Aptamer-based assay for high-throughput substrate profiling of RNA decapping enzymes.

Recent years have led to the identification of a number of enzymes responsible for RNA decapping. This has provided a basis for further research to identify their role, dependency and substrate specificity. However, the multiplicity of these enzymes and the complexity of their functions require advanced tools to study them. Here, we report a high-throughput fluorescence intensity assay based on RNA aptamers designed as substrates for decapping enzymes. Using a library of differently capped RNA probes we generated a decapping susceptibility heat map, which confirms previously reported substrate specificities of seven tested hydrolases and uncovers novel. We have also demonstrated the utility of our assay for evaluating inhibitors of viral decapping enzymes and performed kinetic studies of the decapping process. The assay may accelerate the characterization of new decapping enzymes, enable high-throughput screening of inhibitors and facilitate the development of molecular tools for a better understanding of RNA degradation pathways.

Trinucleotide cap analogs with triphosphate chain modifications: synthesis, properties, and evaluation as mRNA capping reagents.

The recent COVID-19 pandemics have demonstrated the great therapeutic potential of in vitro transcribed (IVT) mRNAs, but improvements in their biochemical properties, such as cellular stability, reactogenicity and translational activity, are critical for further practical applications in gene replacement therapy and anticancer immunotherapy. One of the strategies to overcome these limitations is the chemical modification of a unique mRNA 5'-end structure, the 5'-cap, which is responsible for regulating translation at multiple levels. This could be achieved by priming the in vitro transcription reaction with synthetic cap analogs. In this study, we combined a highly efficient trinucleotide IVT capping technology with several modifications of the 5' cap triphosphate bridge to synthesize a series of 16 new cap analogs. We also combined these modifications with epigenetic marks (2'-O-methylation and m6Am) characteristic of mRNA 5'-ends in higher eukaryotes, which was not possible with dinucleotide caps. All analogs were compared for their effect on the interactions with eIF4E protein, IVT priming, susceptibility to decapping, and mRNA translation efficiency in model cell lines. The most promising α-phosphorothiolate modification was also evaluated in an in vivo mouse model. Unexpected differences between some of the analogs were analyzed using a protein cell extract pull-down assay.

Trimethylguanosine cap-fluorescent molecular rotor (TMG-FMR) conjugates are potent, specific snurportin1 ligands enabling visualization in living cells.

The trimethylguanosine (TMG) cap is a motif present inter alia at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo, particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'-O-position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar KD (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by ∼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (Fm/F0) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.

Investigating the Effects of the Physicochemical Properties of Cellulose-Derived Biocarbon on Direct Carbon Solid Oxide Fuel Cell Performance.

This paper presents a study of the characteristic effects of the physicochemical properties of microcrystalline cellulose and a series of biocarbon samples produced from this raw material through thermal conversion at temperatures ranging from 200 °C to 850 °C. Structural studies revealed that the biocarbon samples produced from cellulose had a relatively low degree of graphitization of the carbon and an isometric shape of the carbon particles. Based on thermal investigations using the differential thermal analysis/differential scanning calorimeter method, obtaining fully formed biocarbon samples from cellulose feedstock was possible at about 400 °C. The highest direct carbon solid oxide fuel cell (DC-SOFC) performance was found for biochar samples obtained via thermal treatment at 400-600 °C. The pyrolytic gases from cellulose decomposition had a considerable impact on the achieved current density and power density of the DC-SOFCs supplied by pure cellulose samples or biochars derived from cellulose feedstock at a lower temperature range of 200-400 °C. For the DC-SOFCs supplied by biochars synthesised at higher temperatures of 600-850 °C, the "shuttle delivery mechanism" had a substantial effect. The impact of the carbon oxide concentration in the anode or carbon bed was important for the performance of the DC-SOFCs. Carbon oxide oxidised at the anode to form carbon dioxide, which interacted with the carbon bed to form more carbon oxide. The application of biochar obtained from cellulose alone without an additional catalyst led to moderate electrochemical power output from the DC-SOFCs. The results show that catalysts for the reverse Boudouard reactions occurring in a biocarbon bed are critical to ensuring high performance and stable operation under electrical load, which is crucial for DC-SOFC development.

