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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 S P and R P 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 S P -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 S P -alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment., (© 2024. The Author(s).)

Cyclic tetrapeptides c(Pro-Phe-Pro-Phe) obtained by the mechanosynthetic method using a ball mill were isolated in a pure stereochemical form as a homochiral system (all L-amino acids, sample A) and as a heterochiral system with D configuration at one of the stereogenic centers of Phe (sample B). The structure and stereochemistry of both samples were determined by X-ray diffraction studies of single crystals. In DMSO and acetonitrile, sample A exists as an equimolar mixture of two conformers, while only one is monitored for sample B. The conformational space and energetic preferences for possible conformers were calculated using DFT methods. The distinctly different conformational flexibility of the two samples was experimentally proven by Variable Temperature (VT) and 2D EXSY NMR measurements. Both samples were docked to histone deacetylase HDAC8. Cytotoxic studies proved that none of the tested cyclic peptide is toxic., (© 2024. The Author(s).)

Cognitive functions are lost due to the rapid hydrolysis of acetylcholine including Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE). Marine algae-derived compounds were reported for their neuroprotective activities and hence they can be utilised for treating neurodegenerative ailments like Alzheimer's Disease and Parkinson's Disease which are due to the loss of cognitive functions. Major attention is currently paid to seaweeds due to their health benefits and high nutritional values. Sea weeds are of a rich sense of natural bioactive compounds which antioxidants, pharmaceutical compounds, flavonoids and alkaloids. They also contain a high amount of vitamins A, D, E, C and Ca, K, Mg and Fe. Regular consumption of a marine algae-based diet may boost immunities. In searching for natural cholinesterase inhibitors, the present study is focussed on some marine bioactive compounds reported from brown, red and green algae. Molecular docking studies have been carried out along with molecular dynamics simulations studies and binding energy calculations resulting in three best bioactive compounds when AChE is used as the target. The results are compared with cocrystal studies. Two best compounds, namely, Diphlorethohydroxycarmalol and Phlorofucofuroeckol from the brown seaweeds are identified as the potential lead compounds for neurodegenerative diseases, Alzheimer's and Parkinson's.Communicated by Ramaswamy H. Sarma.

In these studies, we designed and investigated the potential anticancer activity of five iron(II) cyclopentadienyl complexes bearing different phosphine and phosphite ligands. All complexes were characterized with spectroscopic analysis viz. NMR, FT-IR, ESI-MS, UV-Vis, fluorescence, XRD (for four complexes) and elemental analyses. For biological studies, we used three types of cells-normal peripheral blood mononuclear (PBM) cells, leukemic HL-60 cells and non-small-cell lung cancer A549 cells. We evaluated cell viability and DNA damage after cell incubation with these complexes. We observed that all iron(II) complexes were more cytotoxic for HL-60 cells than for A549 cells. The complex CpFe(CO)(P(OPh) 3 )(η 1 -N-maleimidato) 3b was the most cytotoxic with IC 50  = 9.09 µM in HL-60 cells, IC 50  = 19.16 µM in A549 and IC 50  = 5.80 µM in PBM cells. The complex CpFe(CO)(P(Fu) 3 )(η 1 -N-maleimidato) 2b was cytotoxic only for both cancer cell lines, with IC 50  = 10.03 µM in HL-60 cells and IC 50  = 73.54 µM in A549 cells. We also found the genotoxic potential of the complex 2b in both types of cancer cells. However, the complex CpFe(CO) 2 (η 1 -N-maleimidato) 1 which we studied previously, was much more genotoxic than complex 2b, especially for A549 cells. The plasmid relaxation assay showed that iron(II) complexes do not induce strand breaks in fully paired ds-DNA. The DNA titration experiment showed no intercalation of complex 2b into DNA. Molecular docking revealed however that complexes CpFe(CO)(PPh 3 ) (η 1 -N-maleimidato) 2a, 2b, 3b and CpFe(CO)(P(OiPr) 3 )(η 1 -N-maleimidato) 3c have the greatest potential to bind to mismatched DNA. Our studies demonstrated that the iron(II) complex 1 and 2b are the most interesting compounds in terms of selective cytotoxic action against cancer cells. However, the cellular mechanism of their anticancer activity requires further research., (© 2024. The Author(s).)

