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It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

Abasic (AP) sites in mRNAs are lesions whose accumulation in cells is linked to various neurodegenerative diseases arising from the appearance of truncated peptides due to the premature cessation of translation of these mRNAs. It is believed that the translation of AP site-containing mRNAs is stopped when the damaged codon arrives to the A site, where it is not decoded. We propose an alternative translation arrest mechanism mediated by the 40S ribosomal subunit protein uS3. Recently, it has been shown that in human 80S ribosomal complexes assembled without translation factors, uS3 cross-links to the AP site at the 3'-terminus of the mRNA, whose undamaged part is bound at the 40S subunit channel, via its peptide 55-64 exposed near the mRNA entry pore. In this study, we examined whether such cross-linking occurs during the translation of mRNA with the AP site. To this end, we used a set of synthetic mRNAs bearing the AP site inserted in the desired location in their sequences. An analysis of 80S ribosomal complexes formed with these mRNAs in a mammalian cell-free protein-synthesizing system demonstrates that AP sites do indeed cross-link to uS3 in the course of the translation. We also show that the cross-linking occurs as soon as the AP site arrives to a common favorable position relative to uS3, which is independent on its location in the mRNA. Our findings suggest that the mechanism of stopping translation of damaged mRNAs involving uS3, along with the one mentioned above, could underlie ribosome-associated mRNA quality control., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin-spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation., Competing Interests: 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., (© 2021 The Author(s).)

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 10 B isotope inside target cells, which are then destroyed after neutron irradiation. Nucleic acid aptamers look like very prospective candidates for carrying 10 B 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 10 B-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.

The ribosomal protein eL38 is a component of the mammalian translation machine. The deletion of the Rpl38 locus in mice results in the Tail-short (Ts) mutant phenotype characterized by a shortened tail and other defects in the axial skeleton development. Here, using the next-generation sequencing of total RNA from HEK293 cells knocked down of eL38 mRNA by transfection with specific siRNAs, we examined the effect of reduced eL38 content on genomic transcription. An approximately 4-fold decrease in the level of eL38 was shown to cause changes in the expression of nearly 1500 genes. Among the down-regulated genes, there were those responsible for p53 activity, Ca2+ metabolism and several signaling processes, as well as genes involved in the organization and functioning of the cytoskeleton. The genes related to rRNA processing and translation, along with many others, including those whose dysregulation is associated with developmental disorders, turned out to be up-regulated. Thus, we demonstrated that the decreased RPL38 expression leads to a significant reorganization of genomic transcription. Our findings suggest a possible link between the balance of eL38 and genes implicated in osteogenesis, thereby contributing to the elucidation of the reasons for the appearance of the above Ts mutant phenotype in animals., 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 © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5'-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone covalent bonds is proposed in this article. The approach relies on introducing a hydrazide of the ligand under aqueous/organic media to a fully protected support-bound oligonucleotide containing aldehyde function at the 5'-end. We demonstrated the proof-of-principle of this approach by synthesizing 5'-lipophilic (e.g., cholesterol and α-tocopherol) conjugates of modified siRNA and non-coding RNAs imported into mitochondria (antireplicative RNAs and guide RNAs for Mito-CRISPR/system). The developed method has the potential to be extended for the synthesis of pH-sensitive conjugates of oligonucleotides of different types (ribo-, deoxyribo-, 2'- O -methylribo-, and others) with ligands of different nature.