Photoactivatable mRNA 5' Cap Analogs for RNA-Protein Crosslinking.

Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA-based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5'-end, called 5'-cap. Depending on the 5'-terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5'-end variants. Here, a series of 8 new synthetic 5'-cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap-protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap-binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap-binding subunit in a multi-protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA-related processes.

Effectiveness of UV-C radiation in inactivation of microorganisms on materials with different surface structures.

INTRODUCTION AND OBJECTIVE: Ultraviolet light in the UV-C band is known as germicidal radiation and was widely used for both sterilization of the equipment and creation of a sterile environment. The aim of the study is to assess the effectiveness of inactivation of microorganisms deposited on surfaces with various textures by UV-C radiation disinfection devices. MATERIAL AND METHODS: Five microorganisms (3 bacteria, virus, and fungus) deposited on metal, plastic, and glass surfaces with smooth and rough textures were irradiated with UV-C light emitted by low-pressure mercury lamp and ultraviolet emitting diodes (LEDs), from a distance of 0.5 m, 1 m, and 1.5 m to check their survivability after 20-minute exposure. RESULTS AND CONCLUSIONS: Both tested UV-C sources were effective in inactivation of microorganisms; however, LED emitter was more efficient in this respect than the mercury lamp. The survival rate of microorganisms depended on the UV-C dose, conditioned by the distance from UV-C source being the highest at 0.5 m and the lowest at 1.5 m. For the tested microorganisms, the highest survival rate after UV-C irradiation was usually visible on glass and plastic surfaces. This observation should be considered in all environments where the type of material (from which the elements of technical equipment are manufactured and may be contaminated by specific activities) is important for maintaining the proper level of hygiene and avoiding the unwanted and uncontrolled spread of microbiological pollution.

Microbiological Risks of Traditional Raw Cow's Milk Cheese (Korycinski Cheeses).

Traditional and regional foods have been increasing in popularity among consumers in Poland for many years. The observed trend of searching for natural and authentic taste encourages many producers to craft products from raw milk, including Korycinski cheeses. The aim of this study was to assess the microbiological hazards resulting from the presence of pathogenic bacteria in Korycinski cheeses available in retail trade. The tests were carried out using accredited methods, including the detection of the presence of Salmonella spp., the enumeration of Listeria monocytogenes, the enumeration of coagulase-positive staphylococci, and the detection of staphylococcal enterotoxins in food when the number of coagulase-positive staphylococci in the sample exceeded the limit of 105 cfu/g. The research material consisted of 45 Korycinski cheeses. The tests conducted revealed that Salmonella spp. was not detected in any of the examined cheeses. However, coagulase-positive staphylococci were present in 68.9% of the samples. In as many as 15 tested cheeses, the level of S. aureus contamination was above 105 cfu/g; therefore, these samples were tested for the presence of staphylococcal enterotoxins. The presence of staphylococcal enterotoxins was found in one Korycinski cheese. In four cheeses, the number of L. monocytogenes exceeded the level of 102 cfu/g, the limit specified in Regulation 2073/2005 on microbiological criteria for foodstuffs. The obtained research results confirm the validity of monitoring the microbiological quality of Korycinski cheeses and the need to increase awareness of ensuring proper hygienic conditions of production, including the increased risk associated with unpasteurized milk products.

HIV-1 Infection Reduces NAD Capping of Host Cell snRNA and snoRNA.

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.

Synthesis, kinetic studies, and QSAR of dinucleoside polyphosphate derivatives as human AK1 inhibitors.

Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.

Prevalence of Bacillus cereus in food products in Poland.

INTRODUCTION AND OBJECTIVE: Bacillus cereus is a foodborne pathogen causing two main types of gastrointestinal diseases: emetic and diarrheal. The aim of this study was to investigate the prevalence of the Bacillus cereus group in ready-to-eat (RTE) food products available in retail in Poland. MATERIAL AND METHODS: Samples were collected by Sanitary and Epidemiological Stations within the framework of the national official control and monitoring sampling programme in Poland. In 2016-2020, a total of 45,358 food samples, such as: 'confectionery products and products with cream', as well as 'cereal grains and cereal and flour products', 'milk and milk products', 'sugar and others', 'meat offal and meat products', 'poultry offal and poultry products', 'eggs and egg products', 'fish, seafood and their preserves', 'vegetables' (including legumes), 'coffee, tea, cocoa, fruit, and herbal teas', 'delicatessen and culinary products', and 'foods for particular nutritional uses' were collected. RESULTS: The presence of the presumptive B. cereus group was monitored mainly in two categories of food products: 'confectionery products and products with uncooked cream' and 'confectionery products and products with heat-treated cream'. The number of samples disqualified due to presumptive B. cereus was 339 (0.75%). CONCLUSIONS: This study provides useful information regarding the contamination of RTE products with the B. cereus group, which may have implications for food safety.

Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

Associations Between Physical Fitness, Cognitive Function, and Depression in Nursing Homes Residents Between 60-100 Years of Age in South-Western Poland.

BACKGROUND Healthy aging depends on physical fitness, cognitive function, and emotional well-being. Reduced physical activity in the elderly impacts daily activities, increasing morbidity risk. Cognitive decline affects learning, attention, and independence. Depression, prevalent among the elderly, correlates with loneliness and affects overall health. Physical fitness positively influences cognitive health and mood. This study examines these associations in Polish nursing homes residents. MATERIAL AND METHODS We assessed 93 people aged 60-100 years living in nursing homes. The Short Physical Performance Battery (SPPB) test was used to assess physical fitness. The Abbreviated Mental Test Score (AMTS) was used to assess cognitive functions. The Geriatric Depression Scale (GDS) was used to assess depression. RESULTS In the SPPB test, the mean score was 4.85 points, indicating moderate limitations. On the AMTS, 55% of subjects had cognitive impairment. On the GDS scale, 44% of respondents had depressive symptoms. Seniors without mood disorders were characterized by faster gait compared to those with suspected depressive disorders (P=0.036). Men performed significantly better in the whole SPPB test (P=0.024) and in the standing up from a chair and gait speed tests (P=0.046, P<0.001) compared to women. We found a negative correlation between the AMTS test scores and the SPPB gait test scores and age (P<0.05) and a positive correlation between the SPPB gait test scores and the GDS scores (P<0.05). CONCLUSIONS Older nursing homes' residents in better emotional and cognitive state tended to have faster gait. Men tended to have a higher level of physical fitness compared to women. Older age was associated with worse cognitive state of the examined seniors.

Evaluation of the functional outcome and mobility of patients after stroke depending on their cognitive state.

The study aimed to analyze the functional outcome and mobility in stroke patients depending on their cognitive state. 180 patients after first stroke were divided into four groups: 48 patients without symptoms of cognitive impairment (G1); 38 with mild cognitive impairment without dementia (G2); 47 with mild dementia (G3); 47 with moderate dementia (G4). The Mini Mental State Examination (MMSE), Barthel Index (BI), Sitting Assessment Scale (SAS), Berg Balance Scale, Trunk Control Test and Test Up & Go were used. The tests were carried out at the time of admission to the ward (T1) and at the time of discharge (T2). A statistically significant improvement was demonstrated in all parameters in almost all groups. No significant difference was observed only in groups G1 and G4 in SAS head. Statistically significant differences in BI results in T2 between groups G1 and G4 were noted. The lowest change in BI was observed in the G4. Regression analysis showed that MMSE and BI at T1 and MMSE score at T2 explained the functional status at T2. Cognitive dysfunction at the time of admission to the ward and discharge may determining the patient's functional status at the time of discharge from the ward.

Sample preparation - A crucial step to distinguish metallic and ionic platinum forms and their mobility in soil.

The study is focused on one of the technology-critical elements (TCEs), Pt in the context of distinguishing ionic from metallic forms and assessing sorption capacity of soil. Solid-liquid extraction, cross-comparison of the results of two determination techniques and two decomposition methods enable quantitative determination of the ionic form and, indirectly, NPs in soil and plants. Information about ionic form is obtained after sample digestion with conc. HNO3 and AdSV determination, and total Pt content is determined after digestion using mixture of conc. HNO3 and conc. HCl. The recovery of Pt was in the range 99-110 % for both forms. The mobility (0.43 mol L-1 HAc) of Pt-NPs and Pt (II) was below 1 % even in the presence of citrates (after 2 month incubation). The long-term sorption study indicated that Pt retention (both forms) in the organic soil is related to formation of organic complexes, and the equilibrium is achieved after 2 days. When the soil is enriched in Fe2O3, stronger sorption is observed up to 2 days for both Pt forms (the mobility is 9-14 pp. lower), to finally achieve similar sorption as without modification, reaching 89/90 % for incubation with DI water, and 81/85 % with citrates. The addition of biocarbon/biochar does not play an important role in immobilization of Pt (II) and Pt-NPs.