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 β,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-β,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., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Among lipids, lysophosphatidylcholines (LPCs) with various fatty acyl chains have been identified as potential agonists of G protein-coupled receptors (GPCRs). Recently, targeting GPCRs has been switched to diabetes and obesity. Concomitantly, our last findings indicate the insulin secretagogue properties of cis and trans palmitoleic acid (16:1, n-7) resulting from GPCR activation, however, associated with different signaling pathways. We here report the synthesis of LPCs bearing two geometrical isomers of palmitoleic acids and investigation of their impact on human pancreatic β cells viability, insulin secretion, and activation of four GPCRs previously demonstrated to be targeted by free fatty acids and LPCs. Moreover, molecular modeling was exploited to investigate the probable binding sites of tested ligands and calculate their affinity toward GPR40, GPR55, GPR119, and GPR120 receptors., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

In silico studies were performed to assess the binding affinity of selected organophosphorus compounds toward the acetylcholinesterase enzyme (AChE). Quantum mechanical calculations, molecular docking, and molecular dynamics (MD) with molecular mechanics Generalized-Born surface area (MM/GBSA) were applied to assess quantitatively differences between the binding energies of acetylcholine (ACh; the natural agonist of AChE) and neurotoxic, synthetic correlatives (so-called "Novichoks", and selected compounds from the G- and V-series). Several additional quantitative descriptors like root-mean-square fluctuation (RMSF) and the solvent accessible surface area (SASA) were briefly discussed to give-to the best of our knowledge-the first quantitative in silico description of AChE-Novichok non-covalent binding process and thus facilitate the search for an efficient and effective treatment for Novichok intoxication and in a broader sense-intoxication with other warfare nerve agents as well.

Dietary trans -palmitoleic acid ( trans 16:1n-7, t POA), a biomarker for high-fat dairy product intake, has been associated with a lower risk of type 2 diabetes mellitus (T2DM) in some cross-sectional and prospective epidemiological studies. Here, we investigated the insulin secretion-promoting activity of t POA and compared them with the effects evoked by the cis -POA isomer ( c POA), an endogenous lipokine biosynthesized in the liver and adipose tissue, and found in some natural food sources. The debate about the positive and negative relationships of those two POA isomers with metabolic risk factors and the underlying mechanisms is still going on. Therefore, we examined the potency of both POA isomers to potentiate insulin secretion in murine and human pancreatic β cell lines. We also investigated whether POA isomers activate G protein-coupled receptors proposed as potential targets for T2DM treatment. We show that t POA and c POA augment glucose-stimulated insulin secretion (GSIS) to a similar extent; however, their insulin secretagogue activity is associated with different signaling pathways. We also performed ligand docking and molecular dynamics simulations to predict the preferred orientation of POA isomers and the strength of association between those two fatty acids and GPR40, GPR55, GPR119, and GPR120 receptors. Overall, this study provides insight into the bioactivity of t POA and c POA toward selected GPCR functions, indicating them as targets responsible for the insulin secretagogue action of POA isomers. It reveals that both t POA and c POA may promote insulin secretion and subsequently regulate glucose homeostasis.

In these studies, we designed and investigated cyto- and genotoxic potential of five ruthenium cyclopentadienyl complexes bearing different phosphine and phosphite ligands. All of the complexes were characterized with spectroscopic analysis (NMR, FT-IR, ESI-MS, UV-vis, fluorescence and XRD (for two compounds)). For biological studies, we used three types of cells - normal peripheral blood mononuclear (PBM) cells, leukemic HL-60 cells and doxorubicin-resistance HL-60 cells (HL-60/DR). We compared the results obtained with those obtained for the complex with maleimide ligand CpRu(CO) 2 (η 1 - N -maleimidato) 1, which we had previously reported. We observed that the complexes CpRu(CO)(PPh 3 )(η 1 - N -maleimidato) 2a and CpRu(CO)(P(OEt) 3 )(η 1 - N -maleimidato) 3a were the most cytotoxic for HL-60 cells and non-cytotoxic for normal PBM cells. However, complex 1 was more cytotoxic for HL-60 cells than complexes 2a and 3a (IC 50 = 6.39 μM vs . IC 50 = 21.48 μM and IC 50 = 12.25 μM, respectively). The complex CpRu(CO)(P(OPh) 3 )(η 1 - N -maleimidato) 3b is the most cytotoxic for HL-60/DR cells (IC 50 = 104.35 μM). We found the genotoxic potential of complexes 2a and 3a only in HL-60 cells. These complexes also induced apoptosis in HL-60 cells. Docking studies showed that complexes 2a and CpRu(CO)(P(Fu) 3 )(η 1 - N -maleimidato) 2b have a small ability to degrade DNA, but they may cause a defect in DNA damage repair mechanisms leading to cell death. This hypothesis is corroborated with the results obtained in the plasmid relaxation assay in which ruthenium complexes bearing phosphine and phosphite ligands induce DNA breaks.