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 closo -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 closo -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 closo -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 closo -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 closo -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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: According to current data, an effective Ebola virus vaccine should induce both humoral and T-cell immunity. In this work, we focused our efforts on methods for delivering artificial T-cell immunogen in the form of a DNA vaccine, using generation 4 polyamidoamine dendrimers (PAMAM G4) and a polyglucin:spermidine conjugate (PG)., Methods: Optimal conditions were selected for obtaining complexes of previously developed DNA vaccines with cationic polymers. The sizes, mobility and surface charge of the complexes with PG and PAMAM 4G have been determined. The immunogenicity of the obtained vaccine constructs was investigated in BALB/c mice., Results: It was shown that packaging of DNA vaccine constructs both in the PG envelope and the PAMAM 4G envelope results in an increase in their immunogenicity as compared with the group of mice immunized with the of vector plasmid pcDNA3.1 (a negative control). The highest T-cell responses were shown in mice immunized with complexes of DNA vaccines with PG and these responses significantly exceeded those in the groups of animals immunized with both the combination of naked DNAs and the combination DNAs coated with PAMAM 4G. In the group of animals immunized with complexes of the DNA vaccines with PAMAM 4G, no statistical differences were found in the ability to induce T-cell responses, as compared with the group of mice immunized with the combination of naked DNAs., Conclusions: The PG conjugate can be considered as a promising and safe means to deliver DNA-based vaccines. The use of PAMAM requires further optimization.

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.

The paper describes some biological features of the radioprotective effect of double-stranded RNA preparation. It was found that yeast RNA preparation has a prolonged radioprotective effect after irradiation by a lethal dose of 9.4 Gy. 100 % of animals survive on the 70th day of observation when irradiated 1 hour or 4 days after 7 mg RNA preparation injection, 60 % animals survive when irradiated on day 8 or 12. Time parameters of repair of double-stranded breaks induced by gamma rays were estimated. It was found that the injection of the RNA preparation at the time of maximum number of double-stranded breaks, 1 hour after irradiation, reduces the efficacy of radioprotective action compared with the injection 1 hour before irradiation and 4 hours after irradiation. A comparison of the radioprotective effect of the standard radioprotector B-190 and the RNA preparation was made in one experiment. It has been established that the total RNA preparation is more efficacious than B-190. Survival on the 40th day after irradiation was 78 % for the group of mice treated with the RNA preparation and 67 % for those treated with B-190. In the course of analytical studies of the total yeast RNA preparation, it was found that the preparation is a mixture of single-stranded and double-stranded RNA. It was shown that only double-stranded RNA has radioprotective properties. Injection of 160 μg double-stranded RNA protects 100 % of the experimental animals from an absolutely lethal dose of gamma radiation, 9.4 Gy. It was established that the radioprotective effect of double-stranded RNA does not depend on sequence, but depends on its double-stranded form and the presence of "open" ends of the molecule. It is supposed that the radioprotective effect of double-stranded RNA is associated with the participation of RNA molecules in the correct repair of radiation-damaged chromatin in blood stem cells. The hematopoietic pluripotent cells that have survived migrate to the periphery, reach the spleen and actively proliferate. The newly formed cell population restores the hematopoietic and immune systems, which determines the survival of lethally irradiated animals., (Copyright © AUTHORS.)

The Purpose of the Article: Protection from ionizing radiation is the most important component in the curing malignant neoplasms, servicing atomic reactors, and resolving the situations associated with uncontrolled radioactive pollutions. In this regard, discovering new effective radioprotectors as well as novel principles of protecting living organisms from high-dose radiation is the most important factor, determining the new approaches in medical and technical usage of radiation., Materials and Methods: Experimental animals were irradiated on the γ-emitter (Cs 137 ) with a dose of 9.4 Gy. Radioprotective properties of several agents (total RNA, single-stranded RNA, double-stranded RNA and B-190) were estimated by the survival/death rates of experimental animals within 30-90 d. Pathomorphological examination of internal organs end electron microscope assay was done on days 9-12 after irradiation. Cloning and other molecular procedures were performed accordingly to commonly accepted protocols. For assessment of the internalization of labeled nucleic acid, bone marrow cells were incubated with double-stranded RNA labeled with 6-FAM fluorescent dye. Cells with internalized double-stranded RNA were assayed using Axio Imager M1 microscope. In the other experiment, bone marrow cells after incubation with double-stranded RNA were stained with Cy5-labeled anti-CD34 antibodies and assayed using Axioskop 2 microscope., Results: In this study, several biological features of the radioprotective action of double-stranded RNA are characterized. It was shown that 160 µg of the double-stranded RNA per mouse protect experimental animals from the absolutely lethal dose of γ-radiation of 9.4 Gy. In different experiments, 80-100% of irradiated animals survive and live until their natural death. Radioprotective properties of double-stranded RNA were found to be independent on its sequence, but strictly dependent on its double-stranded form. Moreover, double-stranded RNA must have 'open' ends of the molecule to exert its radioprotective activity., Conclusions: Experiments indicate that radioprotective effect of double-stranded RNA is tightly bound to its internalization into hematopoietic stem cells, which further repopulate the spleen parenchyma of irradiated mice. Actively proliferating progenitors form the splenic colonies, which further serve as the basis for restoration of hematopoiesis and immune function and determine the survival of animals received the lethal dose of radiation.