Stereocontrolled access to thioisosteres of nucleoside di- and triphosphates.

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.

The Plasma Membrane Purinoreceptor P2K1/DORN1 Is Essential in Stomatal Closure Evoked by Extracellular Diadenosine Tetraphosphate (Ap4A) in Arabidopsis thaliana.

Dinucleoside polyphosphates (NpnNs) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, NpnN signal recognition and transduction are still enigmatic. Therefore, the aim of our research was the identification of the NpnN receptor and signal transduction pathways evoked by these nucleotides. Earlier, we proved that purine and pyrimidine NpnNs differentially affect the phenylpropanoid pathway in Vitis vinifera suspension-cultured cells. Here, we report, for the first time, that both diadenosine tetraphosphate (Ap4A) and dicytidine tetraphosphate (Cp4C)-induced stomatal closure in Arabidopsis thaliana. Moreover, we showed that plasma membrane purinoreceptor P2K1/DORN1 (does not respond to nucleotide 1) is essential for Ap4A-induced stomata movements but not for Cp4C. Wild-type Col-0 and the dorn1-3 A. thaliana knockout mutant were used. Examination of the leaf epidermis dorn1-3 mutant provided evidence that P2K1/DORN1 is a part of the signal transduction pathway in stomatal closure evoked by extracellular Ap4A but not by Cp4C. Reactive oxygen species (ROS) are involved in signal transduction caused by Ap4A and Cp4C, leading to stomatal closure. Ap4A induced and Cp4C suppressed the transcriptional response in wild-type plants. Moreover, in dorn1-3 leaves, the effect of Ap4A on gene expression was impaired. The interaction between P2K1/DORN1 and Ap4A leads to changes in the transcription of signalling hubs in signal transduction pathways.

On site separation of inorganic forms of thallium and arsenic in sea water systems followed by ICP-MS determination.

Reduction of Tl(III) and oxidation of As(III), which are unstable speciation forms, start just after sampling as a result of disturbed chemical equilibrium. Separation of inorganic Tl and As species, unchanged, is thus crucial for reliable results of speciation analysis in water systems. Presented here a simple and fast sample pretreatment, based on ion exchange cartridges, which gives the possibility to separate Tl and As species already on the sampling site. Note the reduction of Tl(III) (15%) is in the range of losses typical for standard procedures based on Tl(III) fixation. The use of SCX-3 allows for Tl(III) and SAX for As(III) separation, which are then quantitated in the effluent by ICP-MS. Determination of non-retained species was done after reduction of the sample volume to 2 mL (50-fold preconcentration), which allowed for detection of As concentrations <0.1 ppb and Tl <0.01 ppb. For As, a collision chamber is required. The possibility of direct determination is very important for the forms being in trace amounts in sea water in the vicinity of harbors.

Chemical Modifications of mRNA Ends for Therapeutic Applications.