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Adenosine triphosphate (ATP) is one of the most important molecules of life, present both inside the cells and extracellularly. It is an essential building block for nucleic acids biosynthesis and crucial intracellular energy storage. However, one of the most interesting functions of ATP is the role of a signaling molecule. Numerous studies indicate the involvement of ATP-dependent pathways in maintaining the proper functioning of individual tissues and organs. Herein, the latest data indicating the ATP function in the network of intra- and extracellular signaling pathways including purinergic signaling, MAP kinase pathway, mTOR and calcium signaling are collected. The main ATP-dependent processes maintaining the proper functioning of the nervous, cardiovascular and immune systems, as well as skin and bones, are summarized. The disturbances in the ATP amount, its cellular localization, or interaction with target elements may induce pathological changes in signaling pathways leading to the development of serious diseases. The impact of an ATP imbalance on the development of dangerous health dysfunctions such as neurodegeneration diseases, cardiovascular diseases (CVDs), diabetes mellitus, obesity, cancers and immune pathogenesis are discussed here., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The quest to find new inhibitors of biologically relevant targets is considered an important strategy to introduce new drug candidates for the treatment of neurodegenerative diseases. A series of (aminomethyl)benzylphosphonates 8a - c and their metallocarbonyl iron 9a - c and ruthenium 10a - c complexes were designed, synthesized, and evaluated for their inhibitory potentials against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by determination of IC 50 . Metallocarbonyl derivatives, in general, did not show significant inhibition activity against these enzymes, the most potent inhibitor was the (aminomethyl)benzylphosphonate 8a (IC 50 = 1.215 µM against AChE). Molecular docking analysis of AChE and (aminomethyl)benzylphosphonates 8a - c showed the strongest interactions of 8a and AChE compared to isomers 8b and 8c . Cytotoxicity studies of synthesized compounds towards the V79 cell line were also performed and discussed.

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.

Tissue-nonspecific alkaline phosphatase (TNAP) is known to be involved in the degradation of extracellular ATP via the hydrolysis of pyrophosphate (PPi). We investigated, using three different computational methods, namely molecular docking, thermodynamic integration (TI) and conventional molecular dynamics (MD), whether TNAP may also be involved in the utilization of β,γ-modified ATP analogues. For that, we analyzed the interaction of bisphosphonates with this enzyme and evaluated the obtained structures using in silico studies. Complexes formed between pyrophosphate, hypophosphate, imidodiphosphate, methylenediphosphonic acid monothiopyrophosphate, alendronate, pamidronate and zoledronate with TNAP were generated and analyzed based on ligand docking, molecular dynamics and thermodynamic integration. The obtained results indicate that all selected ligands show high affinity toward this enzyme. The forming complexes are stabilized through hydrogen bonds, electrostatic interactions and van der Waals forces. Short- and middle-term molecular dynamics simulations yielded very similar affinity results and confirmed the stability of the protein and its complexes. The results suggest that certain effectors may have a significant impact on the enzyme, changing its properties.

Bisphosphonates constitute a group of pyrophosphate analogues therapeutically active against bone diseases. Numerous studies confirm their anticancer and antimetastatic potential as well as ability to relieve pathological pain. Although this is a known class of compounds, many aspects of their action remain unexplained and their new interaction partners are still being discovered. Due to the structural similarity to pyrophosphate, their interaction with pyrophosphate-recognizing enzymes seems to be feasible. In current work, the placental alkaline phosphatase (PLAP) is considered as a potential target for these class of compounds. PLAP is one of the enzymes responsible for degradation of pyrophosphate with high clinical significance. An elevation of PLAP level are considered as a potential cancer marker. An in silico study of complexes formed between selected phosphate derivatives and PLAP was performed. It indicates that all tested compounds: alendronic acid, clodronic acid, etidronic acid, zoledronic acid, imidodiphosphoric acid, pyrophosphoric acid, medronic acid, chloromethylenediphosphonic acid and hypophosphoric acid form a complexes with PLAP, stabilized by hydrogen bonds, hydrophobic and van der Waals interactions. Zoledronic acid, drug used in prevention of bone complications during cancer treatment was found to have the lowest estimated energy of binding (-6.6 kcal/mol). In silico study yielded very low energy of binding also for hypophosphate, equal -6.4 kcal/mol, despite having no identified hydrogen bonds. Subsequent molecular dynamic simulations, followed by molecular mechanics generalized-born surface area with pairwise decomposition calculations confirmed the stability of protein-ligand complexes. The results indicate that selected phosphate derivatives may potentially interact with the enzyme, changing its function, what should be investigated during in vitro studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Two, well defined binary crystals with 1-Hydroxy-4,5-Dimethyl-Imidazole 3-Oxide (HIMO) as coformer and thiobarbituric acid (TBA) as well barbituric acid (BA) as Active Pharmaceutical Ingredients (APIs) were obtained by cocrystallization (from methanol) or mechanochemically by grinding. The progress of cocrystal formation in a ball mill was monitored by means of high-resolution, solid state NMR spectroscopy. The 13 C CP/MAS, 15 N CP/MAS and 1 H Very Fast (VF) MAS NMR procedures were employed to inspect the tautomeric forms of the APIs, structure elucidation of the coformer and the obtained cocrystals. Single crystal X-ray studies allowed us to define the molecular structure and crystal packing for the coformer as well as the TBA/HIMO and BA/HIMO cocrystals. The intermolecular hydrogen bonding, π-π interactions and CH-π contacts responsible for higher order organization of supramolecular structures were determined. Biological studies of HIMO and the obtained cocrystals suggest that these complexes are not cytotoxic and can potentially be considered as therapeutic materials.

