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 Ca2+-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.
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.
► siRNAs destabilized by mismatches introduced to the central part of the duplex. ► Selective protection of nuclease-sensitive sites in siRNA by 2′-O-methyl analogs. ► Wobble pairs are better ...tolerated by RNAi machinery than mismatches. ► GU wobble pairs in the central part of the duplex can enhance silencing activity.
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 (7–10nt counting from the 5′-end of the antisense strand) of the nuclease-resistant siRNA on its silencing activity.
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.
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.
The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible ...subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2′-O-methyl modifications into nuclease-sensitive sites. This led to 90–95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases.
•2′-O-methyl modified siRNAs can efficiently silence LMP2, LMP7 and MECL-1 gene expression.•The mismatches introduced into the 3′ end of the sense strand of the siRNA increased the silencing activity of the siRNAs.•Incorporation of cholesterol residues into siRNAs is a promising approach for efficient siRNA delivery into cells.
We designed a multimeric nuclease‐resistant 63‐bp trimeric small‐interfering
RNA
(tsi
RNA
) comprising in one duplex the sequence of si
RNA
s targeting
mRNA
s of
MDR
1
,
LMP
2
, and
LMP
7
genes. We ...show that such tsi
RNA
is able to suppress the expression of all the target genes independently and with high efficiency, acting
via
a Dicer‐dependent mechanism. tsi
RNA
is diced into 42‐ and 21‐bp duplexes inside the cell. tsi
RNA
‐induced gene silencing is characterized by kinetics similar to that of canonical si
RNA
, while the silencing efficiency is significantly higher than that of canonical si
RNA
with the same sequence.
New conjugates of triplex‐forming pyrimidine oligo(2′‐O‐methylribonucleotides) with one or two ‘head‐to‐head’ hairpin oligo(N‐methylpyrrole carboxamide) minor‐groove binders (MGBs) attached to the ...terminal phosphate of the oligonucleotides with a oligo(ethylene glycol) linker were synthesized. It was demonstrated that, under appropriate conditions, the conjugates form stable complexes with double‐stranded DNA (dsDNA) similarly to triplex‐forming oligo(deoxyribonucleotide) (TFO) conjugates containing 5‐methylated cytosines. Kinetic and thermodynamic parameters of the complex formation were evaluated by gel‐shift assay and thermal denaturation. Higher melting temperatures (Tm), faster complex formation, and lower dissociation constants (Kd) of the triple helices (6–7 nM) were observed for complexes of MGB‐oligo(2′‐O‐methylribonucleotide) conjugates with the target dsDNA compared to the nonconjugated individual components. Interaction of MGB moieties with the HIV proviral DNA fragment was indicated by UV/VIS absorption changes at 320 nm in the melting curves. The introduction of thymidine via a 3′,3′‐type ‘inverted’ phosphodiester linkage at the 3′‐end of oligo(2′‐O‐methylribonucleotide) conjugates (3′‐protection) had no strong influence on triplex formation, but slightly affected complex stability. At pH 6.0, when one or two hairpin MGBs were attached to the oligonucleotide, both triplex formation and minor‐groove binding played important roles in complex formation. When two ‘head‐to‐head’ oligo(N‐methylpyrrole) ligands were attached to the same terminal phosphate of the oligonucleotide or the linker, binding was observed at pH >7.5 and at high temperatures (up to 74°). However, under these conditions, binding was retained only by the MGB part of the conjugate.
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.
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.
This paper describes a simple approach to obtain hybrids of single-walled carbon nanotubes with therapeutically relevant oligonucleotides that are able to be released upon glutathione treatment at physiological concentrations.
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