Messenger ribonucleic acid (mRNA) is the universal cellular instruction for ribosomes to produce proteins. Proteins are responsible for most of the functions of living organisms, and their abnormal structure or activity is the cause of many diseases. mRNA, which is expressed in the cytoplasm and, unlike DNA, does not need to be delivered into the nucleus, appears to be an ideal vehicle for pursuing the idea of gene therapy in which genetic information about proteins is introduced into an organism to exert a therapeutic effect. mRNA molecules of any sequence can be synthesized using the same set of reagents in a cell-free system via a process called in vitro transcription (IVT), which is very convenient for therapeutic applications. However, this does not mean that the path from the idea to the first mRNA-based therapeutic was short and easy. It took 30 years of trial and error in the search for solutions that eventually led to the first mRNA vaccines created in record time during the SARS-CoV-2 pandemic. One of the fundamental problems in the development of RNA-based therapeutics is the legendary instability of mRNA, due to the transient nature of this macromolecule. From the chemical point of view, mRNA is a linear biopolymer composed of four types of ribonucleic subunits ranging in length from a few hundred to hundreds of thousands of nucleotides, with unique structures at its ends: a 5'-cap at the 5'-end and a poly(A) tail at the 3'-end. Both are extremely important for the regulation of translation and mRNA durability. These elements are also convenient sites for sequence-independent labeling of mRNA to create probes for enzymatic assays and tracking of the fate of mRNA in cells and living organisms. Synthetic 5'-cap analogs have played an important role in the studies of mRNA metabolism, and some of them have also been shown to significantly improve the translational properties of mRNA or affect mRNA stability and reactogenicity. The most effective of these is used in clinical trials of mRNA-based anticancer vaccines. Interestingly, thanks to the knowledge gained from the biophysical studies of cap-related processes, even relatively large modifications such as fluorescent tags can be attached to the cap structure without significant effects on the biological properties of the mRNA, if properly designed cap analogs are used. This has been exploited in the development of molecular tools (fluorescently labeled mRNAs) to track these macromolecules in complex biological systems, including organisms. These tools are extremely valuable for better understanding of the cellular mechanisms involved in mRNA metabolism but also for designing therapeutic mRNAs with superior properties. Much less is known about the usefulness/utility of poly(A) tail modifications in the therapeutic context, but it is clear that chemical modifications of poly(A) can also affect biochemical properties of mRNA. This Account is devoted to chemical modifications of both the 5'- and 3'-ends of mRNA aimed at improving the biological properties of mRNA, without interfering with its translational function, and is based on the authors' more than 20 years of experience in this field.

Phosphoryl Trichloride-Method of Determination in Workplace Air.

The aim of this study was to develop and validate a method for determining phosphoryl trichloride in workplace air. The method is based on passing the tested air through a sodium carbonate-impregnated quartz fiber sampling filter. The substance collected on the sampling filter is extracted with ultrapure water. Phosphoryl trichloride is determined as chloride ions (the product of the hydrolysis of phosphoryl trichloride) in the obtained aqueous solutions by ion chromatography with conductometric detection. The developed method enables the determination of phosphoryl trichloride in the air in the concentration range from 0.004 to 0.160 mg/m3. The method is not applicable in the presence of phosphorus trichloride, hydrochloric acid, and its salts in the air. Good validation results were obtained. All requirements of the norm PN-EN 482 were met while developing and validating the method. This method can be used to measure workplace air in order to assess workers' occupational exposure.

Synthesis and Evaluation of Diguanosine Cap Analogs Modified at the C8-Position by Suzuki-Miyaura Cross-Coupling: Discovery of 7-Methylguanosine-Based Molecular Rotors.

Chemical modifications of the mRNA cap structure can enhance the stability, translational properties, and half-life of mRNAs, thereby altering the therapeutic properties of synthetic mRNA. However, cap structure modification is challenging because of the instability of the 5'-5'-triphosphate bridge and N7-methylguanosine. The Suzuki-Miyaura cross-coupling reaction between boronic acid and halogen compound is a mild, convenient, and potentially applicable approach for modifying biomolecules. Herein, we describe two methods to synthesize C8-modified cap structures using the Suzuki-Miyaura cross-coupling reaction. Both methods employed phosphorimidazolide chemistry to form the 5',5'-triphosphate bridge. However, in the first method, the introduction of the modification via the Suzuki-Miyaura cross-coupling reaction at the C8 position occurs postsynthetically, at the dinucleotide level, whereas in the second method, the modification was introduced at the level of the nucleoside 5'-monophosphate, and later, the triphosphate bridge was formed. Both methods were successfully applied to incorporate six different groups (methyl, cyclopropyl, phenyl, 4-dimethylaminophenyl, 4-cyanophenyl, and 1-pyrene) into either the m7G or G moieties of the cap structure. Aromatic substituents at the C8-position of guanosine form a push-pull system that exhibits environment-sensitive fluorescence. We demonstrated that this phenomenon can be harnessed to study the interaction with cap-binding proteins, e.g., eIF4E, DcpS, Nudt16, and snurportin.