Fluorescent imaging, which is an important interdisciplinary field bridging research from organic chemistry, biochemistry and cell biology has been applied for multi-dimensional detection, visualization and characterization of biological structures and processes. Especially valuable is the possibility to monitor cellular processes in real time using fluorescent probes. In this work, conjugated oligoelectrolytes and neutral derivatives with the distyrylnaphthalene core (SN-COEs) were designed, synthetized and tested for biological properties as membrane-specific fluorescent dyes for the visualization of membrane-dependent cellular processes. The group of tested compounds includes newly synthesized distyrylnaphthalene derivatives (DSNNs): a trimethylammonium derivative (DSNN-NMe 3 + ), a phosphonate derivative (DSNN-P), a morpholine derivative (DSNN-Mor), a dihydroxyethylamine derivative (DSNN-DEA), a phosphonate potassium salt (DSNN-POK), an amino derivative (DSNN-NH 2 ) and pyridinium derivative (DSNN-Py+). All compounds were tested for their biological properties, including cytotoxicity and staining efficiency towards mammalian cells. The fluorescence intensity of SN-COEs incorporated into cellular structures was analyzed by fluorescence activated cell sorting (FACS) and photoluminescence spectroscopy. The cytotoxicity results have shown that all tested SN-COEs can be safely used in the human and animal cell studies. Fluorescence and confocal microscopy observations confirm that tested COEs can be applied as fluorescent probes for the visualization of intracellular membrane components in a wide range of different cell types, including adherent and suspension cells. The staining procedure may be performed under both serum free and complete medium conditions. The presented studies have revealed the interesting biological properties of SN-COEs and confirmed their applicability as dyes for staining the membranous structures of eukaryotic cells, which may be useful for visualization of wide range of biological processes dependent of the extra-/intracellular communications and/or based on the remodeling of cellular membranes.

This research aims to utilize sericin, which is the waste from boiling silk cocoon, for the supramolecular scaffold preparation with chitosan. A suitable method for the self-assembled scaffold formation of sericin and chitosan at 1:1 stoichiometry is presented and the morphological and physical properties of the scaffold are studied. The effect of an alcohol/NaOH solution on the secondary structure of sericin protein within the sericin-chitosan scaffold, with adjusted pH, was investigated. Additionally, the scaffold was tested in a native phosphate buffer solution (PBS). The results show that sericin increases the porosity of scaffold while chitosan increases the rigidity. The self-assembled sericin and chitosan material is nontoxic to human cells and which can adhere and spread well on such support. For the effect of the molecular weight of chitosan (15,000 and 100,000 g/mol), the scaffold made from lower molecular weight (MW) chitosan provides a somewhat smaller porosity, but a similar swelling ratio and water uptake. On the basis of this research, sericin, which is a silk waste from the textile industry, can be utilized to produce a self-assembled scaffold with chitosan in order to increase the porosity of the scaffold. This type of scaffold is not toxic and can be used for the adhesion of fibroblast cells.

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.

Purpose: Conjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2., Methods: The 1 H NMR, 13 C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively., Results: The PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only., Conclusions: Our results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel.

