Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP.

Modified nucleotides are commonly used in molecular biology as substrates or inhibitors for several enzymes but also as tools for the synthesis of modified DNA and RNA fragments. Introduction of modification into RNA, such as phosphorothioate (PS), has been demonstrated to provide higher stability, more effective transport, and enhanced activity of potential therapeutic molecules. Hence, in order to achieve widespread use of RNA molecules in medicine, it is crucial to continuously refine the techniques that enable the effective introduction of modifications into RNA strands. Numerous analogues of nucleotides have been tested for their substrate activity with the T7 RNA polymerase and therefore in the context of their utility for use in in vitro transcription. In the present studies, the substrate preferences of the T7 RNA polymerase toward ß,γ-hypophospho-modified ATP derivatives for the synthesis of unmodified RNA and phosphorothioate RNA (PS) are presented. The performed studies revealed the stereoselectivity of this enzyme for α-thio-ß,γ-hypo-ATP derivatives, similar to that for α-thio-ATP. Additionally, it is demonstrated herein that hypodiphosphoric acid may inhibit in vitro transcription catalyzed by T7 RNA polymerase.

Modified Nucleotides for Chemical and Enzymatic Synthesis of Therapeutic RNA.

In recent years, RNA has emerged as a medium with a broad spectrum of therapeutic potential, however, for years, a group of short RNA fragments was studied and considered therapeutic molecules. In nature, RNA plays both functions, with coding and non-coding potential. For RNA, like any other therapeutic, to be used clinically, certain barriers must be crossed. Among them, there are biocompatibility, relatively low toxicity, bioavailability, increased stability, target efficiency and low off-target effects. In the case of RNA, most of these obstacles can be overcome by incorporating modified nucleotides into its structure. This may be achieved by both, in vitro and in vivo biosynthetic methods, as well as chemical synthesis. Some advantages and disadvantages of each approach are summarized here. The wide range of nucleotide analogues has been tested for their utility as monomers for RNA synthesis. Many of them have been successfully implemented, and a lot of pre-clinical and clinical studies involving modified RNA have been carried out. Some of these medications have already been introduced into clinics. After the huge success of RNA-based vaccines that were introduced into widespread use in 2020, and the introduction to the market of some RNA-based drugs, RNA therapeutics containing modified nucleotides appear to be the future of medicine.

Numerical Simulation of the Impact of Water Vapour and Moisture Blockers in Energy Diagnostics of Ventilated Partitions.

Current trends towards saving energy and designing sustainable buildings result in most designers focusing on achieving the best thermal parameters, thereby neglecting a careful moisture analysis. Excessive moisture content in building partitions degrades the mechanical properties of materials, reduces thermal insulation properties (which leads to an increase in the demand for thermal energy) and worsens the microclimate in rooms. Modern digital solutions help create appropriate models of partitions that work for many years in good environmental conditions. According to the analysis of air parameters, 1 m3 of air at 20 °C contains approx. 17.3 g of water. When the temperature of the air reaches the dew point temperature, water vapour condenses. The dew point depends on air temperature and relative air humidity; for instance, at the same air temperature of 20 °C, the dew point temperature at 40% relative air humidity is 6 °C, whereas at 90% relative humidity, it is over 18 °C. This means that the higher the value of relative humidity in the room at a certain temperature, the lower the temperature that will cause condensation. The article presents a numerical analysis of the insulation work of flexible materials within the layers of ventilated partitions in an 8-year simulated period of varying environmental conditions. The aim of the article is to analyze different models and variants of ventilated partition operation with respect to the advisability of using a vapour barrier to avoid the problem of destruction of thermal insulation and finishing layers of a ventilated roof.

Solid Truss to Shell Numerical Homogenization of Prefabricated Composite Slabs.

