Publications by authors named "Edward Darzynkiewicz"
All cells in our body are equipped with receptors to recognize pathogens and trigger a rapid defense response. As a result, foreign molecules are blocked, and cells are alerted to the danger. Among the many molecules produced in response to viral infection are interferon-induced proteins with tetratricopeptide repeats (IFITs).
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- mRNA-based drugs are rapidly advancing, particularly as viral vaccines, with potential uses in various diseases due to their easy and adaptable production process.
- The structure of mRNA components, especially modifications at the 5' cap, significantly influences its effectiveness and longevity in medical applications.
- This study focuses on synthesizing modified cap analogues and testing these compounds for their ability to act as translation inhibitors and improve mRNA preparation.
Eukaryotic initiation factor 4E (eIF4E) is a pivotal protein involved in the regulatory mechanism for global protein synthesis in both physiological and pathological conditions. MicroRNAs (miRNAs) play a significant role in regulating gene expression by targeting mRNA. However, the ability of miRNAs to regulate eIF4E and its phosphorylation remains relatively unknown.
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- Nudt15 is a well-studied enzyme from the NUDIX protein family, primarily for its effects on thiopurine drugs used in cancer and inflammatory disease treatment.
- Besides its known activity on thiopurines, Nudt15 also acts on various nucleotide substrates, and there's speculation about its role in the degradation of mRNA through hydrolysis of mGDP.
- Recent research showed that Nudt15 has moderate activity with methylated forms of GDP and GTP, suggesting these can inform the design of new small molecule inhibitors targeting Nudt15.
Mesenchymal stem cell engineering by ARCA analog-capped mRNA.
Mol Ther Nucleic Acids
September 2023
Article Synopsis
- The study examines factors affecting mRNA engineering of mesenchymal stem cells (MSCs), focusing on transfection methods, mRNA purification, and capping types.
- Results show that Lipofectamine 2000 is superior to TransIT for MSC transfection, while HPLC purification is essential for maintaining MSC health.
- It concludes that using Lipofectamine 2000 with HPLC-purified mRNA and β-S-ARCA D1 capping achieves enhanced protein production, albeit with reduced MSC metabolic activity.
Recent findings have substantially broadened our knowledge about the diversity of modifications of the 5'end of RNAs, an issue generally attributed to mRNA cap structure (mGpppN). Nudt12 is one of the recently described new enzymatic activities involved in cap metabolism. However, in contrast to its roles in metabolite-cap turnover (e.
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- mRNA-based vaccines are innovative technologies that have recently gained attention from research centers and pharmaceutical companies, offering advantages over DNA-based vaccines due to their safety and flexibility.
- These vaccines avoid risks of genomic integration and can be easily engineered to improve their efficiency and stability.
- The study discusses the use of N2 modified dinucleotide cap analogs in mRNA, which enhance translation efficiency and proper attachment, showing promising results in both in vitro and human cell experiments.
Article Synopsis
- Transcribed synthetic mRNAs (IVT mRNAs) are in high demand for therapeutic applications due to their efficient and scalable production using bacteriophage RNA polymerases (RNAP).
- However, IVT mRNA preparations often have contaminants like double-stranded RNA (dsRNA) that trigger unwanted immune responses when introduced into cells, making it essential to remove these contaminants.
- The study found that using a genetically modified polymerase (HiT7 RNAP) at higher temperatures reduced dsRNA levels and immune responses compared to a standard polymerase (SP6 RNAP), and incorporating pseudouridine nucleotides further improved translation efficiency and reduced immune detection.
Article Synopsis
- Nudt16, part of the NUDIX hydrolase family, selectively works on nucleoside diphosphates but there are conflicting reports on its substrates and biological roles.* -
- A study found that hNudt16 has the strongest affinity for IDP and GppG, with K values under 100 nM, while other substrates displayed significantly weaker binding.* -
- The research identified GppG as a new substrate for hNudt16 and suggested the enzyme's strong binding is enhanced by interactions with specific amino acids, hinting at its regulatory functions.*
Article Synopsis
- The m7G cap is crucial for RNA produced by RNA Polymerase II and is important for gene expression in eukaryotes, but its specific function in mammals was previously unclear.
- Researchers found that the methyltransferase RNMT plays a significant role in T cell activation by regulating the production of mRNA and ribosomes, which are essential for metabolic changes and rapid cell division.
- RNMT's induction during T cell receptor stimulation leads to increased expression of certain mRNAs and snoRNAs, vital for ribosome biogenesis, and its absence results in decreased ribosome production and impaired T cell proliferation.
Article Synopsis
- IFITs are RNA-binding proteins that are crucial for the immune system's antiviral response by recognizing and binding to foreign viral RNA particles.
- They form stable complexes with the RNA, which leads to a shutdown of translation for non-self transcripts.
- The study developed a fluorescent assay to explore the interaction between RNA and IFIT proteins, focusing on IFIT1 and IFIT5, and found a probe that helps compare the binding affinities of various mRNAs based on their 5' end structures.