In this study we have investigated the effects of pH and surfactant on the internal charge transfer (ICT) process in the DSNN derivative, DSNN-NMe + 3 (4,4'-bis(4'-(N,N-bis(6″-(N,N,N-trimethylammonium)hexyl)amino)-styryl) naphthalene tetraiodide) with the aim to show that environmentally-induced changes in the degree of ICT process determine the spectral response of the DSNN chromophore. Obtained results showed that DSNN chromophore exhibits evident changes in linear optical properties (absorption/emission wavelengths, quantum yield) upon protonation. These changes are a manifestation of the attenuation of the internal charge transfer processes, which accompanies binding of proton to the nitrogen atoms of the dialkylamino groups at the termini of DSNN chromophore. The results obtained in this study clearly demonstrated the sensitivity of the ICT process in DSNN upon protonation, which, together with the affinity of DSNN towards biological and artificial membranes, may open new perspectives for its utility in fluorescence-based sensing. Moreover, the studied compound showed substantial surfactochromic effects in the ionic and non-ionic surfactant solutions, which indicate the formation of various self-organized DSNN-surfactant aggregates. The structure of these aggregates is determined by the type of specific intermolecular interactions between the chromophore and surfactant molecules. The knowledge of the nature of these interactions may be substantial in the future development of DSNN-based sensing platforms with suitable optical properties., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the search for enzymatically processed RNA fragments, we found the novel three-way junction motif. The structure prediction suggested the arrangement of helices at acute angle approx. 60°. This allows the design of a trimeric RNA nanoparticle that can be functionalized with multiple regulatory fragments. Such RNA nano-object of equilateral triangular shape was applied for gene expression regulation studies in two independent cellular systems. Biochemical and functional studies confirmed the predicted shape and structure of the nanoparticle. The regulatory siRNA fragments incorporated into the nanoparticle were effectively released and triggered gene silencing. The regulatory effect was prolonged when induced with structuralized RNA compared to unstructured siRNAs. In these studies, the enzymatic processing of the motif was utilized for function release from the nanoparticle, enabling simultaneous delivery of different regulatory functions. This methodology of sequence search, RNA structural prediction, and application for rational design opens a new way for creating enzymatically processed RNA nanoparticles.

The strategy utilizing trastuzumab, a humanized monoclonal antibody against human epidermal growth receptor 2 (HER-2), as a therapeutic agent in HER-2 positive breast cancer therapy seems to have advantage over traditional chemotherapy, especially when given in combination with anticancer drugs. However, the effectiveness of single antibody or antibody conjugated with chemotherapeutics is still far from ideal. Antibody⁻dendrimer conjugates hold the potential to improve the targeting and release of active substance at the tumor site. In the present study, we developed and synthesized PAMAM dendrimer⁻trastuzumab conjugates carrying doxorubicin (dox) specifically to cells overexpressing HER-2. ¹HNMR, FTIR and RP-HPLC were used to characterize the products and analyze their purity. Toxicity of PAMAM⁻trastuzumab and PAMAM⁻dox⁻trastuzumab conjugates compared with free trastuzumab and doxorubicin towards HER-2 positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines was determined using MTT assay. Furthermore, the cellular uptake and cellular localization were studied by flow cytometry and confocal microscopy, respectively. A cytotoxicity profile of above mentioned compounds indicated that conjugate PAMAM⁻dox⁻trastuzumab was more effective when compared to free drug or the conjugate PAMAM⁻trastuzumab. Moreover, these results reveal that trastuzumab can be used as a targeting agent in PAMAM⁻dox⁻trastuzumab conjugate. Therefore PAMAM⁻dox⁻trastuzumab conjugate might be an interesting proposition which could lead to improvements in the effectiveness of drug delivery systems for tumors that overexpress HER-2., Competing Interests: The authors declare no conflict of interest.

Natural stable RNAs fold and assemble into complex three-dimensional architectures by relying on the hierarchical formation of intricate, recurrent networks of noncovalent tertiary interactions. These sequence-dependent networks specify RNA structural modules enabling orientational and topological control of helical struts to form larger self-folding domains. Borrowing concepts from linguistics, we defined an extended structural syntax of RNA modules for programming RNA strands to assemble into complex, responsive nanostructures under both thermodynamic and kinetic control. Based on this syntax, various RNA building blocks promote the multimolecular assembly of objects with well-defined three-dimensional shapes as well as the isothermal folding of long RNAs into complex single-stranded nanostructures during transcription. This work offers a glimpse of the limitless potential of RNA as an informational medium for designing programmable and functional nanomaterials useful for synthetic biology, nanomedicine, and nanotechnology.

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining was determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The rapid development of synthetic biology and nanobiotechnology has led to the construction of various synthetic RNA nanoparticles of different functionalities and potential applications. As they occur naturally, nucleic acids are an attractive construction material for biocompatible nanoscaffold and nanomachine design. In this review, we provide an overview of the types of RNA and nucleic acid's nanoparticle design, with the focus on relevant nanostructures utilized for gene-expression regulation in cellular models. Structural analysis and modeling is addressed along with the tools available for RNA structural prediction. The functionalization of RNA-based nanoparticles leading to prospective applications of such constructs in potential therapies is shown. The route from the nanoparticle design and modeling through synthesis and functionalization to cellular application is also described. For a better understanding of the fate of targeted RNA after delivery, an overview of RNA processing inside the cell is also provided.

