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RSC Advances May 2024Two novel bicyclo[6.1.0]nonyne (BCN) linker derivatives, which can be directly incorporated into oligonucleotide sequences during standard automated solid-phase...
Two novel bicyclo[6.1.0]nonyne (BCN) linker derivatives, which can be directly incorporated into oligonucleotide sequences during standard automated solid-phase synthesis, are reported. Stabilities of BCN-carbinol and two BCN-oligonucleotides are evaluated under acidic conditions. In addition, derivatized BCN linkers (non-acidic and acid treated) are evaluated for strain-promoted alkyne-azide cycloaddition (SPAAC).
PubMed: 38813131
DOI: 10.1039/d3ra08732h -
Scientific Reports May 2024The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal...
The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)] on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.
Topics: Biosensing Techniques; Citrus; Plant Diseases; DNA Probes; Bacterial Outer Membrane Proteins; Electrochemical Techniques; Electrodes; Nucleic Acid Hybridization; Dielectric Spectroscopy; Limit of Detection; Rhizobiaceae; Liberibacter
PubMed: 38806617
DOI: 10.1038/s41598-024-63112-w -
Nature Communications May 2024The chemokine CXCL12 promotes glioblastoma (GBM) recurrence after radiotherapy (RT) by facilitating vasculogenesis. Here we report outcomes of the dose-escalation part...
The chemokine CXCL12 promotes glioblastoma (GBM) recurrence after radiotherapy (RT) by facilitating vasculogenesis. Here we report outcomes of the dose-escalation part of GLORIA (NCT04121455), a phase I/II trial combining RT and the CXCL12-neutralizing aptamer olaptesed pegol (NOX-A12; 200/400/600 mg per week) in patients with incompletely resected, newly-diagnosed GBM lacking MGMT methylation. The primary endpoint was safety, secondary endpoints included maximum tolerable dose (MTD), recommended phase II dose (RP2D), NOX-A12 plasma levels, topography of recurrence, tumor vascularization, neurologic assessment in neuro-oncology (NANO), quality of life (QOL), median progression-free survival (PFS), 6-months PFS and overall survival (OS). Treatment was safe with no dose-limiting toxicities or treatment-related deaths. The MTD has not been reached and, thus, 600 mg per week of NOX-A12 was established as RP2D for the ongoing expansion part of the trial. With increasing NOX-A12 dose levels, a corresponding increase of NOX-A12 plasma levels was observed. Of ten patients enrolled, nine showed radiographic responses, four reached partial remission. All but one patient (90%) showed at best response reduced perfusion values in terms of relative cerebral blood volume (rCBV). The median PFS was 174 (range 58-260) days, 6-month PFS was 40.0% and the median OS 389 (144-562) days. In a post-hoc exploratory analysis of tumor tissue, higher frequency of CXCL12 endothelial and glioma cells was significantly associated with longer PFS under NOX-A12. Our data imply safety of NOX-A12 and its efficacy signal warrants further investigation.
Topics: Humans; Glioblastoma; Aptamers, Nucleotide; Chemokine CXCL12; Male; Female; Middle Aged; Aged; Brain Neoplasms; Adult; Maximum Tolerated Dose; Quality of Life; Neoplasm Recurrence, Local
PubMed: 38806504
DOI: 10.1038/s41467-024-48416-9 -
Proceedings of the National Academy of... Jun 2024Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive...
Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., , , and ), upregulation of genes associated with effector T cells (e.g., and ), and loss of Treg-mediated suppression.
Topics: Humans; RNA, Long Noncoding; T-Lymphocytes, Regulatory; Forkhead Transcription Factors; Interleukin-2 Receptor alpha Subunit; Epigenesis, Genetic; Gene Expression Regulation; Cell Differentiation
PubMed: 38805281
DOI: 10.1073/pnas.2315363121 -
Molecular Therapy. Nucleic Acids Jun 2024Huntington's disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the () gene. Reducing...
Huntington's disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the () gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal, but reduction of wild-type HTT (wtHTT) is undesirable, as it compromises gene function and potential therapeutic efficacy. One promising allele-selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing (RNA-seq) to monitor the processing precision of a limited set of CAG repeat-targeted small RNAs expressed from multiple scaffold contexts. Small RNA-seq identified expression constructs with high-guide strand 5' processing precision and promising allele-selective inhibition of mutHTT. Transcriptome-wide mRNA-seq also identified an allele-selective small RNA with a favorable off-target profile. These results support continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process.
PubMed: 38803421
DOI: 10.1016/j.omtn.2024.102206 -
Molecular Therapy. Nucleic Acids Jun 2024
PubMed: 38803420
DOI: 10.1016/j.omtn.2024.102208 -
Scientific Reports May 2024Emergence of Coronavirus disease 2019 (COVID-19) pandemic has posed a huge threat to public health. Rapid and reliable test to diagnose infected subjects is crucial for...