Article Synopsis
- DcpS enzymes are crucial for mRNA degradation in eukaryotes and are linked to conditions like spinal muscular atrophy and acute myeloid leukemia, with recent connections to intellectual disability.* -
- This study is the first to analyze the effects of different tags (N-terminus vs. C-terminus) on DcpS enzymes from humans and other species, noting that native forms were created through tag removal.* -
- While the thermal stability of most DcpS versions remained stable, the catalytic activity showed that tagged DcpSs had enhanced efficiency with specific substrates compared to their native counterparts, except for mGDP which was resistant to cleavage.*
Eukaryotic messenger RNA (mRNA) is modified by the addition of an inverted guanosine cap to the 5' triphosphate. The cap guanosine and initial transcribed nucleotides are further methylated by a series of cap methyltransferases to generate the mature cap structures which protect RNA from degradation and recruit proteins involved in RNA processing and translation. Research demonstrating that the cap methyltransferases are regulated has generated interest in determining the methylation status of the mRNA cap structures present in cells.
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- Cellular antiviral responses increase the levels of interferon-induced proteins with tetratricopeptide repeats (IFITs) that bind to the 5' end of mRNA, which has a unique cap structure.* -
- Research focused on IFIT1 and IFIT5 showed that the stability of their binding to various capped and uncapped mRNAs is affected by the modifications to the cap structure.* -
- Interaction studies revealed that certain modified synthetic cap analogs can somewhat protect mRNA from translational inhibition by IFIT1, highlighting their potential in biotechnology and medicine.*
Article Synopsis
- mRNA degradation plays a crucial role in regulating gene expression, specifically through the 3' → 5' decay pathway involving the exosome complex and decapping enzymes.
- The study focuses on the decapping scavenger enzyme DcpS from humans, Caenorhabditis elegans (CeDcpS), and Aspergillus species (AsDcpS), examining their activity toward modified cap analogues.
- Results showed that longer alkyl chains on cap analogues led to resistance against hydrolysis by hDcpS and CeDcpS, likely due to weaker binding interactions between the modified caps and the enzymes.
Article Synopsis
- Advances in gene manipulation techniques, particularly DNA therapy, are being significantly enhanced by new mRNA technologies that improve particle stability and translation efficiency.
- Recent studies focused on modifying mRNA cap structures, specifically at the N2 position of 7-methylguanosine, which increased translation inhibition and led to the design of new dinucleotide cap analogs.
- In testing these new cap analogs in rabbit reticulocyte lysate and HEK293 cell lines, the results showed improved translational properties and stability over conventional caps, with one analog demonstrating strong translation inhibition.
Article Synopsis
- - Human Nudt16 (hNudt16) is an enzyme that breaks down various RNA-related substrates, with a particular focus on its decapping activity in the nucleolus, specifically targeting U8 snoRNA.
- - Recent findings show that hNudt16 localizes to the cytoplasm and plays a role in RNA turnover, similar to the enzyme Dcp2, by hydrolyzing cap structures in RNA.
- - The study reveals that hNudt16 is more effective at breaking down dinucleotide cap analogs and short capped oligonucleotides that contain guanine, indicating its broader specificity than previously understood.
mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.
Nucleic Acids Res
September 2017
Analogues of the mRNA 5'-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e.
View Article and Find Full Text PDFmRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry.
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- There's increasing interest in mRNA-based gene therapies, but a major challenge is achieving sufficient expression of delivered mRNA in the body.
- Researchers developed a new class of cap analogs called 2S analogs, which are designed to modify mRNA's cap structure for better functionality.
- These 2S analogs improve translation efficiency in human cells and resist degradation, showing promise for enhancing mRNA therapies, including those for cancer immunization.
The mRNA 5' cap structure plays a pivotal role in coordination of eukaryotic translation and mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a dimeric exoribonuclease that efficiently degrades mRNA 3' poly(A) tails while also simultaneously interacting with the mRNA 5' cap. The cap binding amplifies the processivity of PARN action.
View Article and Find Full Text PDFThe synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m(7)GTP derivatives, whereas 11 are 'two headed' tetraphosphate dinucleotides based on a m(7)Gppppm(7)G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between β and γ phosphorus atoms.
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- - Snurportin 1 is a protein that helps transport specific small nuclear RNAs into the nucleus, influencing how these RNA complexes function in the cell.
- - Researchers created new cap analogues to better understand how snurportin recognizes and binds to the TMG (trimethylguanosine) structure on RNA.
- - The study found that snurportin is very selective for the TMG-cap, favoring its specific structure due to its stiffness and compactness, which are crucial for binding.
Effect of different N7 substitution of dinucleotide cap analogs on the hydrolytic susceptibility towards scavenger decapping enzymes (DcpS).
Biochem Biophys Res Commun
August 2015
Article Synopsis
- Scavenger decapping enzymes (DcpS) play a crucial role in breaking down mRNA by targeting the cap structure m(7)GpppN, which is a common feature in eukaryotic mRNA.
- The study explores how different substitutions at the N7 position of the guanine in the cap structure affect DcpS from various organisms, including humans and some nematodes, focusing on their rate of hydrolysis and binding affinities.
- Findings indicate that the DcpS enzyme has a flexible cap-binding pocket, allowing it to accommodate various substrates with different N7 structural modifications for effective hydrolysis.
Article Synopsis
- eIF4E1b is a cap-binding protein similar to eIF4E1a, and is primarily found in oocytes of mouse, Xenopus, and zebrafish, functioning as part of a translation repressor complex.
- Researchers examined the interactions of eIF4E1b with cap analogues using fluorescence techniques and structural modeling, revealing that it binds to caps less effectively than eIF4E1a due to specific amino acid differences.
- Key findings indicate that eIF4E1b's cap-binding ability is enhanced by certain chemical modifications and that mutations in its distinguishing amino acids can significantly affect its cap-binding strength compared to eIF4E1a.