RNA architectonics offers the possibility to design and assemble RNA into specific shapes, such as nanoscale 3D solids or nanogrids. Combining the minute size of these programmable shapes with precise positioning on a surface further enhances their potential as building blocks in nanotechnology and nanomedicine. Here we describe a bottom-up approach to direct the arrangement of nucleic acid nanostructures by using a supported fluid lipid bilayer as a surface scaffold. The strong attractive electrostatic interactions between RNA polyanions and cationic lipids promote RNA adsorption and self-assembly. Protocol steps for the characterization of assembled RNA complexes via several complementary methods (QCM-D, ellipsometry, confocal fluorescence microscopy, AFM) are also provided. Due to their tunable nature, lipid bilayers can be used to organize RNA laterally on the micrometer scale and thus facilitate the building of more complex 3D structures. The bilayer-based approach can be extended to other programmable RNA or DNA objects to construct intricate structures, such as 2D grids or 3D cages, with high precision.

T4 DNA ligase is one of the most commonly used enzymes for in vitro molecular research and a useful model for testing the ligation mechanism of ATP-dependent DNA ligation. To better understand the influence of phosphate group modifications in the ligation process, a series of ATP analogs were tested as cofactors. P-diastereomers of newly developed β,γ-hypo-ATPαS (thio) and β,γ-hypo-ATP (oxo) were synthesized and their activity was compared to ATPαS and their natural precursors. The evaluation of presented ATP analogs revealed the importance of the α-phosphate stereogenic center in ATPαS for the T4 DNA ligase activity and sheds new light on the interaction between ATP-dependent DNA ligases and cofactors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In times, when drug seeking assays focus on the natural molecular triggers and their analogs, a deeper insight into molecular mechanisms governing the initial step of intrinsic apoptosis (cytochrome c release) is essential to suppress the immortality of pathologically changed cells. In this study, we examined RNA molecules mimicking mitochondrial tRNAs interacting with cytochrome c and possibly affecting its cellular function. tRNA analogs were designed and synthesized prior to the conformational analysis and gel assays clearly stating the nucleic acid-protein complex formation. The circular dichroism spectroscopic (CD) and microscale thermophoresis examination revealed the structural and conformational differences between four tRNA analogs in their interactions with cytochrome c. Obtained CD spectra and gel studies resulted in the complex ratio estimation and conclusion that not only the complex formation may be preferential towards specific tRNAs present in the cell, but nucleobase modifications are not essential for such interaction.

The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.

Conjugated phenylenevinylene oligoelectrolytes, which consist of a phenylenevinylene core equipped at each end with hydrophilic pendent groups, are shown to be good candidates for mammalian cell membrane staining. When used in the micromolar concentration range, they express low to moderate cell toxicity for selected regular and cancerous cell lines as tested for adherent and suspension cells.

Conjugated oligoelectrolytes are of emerging technological interest due to their recent function in the fabrication of optoelectronic devices, application in biosensors, and as species that facilitate transmembrane charge migration. Solubility in aqueous, or highly polar, solvents is important for many of these applications; however, there are few studies on how the self-assembly of conjugated oligoelectrolytes into multichromophore species influences linear and nonlinear optical properties. Here, we examine 1,4-bis(4'-(N,N-bis(6''-(N,N,N-trimethylammonium)hexyl)amino)-styryl)benzene tetraiodide (DSBNI) in water, a conjugated oligoelectrolyte based on the distyrylbenzene framework. We find that DSBNI aggregation leads to increased fluorescence lifetimes, coupled with hypsochromic shifts, and larger two-photon absorption cross sections. Liquid atomic force microscopy (AFM) and cryogenic transmission electron microscopy (cryo-TEM) were used to image DSBNI aggregates and to confirm that the planar molecules stack to form nanocylinders above a critical aggregation concentration. Finally, small-angle neutron scattering (SANS) was used to quantify the aggregate dimensions in situ. Comparison of the results highlights that the hydrophilic mica surface used to image via liquid AFM and the high concentrations required for cryo-TEM facilitate the propagation of the cylinders into long fibers. SANS experiments are consistent with equivalent molecular packing geometry but lower aspect ratios. It is therefore possible to understand the evolution of optical properties as a function of concentration and aggregation and the general geometric features of the resulting supramolecular structures., (© 2011 American Chemical Society)

Bottom-up fabrication of self-assembled nanomaterials requires control over forces and interactions between building blocks. We report here on the formation and architecture of supramolecular structures constructed from two different peptide amphiphiles. Inclusion of four alanines between a 16-mer peptide and a 16 carbon long aliphatic tail resulted in a secondary structure shift of the peptide headgroups from α helices to β sheets. A concomitant shift in self-assembled morphology from nanoribbons to core-shell worm-like micelles was observed by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In the presence of divalent magnesium ions, these a priori formed supramolecular structures interacted in distinct manners, highlighting the importance of peptide amphiphile design in self-assembly.

