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Nucleoside diphosphates and triphosphates impact nearly every aspect of biochemistry; however, the use of such compounds as tools or medicinal leads for nucleotide-dependent enzymes and receptors is hampered by their rapid in vivo metabolism. Although a successful strategy to address the instability of the monophosphate moiety in oligonucleotide therapeutics has been accomplished by their isosteric replacement with phosphorothioates, no practical methods exist to rapidly and controllably access stereopure di- and triphosphate thioisosteres of both natural and unnatural nucleosides. Here we show how a modular, reagent-based platform can enable the stereocontrolled and scalable synthesis of a library of such molecules. This operationally simple approach provides access to pure stereoisomers of nucleoside α-thiodiphosphates and α-thiotriphosphates, as well as symmetrical or unsymmetrical dinucleoside thiodiphosphates and thiotriphosphates (including RNA cap reagents). We demonstrate that ligand-receptor interactions can be dramatically influenced by P-stereochemistry, showing that such thioisosteric replacements can have profound effects on the potency and stability of lead candidates., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Poly(A)-binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate mRNA stability and translational potential, and that oligoadenylate-derived PABP ligands can act as effective translational inhibitors with potential applications in pain management. Although extensive research has focused on protein-RNA and protein-protein interactions involving PABPs, further studies are required to examine the ligand specificity of PABP. In this study, we developed a microscale thermophoresis-based assay to probe the interactions between PABP and oligoadenylate analogs containing different chemical modifications. Using this method, we evaluated oligoadenylate analogs modified with nucleobase, ribose, and phosphate moieties to identify modification hotspots. In addition, we determined the susceptibility of the modified oligos to CNOT7 to identify those with the potential for increased cellular stability. Consequently, we selected two enzymatically stable oligoadenylate analogs that inhibit translation in rabbit reticulocyte lysates with a higher potency than a previously reported PABP ligand. We believe that the results presented in this study and the implemented methodology can be capitalized upon in the future development of RNA-based biological tools., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Gold nanoparticles (AuNPs) decorated with biologically relevant molecules have variety of applications in optical sensing of bioanalytes. Coating AuNPs with small nucleotides produces particles with high stability in water, but functionality-compatible strategies are needed to uncover the full potential of this type of conjugates. Here, we demonstrate that lipoic acid-modified dinucleotides can be used to modify AuNPs surfaces in a controllable manner to produce conjugates that are stable in aqueous buffers and biological mixtures and capable of interacting with nucleotide-binding proteins. Using this strategy we obtained AuNPs decorated with 7-methylguanosine mRNA 5' cap analogs and showed that they bind cap-specific protein, eIF4E. AuNPs decorated with non-functional dinucleotides also interacted with eIF4E, albeit with lower affinity, suggesting that eIF4E binding to cap-decorated AuNPs is partially mediated by unspecific ionic interactions. This issue was overcome by applying lipoic-acid-Tris conjugate as a charge-neutral diluting molecule. Tris-Lipo-diluted cap-AuNPs conjugates interacted with eIF4E in fully specific manner, enabling design of functional tools. To demonstrate the potential of these conjugates in protein sensing, we designed a two-component eIF4E sensing system consisting of cap-AuNP and 4E-BP1-AuNP conjugates, wherein 4E-BP1 is a short peptide derived from 4E-BP protein that specifically binds eIF4E at a site different to that of the 5' cap. This system facilitated controlled aggregation, in which eIF4E plays the role of the agent that crosslinks two types of AuNP, thereby inducing a naked-eye visible absorbance redshift. The reported AuNPs-nucleotide conjugation method based on lipoic acid affinity for gold, can be harnessed to obtain other types of nucleotide-functionalized AuNPs, thereby paving the way to studying other nucleotide-binding proteins., (© 2021. The Author(s).)

Cancer vaccines have garnered attention as a potential treatment for cancer metastases. Nevertheless, the clinical response rate to vaccines remains < 30%. Nanoparticles stabilize vaccines and improve antigen recognition and presentation, resulting in high tumor penetration or accumulation, effective co-distribution of drugs to the secondary lymphatic system, and adaptable antigen or adjuvant administration. Such vaccine-like nanomedicines have the ability to eradicate the primary tumors as well as to prevent or eliminate metastases. This review examines state-of-the-art nanocarriers developed to deliver tumor vaccines to metastases, including synthetic, semi-biogenic, and biogenic nanosystems. Moreover, it highlights the physical and pharmacological properties that enhance their anti-metastasis efficiency. This review also addresses the combination of nanovaccines with cancer immunotherapy to target various steps in the metastatic cascade, drawing insights from preclinical and clinical studies. The review concludes with a critical analysis of the challenges and frameworks linked to the clinical translation of cancer nanovaccines. [ABSTRACT FROM AUTHOR]

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7's RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid–liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation. [ABSTRACT FROM AUTHOR]

Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue—alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure–activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist—AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial–mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment. [ABSTRACT FROM AUTHOR]

Recently, chemically synthesized minimal mRNA (CmRNA) has emerged as a promising alternative to in vitro transcribed mRNA (IVT-mRNA) for cancer therapy and immunotherapy. CmRNA lacking the untranslated regions and polyadenylation exhibits enhanced stability and efficiency. Encapsulation of CmRNA within lipid-polymer hybrid nanoparticles (LPPs) offers an effective approach for personalized neoantigen mRNA vaccines with improved control over tumor growth. LPP-based delivery systems provide superior pharmacokinetics, stability, and lower toxicity compared to viral vectors, naked mRNA, or lipid nanoparticles that are commonly used for mRNA delivery. Precise customization of LPPs in terms of size, surface charge, and composition allows for optimized cellular uptake, target specificity, and immune stimulation. CmRNA-encoded neo-antigens demonstrate high translational efficiency, enabling immune recognition by CD8+ T cells upon processing and presentation. This perspective highlights the potential benefits, challenges, and future directions of CmRNA neoantigen vaccines in cancer therapy compared to Circular RNAs and IVT-mRNA. Further research is needed to optimize vaccine design, delivery, and safety assessment in clinical trials. Nevertheless, personalized LPP-CmRNA vaccines hold great potential for advancing cancer immunotherapy, paving the way for personalized medicine. [ABSTRACT FROM AUTHOR]