The need for quick and easy deflection calculations of various prefabricated slabs causes simplified procedures and numerical tools to be used more often. Modelling of full 3D finite element (FE) geometry of such plates is not only uneconomical but often requires the use of complex software and advanced numerical knowledge. Therefore, numerical homogenization is an excellent tool, which can be easily employed to simplify a model, especially when accurate modelling is not necessary. Homogenization allows for simplifying a computational model and replacing a complicated composite structure with a homogeneous plate. Here, a numerical homogenization method based on strain energy equivalence is derived. Based on the method proposed, the structure of the prefabricated concrete slabs reinforced with steel spatial trusses is homogenized to a single plate element with an effective stiffness. There is a complete equivalence between the full 3D FE model built with solid elements combined with truss structural elements and the simplified homogenized plate FE model. The method allows for the correct homogenization of any complex composite structures made of both solid and structural elements, without the need to perform advanced numerical analyses. The only requirement is a correctly formulated stiffness matrix of a representative volume element (RVE) and appropriate formulation of the transformation between kinematic constrains on the RVE boundary and generalized strains.

Gold Nanoparticles as Carriers for Functional RNA Nanostructures.

Conjugates of gold nanoparticles and ribonucleic acid are particularly interesting for biological applications to serve as therapeutics or biosensors. In this paper we present, for the first time, a conjugate of gold nanoparticles and structural RNA (tectoRNA), which serves as a tool for gene expression regulation. The tectoRNA trimer was modified to facilitate the introduction of a thiol linker, which aids the formation of stable RNA:AuNP conjugates. We demonstrated that these complexes can penetrate cells, which were observed in TEM analysis and are effective in gene expression regulation evident in GFP expression studies with fluorescence methods. The presented compounds have the potential to become a new generation of therapeutics that utilize the power of self-assembling, biologically active RNAs and gold nanoparticles, with their diagnostically useful optical properties and biocompatibility advantages.

Gold Nanoparticles in Conjunction with Nucleic Acids as a Modern Molecular System for Cellular Delivery.

Development of nanotechnology has become prominent in many fields, such as medicine, electronics, production of materials, and modern drugs. Nanomaterials and nanoparticles have gained recognition owing to the unique biochemical and physical properties. Considering cellular application, it is speculated that nanoparticles can transfer through cell membranes following different routes exclusively owing to their size (up to 100 nm) and surface functionalities. Nanoparticles have capacity to enter cells by themselves but also to carry other molecules through the lipid bilayer. This quality has been utilized in cellular delivery of substances like small chemical drugs or nucleic acids. Different nanoparticles including lipids, silica, and metal nanoparticles have been exploited in conjugation with nucleic acids. However, the noble metal nanoparticles create an alternative, out of which gold nanoparticles (AuNP) are the most common. The hybrids of DNA or RNA and metal nanoparticles can be employed for functional assemblies for variety of applications in medicine, diagnostics or nano-electronics by means of biomarkers, specific imaging probes, or gene expression regulatory function. In this review, we focus on the conjugates of gold nanoparticles and nucleic acids in the view of their potential application for cellular delivery and biomedicine. This review covers the current advances in the nanotechnology of DNA and RNA-AuNP conjugates and their potential applications. We emphasize the crucial role of metal nanoparticles in the nanotechnology of nucleic acids and explore the role of such conjugates in the biological systems. Finally, mechanisms guiding the process of cellular intake, essential for delivery of modern therapeutics, will be discussed.

[Safe usage of chemicals in European Union countries]. / Bezpieczne stosowanie chemikaliów w krajach Unii Europejskiej.

New legal provisions have changed the rules of production, import and use of chemicals in the European Union Member States. Chemical substances present on the common market are covered by registration duty. Manufacturers and importers of substances are obliged to ensure that the risk posed by a given substance is adequately controlled on each stage. Information on substances is to be transferred both downstream and upstream in a supply chain. Substances of very high concern are the subject of authorisation procedure and will be gradually replaced by their safer equivalents. Information on substances of very high concern is transferred to downstream users and consumers. The European Chemicals Agency (ECHA) and Member States Competent Authorities have been established. National Helpdesks on REACH are operating in Member States, giving help to enterprises and other stakeholders.