RNA is an attractive biopolymer for nanodesign of self-assembling particles for nanobiotechnology and synthetic biology. Here, we experimentally characterize by biochemical and biophysical methods the formation of thermostable and ribonuclease resistant RNA nanorings previously proposed by computational design. High yields of fully programmable nanorings were produced based on several RNAI/IIi kissing complex variants selected for their ability to promote polygon self-assembly. This self-assembly strategy relying on the particular geometry of bended kissing complexes has potential for developing short interfering RNA delivery agents.

A common affinity tag used to express and purify fusion proteins is glutathione S-transferase. However, many researchers have reported difficulty eluting GST-tagged proteins from the affinity matrix. This report demonstrates that the problem likely is due to the propensity of glutathione S-transferase to dimerize combined with a propensity of the tagged protein to oligomerize, which results in formation of large oligomers of fusion protein that are chelated by the affinity matrix. The solution to the problem is to use S-butylglutathione instead of glutathione to elute, as S-butylglutathione binds more tightly to glutathione S-transferase and overcomes the chelate effect. Moreover, in contrast to glutathione, S-butylglutathione has no reducing capability that might inactivate a tagged protein., (Copyright © 2010 Elsevier Inc. All rights reserved.)

RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.

Electrostatic complexes containing a cationic conjugated oligoelectrolyte (COE) and fluorescein (FAM)-labeled single-stranded DNA (ssDNA) serve as the basis for identifying various bacteria. The approach involves the preparation of five COE/ssDNA(x)-FAM electrostatic complexes, which differ in the ssDNA composition and which provide different photoluminescence (PL) spectra as a result of different degrees of energy transfer efficiency from the COE to FAM. Changes in the PL spectra upon addition of the bacteria can be quantified, and the differential response from the five ssDNAs gives rise to a multicomponent array response that allows identification of the microorganism under investigation.

Supramolecular assembly is a powerful strategy used by nature to build nanoscale architectures with predefined sizes and shapes. With synthetic systems, however, numerous challenges remain to be solved before precise control over the synthesis, folding and assembly of rationally designed three-dimensional nano-objects made of RNA can be achieved. Here, using the transfer RNA molecule as a structural building block, we report the design, efficient synthesis and structural characterization of stable, modular three-dimensional particles adopting the polyhedral geometry of a non-uniform square antiprism. The spatial control within the final architecture allows the precise positioning and encapsulation of proteins. This work demonstrates that a remarkable degree of structural control can be achieved with RNA structural motifs for the construction of thermostable three-dimensional nano-architectures that do not rely on helix bundles or tensegrity. RNA three-dimensional particles could potentially be used as carriers or scaffolds in nanomedicine and synthetic biology.

We report on the modification of membranes by incorporation of phenylenevinylene oligoelectrolytes with the goal of tailoring their optical and electronic properties and their applications. A water-soluble distyrylstilbene oligoelectrolyte (DSSN+), capped at each end with nitrogen bound, terminally charged pendant groups, was synthesized. The photophysical and solvatochromatic properties of DSSN+ and the shorter distyrylbenzene analogue DSBN+ were probed and found to be useful for characterizing insertion into membranes based on phospholipid vesicle systems. A combination of UV/visible absorbance and photoluminescence spectroscopies, together with confocal microscopy, were employed to confirm membrane incorporation. Examination of the emission intensity profile in stationary multilamellar vesicles obtained with a polarized excitation source provides insight into the orientation of these chromophores within lipid bilayers and indicates that these molecules are highly ordered, such that the hydrophobic electronically delocalized region positions within the inner membrane with the long molecular axis perpendicular to the bilayer plane. Cyclic voltammetry experiments provide evidence that DSSN+ and DSBN+ facilitate transmembrane electron transport across lipid bilayers supported on glassy carbon electrodes. Additionally, the interaction with living microorganisms was probed. Fluorescence imaging indicates that DSSN+ and DSBN+ preferentially accumulate within cell membranes. Furthermore, notable increases in yeast microbial fuel cell performance were observed when employing DSSN+ as the electron transport mediator.

Tripod-shaped molecules were designed for chemical modification of the surface of probes used for atomic force microscopy (AFM). These chemically functionalized tips were used for chemical force spectroscopy (CFS) measurements of the ligand-protein receptor interaction in a biotin-NeutrAvidin model system. We demonstrate that by using this unique tripodal system, we can achieve significantly lower density of ligand on the AFM tip apex, which is optimal for true single molecule measurements. Furthermore, the molecular tripods form highly stable bonds to the AFM probes, leading to more robust and reproducible unbinding force data, thereby addressing one of the challenges in CFS studies. Histogram analysis of the hundreds of collected unbinding forces showed a specific distribution with a peak force maximum at approximately 165 pN, in good agreement with the previously reported data of single rupture events of biotin-avidin. We compared these molecular tripod tips with a molecular monopod. The results showed that the molecular tripods are more robust for repeated measurements. The distinct biotin-avidin force maximum was not observed in the control experiments. This indicated that the force distribution observed for molecular tripods corresponds to the specific rupture force between biotin and avidin. The improved robustness of molecular tripods for CFS will provide benefits in other ligand-receptor unbinding studies, including those of transmembrane receptor systems, which require high resolution, sensitivity, and reproducibility in force spectroscopy measurements.