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2' position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2'- O -protecting groups, namely 2'- O -thiomorpholine-carbothioate (TC, as "permanent") and 2'- O - tert -butyl(dimethyl)silyl ( t BDMS, as "temporary"), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2' functionalization of the naked hydroxyl group. This convenient method to tailor RNA, utilizing the advantages of solid phase approaches, gives an opportunity to introduce site-specifically a wide range of linkers and functional groups. By this strategy, a series of RNAs containing diverse 2' functionalities were synthesized and studied with respect to their physicochemical properties.

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

Progress in synthesis of novel fluorescent oligonucleotides has provided effective instruments for nucleic acid detection. Pyrene conjugated oligonucleotides have demonstrated their effectiveness as fluorescent hybridization probes. Here we describe the synthesis, isolation, and analysis of 5'-monopyrene and 5'-bispyrene conjugates of oligo(2'-O-methylribonucleotides) and their application as probes for fluorescent detection of mismatches in RNA targets.

The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3'-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2'-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A novel and convenient approach for the solid-phase 5'-functionalization of oligonucleotides is proposed in this article. The approach is based on the activation of free 5'-hydroxyl of polymer support-bound protected oligonucleotides by N , N '-disuccinimidyl carbonate followed by interaction with amino-containing ligands. Novel amino-containing derivatives of closo -dodecaborate, estrone, cholesterol, and α-tocopherol were specially prepared. A wide range of oligonucleotide conjugates bearing closo -dodecaborate, short peptide, pyrene, lipophilic residues (cholesterol, α-tocopherol, folate, estrone), aliphatic diamines, and propargylamine were synthesized and characterized to demonstrate the versatility of the approach. The developed method is suitable for the conjugate synthesis of oligonucleotides of different types (ribo-, deoxyribo-, 2'- O -methylribo-, and others).

Bioluminescent solid-phase sandwich-type microassay was developed to detect multiple sclerosis (MS)-associated autoantibodies in human sera. The assay is based on two different 2'-F-Py RNA aptamers against the target autoantibodies as biospecific elements, and Ca 2+ -regulated photoprotein obelin as a reporter. The paper describes elaboration of the assay and its application to 91 serum samples from patients with clinically definite MS and 86 ones from individuals healthy in terms of MS. Based on the receiver-operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers sensitivity of 63.7% and specificity of 94.2%. The area under the ROC curve (AUC) value of 0.87 shows a good difference between the groups under investigation. The likelihood ratio of 10.97 proves the diagnostic value of the assay and its potential as one of the laboratory MS-tests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The small subunit ribosomal protein uS3 is a critically important player in the ribosome-mRNA interactions during translation and has numerous functions not directly related to protein synthesis in eukaryotes. A peculiar feature of the human uS3 protein is the ability of its fragment 55-64 exposed on the 40S subunit surface near the mRNA entry channel to form cross-links with 3'-terminal dialdehyde derivatives of various unstructured RNAs and with abasic sites in single-stranded DNAs. Here we showed that the ability of the above uS3 fragment to cross-link to abasic sites in DNAs is inherent only in mature cytoplasmic 40S subunits, but not nuclear pre-40S particles, which implies that it may be relevant to the ribosome-mRNA interplay. To clarify this issue, we investigated interactions of human ribosomes with synthetic mRNA analogues bearing an abasic site protected by a photocleavable group at the 3'-termini. We found that these mRNA analogues can form specific complexes with 80S ribosomes and 40S subunits, where the undamaged upstream part of the analogue is fixed in the mRNA binding channel by interaction with the P-site tRNA, and the downstream part located outside the ribosome is cross-linked to the uS3 fragment 55-64. The yield of cross-links of the mRNA analogues was rather high when their undamaged parts were bound to the mRNA channel prior to deprotection of the abasic site enabling its covalent attachment to the 40S subunit via the uS3 protein, but not vice versa. Based on our findings, one can assume that abasic sites, which can occur in mRNAs due to oxidative stress and ageing, are able to interact directly with the uS3 fragment exposed on the 40S subunit surface near the mRNA entry channel during translation. Consequently, the 40S subunit can be considered as a potential mRNA quality controller., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

