One of the key problems in the design of therapeutic and diagnostic oligonucleotides is the attachment of small-molecule ligands for targeted deliveries in such a manner that provides the controlled ...release of the oligonucleotide at a certain moment. Here, we propose a novel, convenient approach for attaching ligands to the 5'-end of the oligonucleotide via biodegradable, acid-labile phosphoramide linkage. The method includes the activation of the 5'-terminal phosphate of the fully protected, support-bound oligonucleotide, followed by interaction with a ligand bearing the primary amino group. This technique is simple to perform, allows for forcing the reaction to completion by adding excess soluble reactant, eliminates the problem of the limited solubility of reagents, and affords the possibility of using different solvents, including water/organic media. We demonstrated the advantages of this approach by synthesizing and characterizing a wide variety of oligonucleotide 5'-conjugates with different ligands, such as cholesterol, aliphatic oleylamine, and
-anisic acid. The developed method suits different types of oligonucleotides (deoxyribo-, 2'-O-methylribo-, ribo-, and others).
Among the great variety of anti-cancer therapeutic strategies, boron neutron capture therapy (BNCT) represents a unique approach that doubles the targeting accuracy due to the precise positioning of ...a neutron beam and the addressed delivery of boron compounds. We have recently demonstrated the principal possibility of using a cell-specific 2'-F-RNA aptamer for the targeted delivery of boron clusters for BNCT. In the present study, we evaluated the amount of boron-loaded aptamer inside the cell via two independent methods: quantitative real-time polymerase chain reaction and inductive coupled plasma-atomic emission spectrometry. Both assays showed that the internalized boron level inside the cell exceeds 1 × 10
atoms/cell. We have synthesized
-dodecaborate conjugates of 2'-F-RNA aptamers GL44 and Waz, with boron clusters attached either at the 3'- or at the 5'-end. The influence of cluster localization was evaluated in BNCT experiments on U-87 MG human glioblastoma cells and normal fibroblasts and subsequent analyses of cell viability via real-time cell monitoring and clonogenic assay. Both conjugates of GL44 aptamer provided a specific decrease in cell viability, while only the 3'-conjugate of the Waz aptamer showed the same effect. Thus, an individual adjustment of boron cluster localization is required for each aptamer. The efficacy of boron-loaded 2'-F-RNA conjugates was comparable to that of
B-boronophenylalanine, so this type of boron delivery agent has good potential for BNCT due to such benefits as precise targeting, low toxicity and the possibility to use boron clusters made of natural, unenriched boron.
Immunoassays based on antibodies as recognizing elements and enzymes as signal-generating modules are extensively used now in clinical lab diagnostics, food, and environmental analyses. However, the ...application of natural enzymes and antibodies has some drawbacks, such as relatively high manufacturing costs, thermal instability, and lot-to-lot variations that lower the reproducibility of results. Oligonucleotide aptamers are able to specifically bind their targets with high affinity and selectivity, so they represent a prospective alternative to protein antibodies for analyte recognition. Their main advantages include thermal stability and long shelf life, cost-efficient chemical synthesis, and negligible batch-to-batch variations. At the same time, a wide variety of non-protein peroxidase mimics are now available that show strong potential to replace protein enzymes. Here, we review and analyze non-protein biosensors that represent a nexus of these two concepts: aptamer-based sensors (aptasensors) with optical detection (colorimetric, luminescent, or fluorescent) based on different peroxidase mimics, such as DNAzymes, nanoparticles, or metal-organic frameworks.
Boron neutron capture therapy (BNCT) is a binary radiotherapeutic approach to the treatment of malignant tumors, especially glioblastoma, the most frequent and incurable brain tumor. For successful ...BNCT, a boron-containing therapeutic agent should provide selective and effective accumulation of 10B isotope inside target cells, which are then destroyed after neutron irradiation. Nucleic acid aptamers look like very prospective candidates for carrying 10B to the tumor cells. This study represents the first example of using 2′-F-RNA aptamer GL44 specific to the human glioblastoma U-87 MG cells as a boron delivery agent for BNCT. The closo-dodecaborate residue was attached to the 5′-end of the aptamer, which was also labeled by the fluorophore at the 3′-end. The resulting bifunctional conjugate showed effective and specific internalization into U-87 MG cells and low toxicity. After incubation with the conjugate, the cells were irradiated by epithermal neutrons on the Budker Institute of Nuclear Physics neutron source. Evaluation of the cell proliferation by real-time cell monitoring and the clonogenic test revealed that boron-loaded aptamer decreased specifically the viability of U-87 MG cells to the extent comparable to that of 10B-boronophenylalanine taken as a control. Therefore, we have demonstrated a proof of principle of employing aptamers for targeted delivery of boron-10 isotope in BNCT. Considering their specificity, ease of synthesis, and large toolkit of chemical approaches for high boron-loading, aptamers provide a promising basis for engineering novel BNCT agents.
Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, ...siRNA components, and antisense agents. Particularly, the
-dodecaborate anion represents a high-boron-containing residue with remarkable chemical stability and low toxicity, and is suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of
-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of
-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of
-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, whereas analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized the DNA/DNA duplex but gave a strong stabilization of the duplex with an RNA target. According to circular dichroism spectroscopy results, two terminal
-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from the B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.