The structural information encoding specific conformations of natural RNAs can be implemented within artificial RNA sequences to control both three-dimensional (3D) shape and self-assembling interfaces for nanotechnology and synthetic biology applications. We have identified three natural RNA motifs known to direct helical topology into approximately 90 degrees bends: a five-way tRNA junction, a three-way junction, and a two-helix bend. These three motifs, embedded within rationally designed RNAs (tectoRNA), were chosen for generating square-shaped tetrameric RNA nanoparticles. The ability of each motif to direct the formation of supramolecular assemblies was compared by both native gel assays and atomic force microscopy. While there are multiple structural solutions for building square-shaped RNA particles, differences in the thermodynamics and molecular dynamics of the 90 degrees motif can lead to different biophysical behaviors for the resulting supramolecular complexes. We demonstrate via structural assembly programming how the different 90 degrees motifs can preferentially direct the formation of either 2D or 3D assemblies.

We detail a method originally described by Okahata et al. (Macromol. Rapid Commun. 2002, 23, 252-255) to prepare noncovalent self-assembling films by exchanging the counter-ions of the nucleic acid phosphate moieties with those of cationic lipid amphiphiles. We are able to control the strength and surface properties of these films by varying the composition between blends of DNA of high molecular weight and RNA of low molecular weight. X-ray and AFM results indicate that these films have a lamellar multilayered structure with layers of nucleic acid separated by layers of cationic amphiphile. The tensile strength of the blended films between DNA and RNA increases elastically with DNA content. The length as well as the molecular structure of nucleic acids can affect the topology and mechanical properties of these films. We suspect that the permeability properties of these films make them good candidates for further biological applications in vivo.

The ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes. Here, preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. Cloning, overexpression and purification resulted in highly pure protein that crystallized readily under many different conditions. In all cases the protein forms thin hexagonal plate-shaped crystals belonging to space group P3 that are of unusually high stability against different solvent conditions. The crystals diffracted to a resolution of 2.0 A using synchrotron radiation but proved to be radiation-sensitive. Preparations of heavy-atom-derivatized crystals for use in determining the three-dimensional structure are under way.

The past several years have witnessed the emergence of a new world of nucleic-acid-based architectures with highly predictable and programmable self-assembly properties. For almost two decades, DNA has been the primary material for nucleic acid nanoconstruction. More recently, the dramatic increase in RNA structural information led to the development of RNA architectonics, the scientific study of the principles of RNA architecture with the aim of constructing RNA nanostructures of any arbitrary size and shape. The remarkable modularity and the distinct but complementary nature of RNA and DNA nanomaterials are revealed by the various self-assembly strategies that aim to achieve control of the arrangement of matter at a nanoscale level.

Using atomic force microscopy, we describe the linear arrangement of cationic gold nanoparticles directed by programmable self-assembling RNA ladders and demonstrate that the regular spacing of nanoparticles is controlled by the RNA architecture acting as nanocrown scaffoldings. Thus, precise positioning of molecular components can be accomplished with RNA not only through electrostatic but also via size and shape recognitions.

One challenge in supramolecular chemistry is the design of versatile, self-assembling building blocks to attain total control of arrangement of matter at a molecular level. We have achieved reliable prediction and design of the three-dimensional structure of artificial RNA building blocks to generate molecular jigsaw puzzle units called tectosquares. They can be programmed with control over their geometry, topology, directionality, and addressability to algorithmically self-assemble into a variety of complex nanoscopic fabrics with predefined periodic and aperiodic patterns and finite dimensions. This work emphasizes the modular and hierarchical characteristics of RNA by showing that small RNA structural motifs can code the precise topology of large molecular architectures. It demonstrates that fully addressable materials based on RNA can be synthesized and provides insights into self-assembly processes involving large populations of RNA molecules.

The study of the biological consequences of oxidative damage to DNA requires the characterization of DNA lesions at a molecular level. The oxidation of guanine in the dinucleoside monophosphate, d(GpT), by a metal-oxo porphyrin produces a dehydro-guanidinohydantoin derivative. The (1)H NMR spectrum of this oxidized guanine derivative is reported. The structure was confirmed by the chemical transformation of the dehydro-guanidinohydantoin derivative into a linear oxaluric acid derivative upon hydrolysis at ambient temperature. The proposed mechanism of formation of the dehydro-guanidinohydantoin derivative is in agreement with the labeling experiments performed with H(2)(18)O.