This paper examines the complexation of anti-cancer small interfering RNAs (siRNAs) by cationic carbosilane dendrimers, and the interaction of the formed complexes with HeLa and HL-60 cancer cells. Stepwise formation of the complexes accompanied by the evolution of their properties has been observed through the increase of the charge ratio (dendrimer/siRNA). The complexes decrease the viability of both "easy-to-transfect" cells (HeLa) and "hard-to transfect" ones (HL-60), indicating a high potential of the cationic carbosilane dendrimers for siRNA delivery into tumor cells.

The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.

In this work, we report the assemblage of hydrogels from phosphorus dendrimers in the presence of biocompatible additives and the study of their interactions with nucleic acids. As precursors for hydrogels, phosphorus dendrimers of generations 1⁻3 based on the cyclotriphosphazene core and bearing ammonium or pyridinium acetohydrazones (Girard reagents) on the periphery have been synthesized. The gelation was done by the incubation of dendrimer solutions in water or phosphate-buffered saline in the presence of biocompatible additives (glucose, glycine or polyethylene glycol) to form physical gels. Physical properties of gels have been shown to depend on the gelation conditions. Transmission electron microscopy revealed structural units and well-developed network structures of the hydrogels. The hydrogels were shown to bind nucleic acids efficiently. In summary, hydrogels of phosphorus dendrimers represent a useful tool for biomedical applications.

Nucleic acid aptamers capable of selectively recognizing their target molecules have nowadays been established as powerful and tunable tools for biospecific applications, be it therapeutics, drug delivery systems or biosensors. It is now generally acknowledged that in vitro selection enables one to generate aptamers to almost any target of interest. However, the success of selection and the affinity of the resulting aptamers depend to a large extent on the nature and design of an initial random nucleic acid library. In this review, we summarize and discuss the most important features of the design of nucleic acid libraries for in vitro selection such as the nature of the library (DNA, RNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. We also compare and contrast different randomization strategies and consider computer methods of library design and some other aspects., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; nor in the decision to publish the results.

The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5'- and 3'-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3'-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1-3 in the HEK293 cell mRNA 3' UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3' UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

We designed a multimeric nuclease-resistant 63-bp trimeric small-interfering RNA (tsiRNA) comprising in one duplex the sequence of siRNAs targeting mRNAs of MDR1, LMP2, and LMP7 genes. We show that such tsiRNA is able to suppress the expression of all the target genes independently and with high efficiency, acting via a Dicer-dependent mechanism. tsiRNA is diced into 42- and 21-bp duplexes inside the cell. tsiRNA-induced gene silencing is characterized by kinetics similar to that of canonical siRNA, while the silencing efficiency is significantly higher than that of canonical siRNA with the same sequence., (© 2017 Federation of European Biochemical Societies.)

In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence to the microenvironment, ability to form excimers and exciplexes, long fluorescence lifetime, high quantum yield), ability to intercalate into the nucleic acid duplex, to act as a π-π-stacking (including anchoring) moiety, and others. These properties of pyrene have been used to construct novel sensitive fluorescent probes for the sequence-specific detection of nucleic acids and the discrimination of single nucleotide polymorphisms (SNPs), aptamer-based biosensors, agents for binding of double-stranded DNAs, and building blocks for supramolecular complexes. Special attention is paid to the influence of the design of pyrene-modified oligonucleotides on their properties, i.e., the structure-function relationships. The perspectives for the applications of pyrene-modified oligonucleotides in biomolecular studies, diagnostics, and nanotechnology are discussed., Competing Interests: The authors declare no conflict of interest.