Biosensors that rely on aptamers as analyte-recognizing elements (also known as aptasensors) are gaining in popularity during recent years for analytical and biomedical applications. Among them, ...colorimetric ELISA-like systems seem very promising for biomarker detection in medical diagnostics. For their development, one should thoroughly consider the characteristics of the aptamers, with a particular focus on the secondary structure. In this study, we performed an in-depth structural study of previously selected hemoglobin-binding 2′-F-RNA aptamers using CD spectroscopy, enzymatic probing, and specific fluorophore binding. Only a combination of different assays allowed us to prove G-quadruplex formation for anti-hemoglobin 2′-F-RNA aptamers. We also demonstrated a possible application of these 2′-F-RNA aptamers for microplate colorimetric detection of human hemoglobin in both direct and sandwich formats.
Display omitted
•Confirmed formation of quadruplexes for three hemoglobin-specific 2'-F-RNA aptamers using a combination of experimental assays.•Establsihed an aptamer-based microplate colorimetric detection of human hemoglobin both in direct and sandwich assays.•Demonstrated that hemoglobin detection system based on two aptamers could generate a nonspecific colorimetric signal.
Nucleic acid aptamers capable of affine and specific binding to their molecular targets have now established themselves as a very promising alternative to monoclonal antibodies for diagnostic and ...therapeutic applications. Although the main focus in aptamers’ research and development for biomedicine is made on cardiovascular, infectious, and malignant diseases, the use of aptamers as therapeutic or diagnostic tools in the context of rheumatic diseases is no less important. In this review, we consider the main features of aptamers that make them valuable molecular tools for rheumatologists, and summarize the studies on the selection and application of aptamers for protein biomarkers associated with rheumatic diseases. We discuss the progress in the development of aptamer-based diagnostic assays and targeted therapeutics for rheumatic disorders, future prospects in the field, and issues that have yet to be addressed.
Due to the large amount of pollution at coal spoil heaps, soil reclamation is slow and requires development and use of microbial preparations, selected based on assessments of the biochemical ...parameters of soil and the extent of its contamination. The material for this study was mine soils sampled in three different zones (Ka, Kb, Kc) of the Korchakol coal spoil heap. The studied sanitary and chemical indicators did not exceed the approximate permissible concentrations (APC) / maximum allowable concentrations (MAC), except for oil products (in the Ka, Kb, Kc zones, the excess averaged 2.2, 1.4, 1.2 times, respectively). The study found a direct correlation between the zinc content and the enzymatic activity of polyphenol oxidase, the level of Ni and peroxidase, as well as an inverse correlation between the As and Cu concentrations with invertase and nitrite reductase, respectively. The index of bacteria of the Escherichia coli group (coliform bacteria) in different zones exceeded the standard values by 61 and 171 times. Even though the sanitary indicators studied do not exceed permissible levels, they can slow down growth and development of plants. Thus, successful reclamation of mine soils requires introduction of biological preparations based on microorganisms that biotransform heavy metals and rhizobacteria.
Due to the large amount of pollution at coal spoil heaps, soil reclamation is slow and requires development and use of microbial preparations, selected based on assessments of the biochemical ...parameters of soil and the extent of its contamination. The material for this study was mine soils sampled in three different zones (Ka, Kb, Kc) of the Korchakol coal spoil heap. The studied sanitary and chemical indicators did not exceed the approximate permissible concentrations (APC) / maximum allowable concentrations (MAC), except for oil products (in the Ka, Kb, Kc zones, the excess averaged 2.2, 1.4, 1.2 times, respectively). The study found a direct correlation between the zinc content and the enzymatic activity of polyphenol oxidase, the level of Ni and peroxidase, as well as an inverse correlation between the As and Cu concentrations with invertase and nitrite reductase, respectively. The index of bacteria of the Escherichia coli group (coliform bacteria) in different zones exceeded the standard values by 61 and 171 times. Even though the sanitary indicators studied do not exceed permissible levels, they can slow down growth and development of plants. Thus, successful reclamation of mine soils requires introduction of biological preparations based on microorganisms that biotransform heavy metals and rhizobacteria.
Spondyloarthritis (SpA) is a group of rheumatic disorders characterized by chronic inflammation of the spine, joints, entheses, and other symptoms outside the musculoskeletal system. SpA affects ...0.5%-2% of the global population and leads to significantly decreased life quality, increased mortality, and substantial health care-related costs, imposing a high burden not only on patients, but also on society. There is robust experimental and clinical evidence supporting the central role of interleukin (IL)-17A-pathway in inflammation, bone erosion and fusion associated with SpA. Currently, several antibody-based therapeutics selective to IL-17 are now applied in clinical practice. Nevertheless, the search is still ongoing for alternative targeted molecules with lower manufacturing costs, minimal batch-to-batch variations and tolerance to wide range of transportation and storage conditions. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) aptamers seem to be very promising candidates for targeted therapy, particularly in the context of rheumatic diseases. In this study, we evaluated the effects of anti-IL-17A 2'-F-RNA aptamer Apt3-4 on a mouse model of proteoglycan-induced spondyloarthritis in comparison with a commonly used anti-inflammatory drug, diclofenac. We have shown that the injection of the aptamer alleviates the motor impairment in SpA mice in the motor field test with minimal side effects.
PDF
