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.
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.
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.
Chemical modifications are an effective way to improve the therapeutic properties of small interfering RNAs (siRNAs), making them more resistant to degradation in serum and ensuring their delivery to ...target cells and tissues. Here, we studied the carrier-free biodistribution and biological activity of a nuclease-resistant anti-MDR1 cholesterol-siRNA conjugate in healthy and tumor-bearing severe combined immune deficiency (SCID) mice. The attachment of cholesterol to siRNA provided its efficient accumulation in the liver and in tumors, and reduced its retention in the kidneys after intravenous and intraperitoneal injection. The major part of cholesterol-siRNA after intramuscular and subcutaneous injections remained in the injection place. Confocal microscopy data demonstrated that cholesterol-siRNA spread deep in the tissue and was present in the cytoplasm of almost all the liver and tumor cells. The reduction of P-glycoprotein level in human KB-8-5 xenograft overexpressing the MDR1 gene by 60% was observed at days 5–6 after injection. Then, its initial level recovered by the eighth day. The data showed that, regardless of the mode of administration (intravenous, intraperitoneal, or peritumoral), cholesterol-siMDR efficiently reduced the P-glycoprotein level in tumors. The designed anti-MDR1 conjugate has potential as an adjuvant therapeutic for the reversal of multiple drug resistance of cancer cells.
•We design selectively 2′-O-methyl modified 42 and 63bp anti-MDR1-siRNAs.•We examine their ability to silence the expression of the target gene and the specificity of their action.•We show that 42 ...and 63bp siRNAs induce more effective RNAi at lower concentrations than classical 21bp siRNA.•These siRNAs act in a Dicer-independent mode and are devoid of immunostimulating properties.
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.
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.
A novel, simple and convenient approach to assemble non-covalent hybrids of carbon nanotubes with therapeutic oligonucleotides immobilized by means of biodegradable linker is described. Model RNA, ...DNA and 2′- O -methyl RNA oligonucleotides were anchored on the surface of single-walled carbon nanotubes using pyrene anchor groups introduced onto the 5′-termini via short linker containing bioreducible disulfide bond or via stable linker of the similar length, as a control. To optimize the conditions of non-covalent functionalization, the adsorption of oligonucleotides' pyrene conjugates on the carbon nanotubes surface has been studied. The exposure of hybrids to glutathione at physiological concentrations has been shown to cause the release of oligonucleotides anchored via bioreducible linker only. The kinetic parameters of the oligonucleotides release have been determined for the first time. The results obtained can be useful for the design of carbon nanotubes-based nanoconstructions and biomaterials.
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•DEER reveals the conformational variability of mRNA at the certain translation steps.•Elongation and termination complexes exist in 2 conformations in dynamic equilibrium.•The ...conformations of mRNA in 40S channel undergo no major change during termination.
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.
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.
We targeted the purine/pyrimidine (R/Y) sequence in the IGF-I promoter using modified triplex‐forming oligonucleotides (TFOs). A fluorescence‐based gel retardation assay that allows simultaneous detection of all three strands was developed and used to monitor triplex formation and to demonstrate protein binding to DNA. We showed that a TFO specifically inhibited IGF-I transcription in cells using a reporter gene assay.
Electrochemically active hybrid electrodes have been obtained by non-covalent immobilization of homo-oligoribonucleotide probes on the surface of multi-walled carbon nanotubes (MWCNTs) grown ...vertically on a silicon substrate. Immobilization has been achieved by interaction of heterocyclic bases of oligonucleotide with MWCNT surface or through a pyrene anchor group pre-conjugated to oligonucleotide. The electrode capacitance signal measured in our model system allowed for the selective recognition of target RNA oligonucleotides. Pyrene-anchoring of oligonucleotide probes to the MWCNT surface was shown to provide the better selectivity and stability of electrochemical response of the hybrid electrode.
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•Electrodes made of the RNA-modified array of aligned MWCNTs were prepared.•Electrochemical capacitance response of electrode was used for the target recognition.•Use of pyrene-anchored probes increases the efficiency of target recognition.
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