A novel family of amphiphilic ionic carbosilane dendrons containing fatty acids at the focal point were synthesized and characterized. They spontaneously self-assembled in aqueous solution into micelles both in the absence and presence of salt, as confirmed by surface tension, conductivity, and DLS measurements. Dendron based micelles have spherical shapes and increase in size on decreasing dendron generation. These dendritic micelles have been demonstrated to be able to form complexes with therapeutic macromolecules such as siRNA and show a high loading capacity for drugs such as procaine, suggesting their potential use as nanocarriers for therapeutics.

In this paper, we report structure-based rational design and physico-chemical and biological studies of novel pyrene excimer forming dual probes for visualization of intracellular RNAs. Herein, the probes based on 2'-O-methyl RNA with linkers of different structure and length between pyrene moiety and ribose are studied with respect to their hybridization and spectral properties. We found optimal linkers that provide more intense excimer emission (at ∼480nm) of RNA-bound probes; particularly, the length of the linker arm of the 3'-component of dual probes plays a key role in formation of pyrene excimer. Calculated molecular dynamics trajectories and probability distributions of pyrene-pyrene dimer formation upon hybridization of the dual probes with RNA target are in agreement with the obtained fluorescence spectroscopy data for the corresponding duplexes. Our study demonstrates the excellent binding properties of new dual probes to structured RNA and their feasibility for the visualization of intracellular RNA targets., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5'-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNA(Phe), which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The synthesis and properties two series of new 2'-O-methyl RNA probes, each containing a single insertion of a 2'-bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21-fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5'-side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3'-side are important: CC, CG, and UC dinucleotide units on the 3'-side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2'-bispyrene-labeled 2'-O-methyl RNA probes might be useful tools for detection of RNAs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Site-directed spin labeling (SDSL) is widely applied for structural studies of biopolymers by electron paramagnetic resonance (EPR). However, SDSL of long RNA sequences still remains a challenging task. Here, we propose a novel SDSL approach potentially suitable for long natural RNAs, which is based on the attachment of a linker containing an aliphatic amino group to the target nucleotide residue followed by selective coupling of a spin label to this amino group. Such a linker can be attached to the desired RNA residue via a sequence-specific reaction with the derivatives of oligodeoxyribonucleotides. To verify this approach, we applied it to model RNA duplex with known structure and expected distance between corresponding residues. A new 2,5-bis(spirocyclohexane)-substituted spin label with advanced stability and relaxation properties has been used, and the distance distribution measured using Q-band (34 GHz) pulsed double electron-electron resonance corresponds well to the expected one. We have additionally validated the obtained results by studying a similar RNA duplex, where the linker with the aliphatic amino group was introduced via solid-phase synthesis. Although this novel SDSL approach does not provide an advantage in precision of molecular distance measurements, we believe that its applicability to long RNAs is a crucial benefit for future structural studies using pulse EPR.

DsRNAs longer than 30bp induce interferon response and global changes in gene expression profile in mammalians. 21bp siRNA and 25/27bp dsiRNA acting via RNA interference mechanism are used for specific gene silencing in this class of organisms. We designed selectively 2'-O-methyl-modified 42 and 63bp anti-MDR1-siRNAs that silence the expression of P-glycoprotein and restore the sensitivity of drug-resistant cancer cells to cytostatic more efficiently than canonical 21bp siRNAs. We also show that they act in a Dicer-independent mode and are devoid of immunostimulating properties. Our findings suggest that 42 and 63bp siRNAs could be used as potential therapeutics., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Nowadays, there are no specific laboratory tests for establishing the diagnosis of multiple sclerosis (MS). The presence of proteolytic autoantibodies against myelin basic protein is now considered as a characteristic feature of MS. New 2'-F-containing RNA aptamer of high affinity and specificity to these antibodies was selected. Covalent conjugate of this aptamer and Ca(2+)-regulated photoprotein obelin was obtained for the first time and applied as a label in bioluminescent microplate assay to detect target antibodies. The developed model solid-phase microassay is simple, fast, and highly sensitive.

Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells., (© 2014 FEBS.)

Novel hybrids of fluorescein-labeled poly(ethylene glycol)-modified single-walled carbon nanotubes (SWCNTs) with nucleic acids were prepared. 5'-Pyrene conjugates of oligodeoxyribonucleotides were used to construct the noncovalent hybrids, with the pyrene residues acting as anchor groups, immobilizing an oligonucleotide on the SWCNT surface. The hybrid formation characteristics were studied using ζ-potential measurements and adsorption isotherm plots. Transmission electron microscopy (TEM) of the samples stained with contrast agents proved that the pyrene conjugates of oligonucleotides were adsorbed onto the surfaces of the functionalized SWCNTs. On the basis of the MTT assay, the functionalized SWCNTs and their hybrids with oligonucleotides exhibited low toxicity toward HeLa, KB-3-1, and KB-8-5 cells. A TEM study of ultrathin sections of cells treated with SWCNTs revealed that the nanotubes directly interacted with the cellular surface.

This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

Conjugates of pyrimidine triplex forming 3'-protected oligo(2'-O-methylribonucleotides) with minor groove binders (MGB) and triplex specific intercalator benzoindoloquinoline (BIQ) at 5'-terminus were synthesized. The conjugates formed stable complexes with target dsDNA by simultaneous binding both in its minor and major grooves and BIQ intercalation. The dissociation constants and thermal stability of the conjugate complexes with model dsDNA corresponding to polypurine tract (PPT) of genes nef and pol from HIV proviral genome were determined. Conjugation of oligo(2'-O-methylribonucleotides) with MGB and intercalator increased the stability of the triple complexes with dsDNA at pH 7.2 and 37 degrees C. Intercalator introduction accelerates the process of complex formation. Dose-dependent arrest of the in vitro transcription was demonstrated when a 780 b.p. DNA fragment containing the polypurine tract was transcribed under the control of T7 promoter in the presence of different concentrations of conjugates of oligo(2'-O-methylribonucleotides) containing MGB and BIQ intercalator.

Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3'- and 5'-mono- and bis-pyrene conjugated oligo(2'-O-methylribonucleotides), protected by 3'-"inverted" thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems.

Small interfering RNAs (siRNA) are able to activate the mammalian innate immune system depending on their structure, sequence, and method of delivery. The immunostimulatory activity of double-stranded RNA can be applied to antiviral and antitumor therapy. Here we identified a set of 19-bp RNA duplexes with 3-nucleotid overhangs in the 3' ends that display immunostimulating activity (here and after immunostimulating RNA, or isRNA) and studied their sequence/activity relationships. It was found that the introduction of substitutions in the middle part of the isRNA sequence (10-16 positions counting from the 5' end of strand 1) does not alter the antiproliferative activity, while substitutions in the 3' end region of isRNA substantially reduce it. isRNAs efficiently inhibit the proliferation of human oral epidermoid carcinoma cells [half-maximal inhibitory concentration (IC(50)) values varied from 10 to 100 nM]. Our research demonstrated that antiproliferative effects of isRNAs are related to cell growth arrest, rather than the induction of apoptosis. These isRNAs strongly stimulate the synthesis of interferon-α (IFN-α), and to a lesser extent the synthesis of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), in adherent peripheral blood mononuclear cells. An intravenous injection of isRNA/Lipofectamine complexes into C57BL mice increases IFN-α and IL-6 levels in the blood serum up to 15-fold and 3-fold, respectively, compared to the control mice. The results obtained clearly demonstrate the pronounced immunostimulatory and antiproliferative properties of the isRNAs under study. Hence, these short double-stranded RNAs can be considered as potential agents for the therapy of oncological and viral diseases.