Emergence of Coronavirus disease 2019 (COVID-19) pandemic has posed a huge threat to public health. Rapid and reliable test to diagnose infected subjects is crucial for disease spread control. We developed a colorimetric test for COVID-19 detection using a Colorimetric Assay based on thiol-linked RNA modified gold nanoparticles (AuNPs) and oligonucleotide probes. This method was conducted on RNA from 200 pharyngeal swab samples initially tested by Real-Time polymerase chain reaction (RT-PCR) as gold standard. A specific oligonucleotide probe designed based on ORF1ab of COVID-19 was functionalized with AuNPs-probe conjugate. The exposure of AuNP-probe to isolated RNA samples was tested using hybridization. In this comparative study, the colorimetric functionalized AuNPs assay exhibited a detection limit of 25 copies/µL. It was higher in comparison to the RT-PCR method, which could only detect 15 copies/µL. The results demonstrated 100% specificity and 96% sensitivity for the developed method. Herein, we developed an incredibly rapid, simple and cost-effective Colorimetric Assay lasting approximately 30 min which could process considerably higher number of COVID-19 samples compared to the RT-PCR. This AuNP-probe conjugate colorimetric method could be considered the optimum alternatives for conventional diagnostic tools especially in over-populated and/or low-income countries.
Topics: Colorimetry; Humans; COVID-19; Metal Nanoparticles; Gold; Nasopharynx; SARS-CoV-2; RNA, Viral; Sensitivity and Specificity; Limit of Detection; Oligonucleotide Probes; COVID-19 Nucleic Acid Testing; Real-Time Polymerase Chain Reaction; COVID-19 Testing
PubMed: 38802360
DOI: 10.1038/s41598-024-53747-0 -
NAR Genomics and Bioinformatics Jun 2024Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs...
Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs contain extensive chemical modifications that improve stability to enzymatic cleavage and modulate binding affinity relative to natural RNA/DNA. Molecular dynamics (MD) simulation can provide valuable insights into how such modifications affect ASO conformational sampling and target binding. However, force field parameters for chemically modified nucleic acids (NAs) are still underdeveloped. To bridge this gap, we developed parameters to allow simulations of ASOs with the widely applied phosphorothioate (PS) backbone modification, and validated these in extensive all-atom MD simulations of relevant PS-modified NA systems representing B-DNA, RNA, and DNA/RNA hybrid duplex structures. Compared to the corresponding natural NAs, single PS substitutions had marginal effects on the ordered DNA/RNA duplex, whereas substantial effects of phosphorothioation were observed in single-stranded RNA and B-DNA, corroborated by the experimentally derived structure data. We find that PS-modified NAs shift between high and low twist states, which could affect target recognition and protein interactions for phosphorothioated oligonucleotides. Furthermore, conformational sampling was markedly altered in the PS-modified ssRNA system compared to that of the natural oligonucleotide, indicating sequence-dependent effects on conformational preference that may in turn influence duplex formation.
PubMed: 38800826
DOI: 10.1093/nargab/lqae058 -
Heliyon May 2024A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully...
A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80 %. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits.
PubMed: 38799737
DOI: 10.1016/j.heliyon.2024.e31213 -
ACS Central Science May 2024Antiviral nucleoside analogues (e.g., Molnupiravir, Remdesivir) played key roles in the treatment of COVID-19 by targeting SARS-CoV-2 RNA-dependent RNA polymerase...
Antiviral nucleoside analogues (e.g., Molnupiravir, Remdesivir) played key roles in the treatment of COVID-19 by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The nucleoside of Molnupiravir, -hydroxycytidine (NHC), exists in two tautomeric forms that pair either with G or A within the RdRp active site, causing an accumulation of viral RNA mutations during replication. Detailed insights into the tautomeric states within base pairs and the structural influence of NHC in RNA are still missing. In this study, we investigate the properties of NHC:G and NHC:A base pairs in a self-complementary RNA duplex by UV thermal melting and NMR spectroscopy using atom-specifically N-labeled versions of NHC that were incorporated into oligonucleotides by solid-phase synthesis. NMR analysis revealed that NHC forms a Watson-Crick base pair with G via its amino form, whereas two equally populated conformations were detected for the NHC:A base pair: a weakly hydrogen-bonded Watson-Crick base pair with NHC in the imino form and another conformation with A shifted toward the minor groove. Moreover, we found a variable influence of NHC:G and NHC:A base pairs on the neighboring duplex environment. This study provides conclusive experimental evidence for the existence of two tautomeric forms of NHC within RNA base pairs.
PubMed: 38799674
DOI: 10.1021/acscentsci.4c00146