diagnosis in early stage with a good prognosis while the patients with locally advanced recurrent or metastatic result in a poor prognosis. Adjuvant therapy could benefit the prognosis of patients with high-risk factors. Unfortunately, the molecular classification of great prognostic value has not yet reached an agreement and need to be further refined. The present study aims to identify new targets that have prognostic value in EC based on the method of EC patient-derived organ-like organs (PDOs), and further investigate their efficacy and mechanism.
METHODS
The Cancer Genome Atlas (TCGA) database was used to determine SNORD14E expression. The effects of SNORD14E were investigated using CCK8, Transwell, wound-healing assays, and a xenograft model experiment; apoptosis was measured by flow cytometry. Antisense oligonucleotide (ASO) targeting SNORD14E was designed and patient-derived organoids (PDO) models in EC patients was established. A xenograft mouse and PDO model were employed to evaluate the effects of ASO targeting SNORD14E. RNA-seq, Nm-seq, and RNA immunoprecipitation (RIP) experiments were employed to confirm the alternative splicing (AS) and modification induced by SNORD14E. A minigene reporter gene assay was conducted to confirm AS and splicing factors on a variable exon. Actinomycin-d (Act-D) and Reverse Transcription at Low deoxy-ribonucleoside triphosphate concentrations followed by PCR (RTL-P) were utilized to confirm the effects of 2'-O methylation modification on FOXM1.
RESULTS
We found that SNORD14E was overexpressed in EC tissues and patients with high expressed SNORD14E were distributed in the TCGA biomolecular classification subgroups without difference. Further, SNORD14E could reduce disease-free survival (DFS) and recurrence free survival (RFS) of EC patients. SNORD14E promoted proliferation, migration, and invasion and inhibited the apoptosis of EC cells in vitro. ASOs targeting SNORD14E inhibited cell proliferation, migration, invasion while promoted cell apoptosis. ASOs targeting SNORD14E inhibited tumor growth in the xenograft mouse model. TCGA-UCEC database showed that the proportion of patients with high expression of SNORD14E in middle-high risk and high-risk patients recommended by EMSO-ESGO-ESTRO guidelines for adjuvant therapy is more than 50%. Next, we enrolled 8 cases of high-risk and high-risk EC patients according to EMSO-ESGO-ESTRO guidelines and successfully constructed EC-PDOs. ASOs targeting SNORD14E inhibited the EC-PDO growth. Mechanistically, SNORD14E could recognize the mRNA of FOXM1 and recruit SRSF1 to promote the shearing of the variable exon VIIa of FOXM1, resulting in the overexpression of the FOXM1 malignant subtypes FOXM1b and FOXM1c. In addition, SNORD14E modified FOXM1 mRNA with 2`-O-methylation, which prolonged the half-life of FOXM1 mRNA. The nucleus accumulation of β-catenin caused by aberrant FOXM1 expression led to EC progression.
CONCLUSIONS
ASO targeting SNORD14E can be an effective treatment for EC.
Topics: Humans; Animals; Mice; Female; Oligonucleotides, Antisense; beta Catenin; Oligonucleotides; Endometrial Neoplasms; Exons; Forkhead Box Protein M1; Serine-Arginine Splicing Factors
PubMed: 37667311
DOI: 10.1186/s13046-023-02801-2 -
Cell Reports Aug 2023Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical disorders that mainly develop from viral respiratory infections, sepsis, and...
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical disorders that mainly develop from viral respiratory infections, sepsis, and chest injury. Antigen-presenting cells play a pivotal role in propagating uncontrolled inflammation and injury through the excess secretion of pro-inflammatory cytokines and recruitment of immune cells. Autophagy, a homeostatic process that involves the degradation of cellular components, is involved in many processes including lung inflammation. Here, we use a polyinosinic-polycytidylic acid (poly(I:C))-induced lung injury mouse model to mimic viral-induced ALI/ARDS and show that disruption of autophagy in macrophages exacerbates lung inflammation and injury, whereas autophagy induction attenuates this process. Therefore, induction of autophagy in macrophages can be a promising therapeutic strategy in ALI/ARDS.
Topics: Animals; Mice; Antigen-Presenting Cells; Macrophages; Autophagy; Acute Lung Injury; Poly I-C; Respiratory Distress Syndrome
PubMed: 37590140
DOI: 10.1016/j.celrep.2023.112990 -
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