Insulin-like growth factor I (IGF-I) and its cognate receptor (IGF-1R) contribute to normal cell function and to tumorigenesis. The role of IGF-I signaling in tumor growth has been demonstrated in vivo using nucleic acid-based strategies. Here, we designed the first 10-23 DNAzymes directed against IGF-I mRNA. Unlike antisense approaches and RNA interference that require protein catalysis, DNAzymes catalyze protein-free RNA cleavage. We identified target sequences and measured catalytic properties of differently designed DNAzymes on short synthetic RNA targets and on in vitro transcribed IGF-I mRNA. The most efficient cleavers were then transfected into cells, and their inhibitory effect was analyzed using reporter gene assays. We found that increasing the size of DNAzyme flanking sequences and modifications of the termini with 2'-O-methyl residues improved cleavage rates of target RNAs. Modification of the catalytic loop with six 2'-O-methyl ribonucleotides at nonessential positions increased or decreased catalytic efficiency depending on the mRNA target site. In cells, DNAzymes with 2'-O-methyl-modified catalytic cores and flanking sequences were able to inhibit reporter gene activity because of specific recognition and cleavage of IGF-I mRNA sequences. Mutant DNAzymes with inactive catalytic cores were unable to block reporter gene expression, demonstrating that the RNA cleaving ability of 10-23 DNAzymes contributed to inhibitory mechanisms. Our results show that nuclease-resistant 2'-O-methyl-modified DNAzymes with high catalytic efficiencies are useful for inhibiting IGF-I gene function in cells.

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2'-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2'-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.

Escort aptamers are DNA or RNA sequences with high affinity to certain cell-surface proteins, which can be used for targeted delivery of various agents into cells of a definite type. The peculiarities of the selection of escort aptamers are discussed in this review. The methods used in selection of escort aptamers via the SELEX technique are considered, including selection against isolated cell-surface proteins, cell fragments, living eukaryotic cells, and bacteria. Particular attention is given to the design and chemical modification of escort aptamers. The different fields of application of escort aptamers are described, including the targeted delivery of siRNAs, nanoparticles, toxins, and photoagents, as well as the identification of specific cell markers and the detection or isolation of cells of a definite type. The potential for the application of escort aptamers in the development of new therapeutic agents and diagnostic systems is also discussed.

The thermodynamic properties of siRNA duplexes are important for their silencing activity. siRNAs with high thermodynamic stability of both the central part of the duplex and in the whole, usually display low silencing activity. Destabilization of the central part of the siRNA duplex could increase its silencing activity. However, mismatches located in the central part of the duplex could substantially decrease the amount of RNAi efficacy, hindering active RISC formation and function. In this study, we examined the impact of duplex destabilization by nucleotide substitutions in the central part (7-10 nt counting from the 5'-end of the antisense strand) of the nuclease-resistant siRNA on its silencing activity., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.

Small interfering RNAs (siRNAs) are considered as potent agents for specific gene silencing; however, nuclease sensitivity of siRNA limits their biomedical applications. Till date, no universal methodology has been developed to improve the nuclease resistance of siRNA, preserving low toxicity and high activity. In this study, we proposed an algorithm for the site-specific modification of siRNAs based on the mapping of their nuclease-sensitive sites in the presence of serum followed by the incorporation of 2'-O-methyl analogs of ribonucleotides at the identified positions of cleavage. We found that the protection of nuclease-sensitive sites considerably enhanced nuclease resistance of siRNA and only slightly reduced the efficiency of silencing. Modification of all nuclease-sensitive sites prolonged the duration of the silencing effect of the siRNA compared to nonmodified, partially modified, or randomly modified siRNA of the same sequence. This study showed that the targeted chemical modification of nuclease-sensitive sites could provide highly efficient siRNA-based therapeutics for the control of disease-related genes.

The approach to the synthesis of 5'-lipophilic conjugates of functional nucleic acids based on a combination of H-phosphonate and phosphoramidite methods is presented.

We designed tandems of oligo(2'-O-methylribonucleotides) conjugates containing two bispyrene (5'-bisPyr and 3'-bisPyr) groups on their junction for detection of C677T polymorphism in the methylenetetrahydrofolate reductase gene (MHTFR). The potential of SNP detection with multipyrene tandems of oligo(2'-O-methylribonucleotides) was demonstrated.