38 research outputs found
Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: Lipophilic conjugates with cleavable bonds:
Defects in mitochondrial DNA often cause neuromuscular pathologies, for which no efficient therapy has yet been developed. MtDNA targeting nucleic acids might therefore be promising therapeutic candidates. Nevertheless, mitochondrial gene therapy has never been achieved because DNA molecules can not penetrate inside mitochondria in vivo. In contrast, some small non-coding RNAs are imported into mitochondrial matrix, and we recently designed mitochondrial RNA vectors that can be used to address therapeutic oligoribonucleotides into human mitochondria. Here we describe an approach of carrier-free targeting of the mitochondrially importable RNA into living human cells. For this purpose, we developed the protocol of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable covalent bonds. Conjugates containing pH-triggered hydrazone bond were stable during the cell transfection procedure and rapidly cleaved in acidic endosomal cellular compartments. RNAs conjugated to cholesterol through a hydrazone bond were characterized by efficient carrier-free cellular uptake and partial co-localization with mitochondrial network. Moreover, the imported oligoribonucleotide designed to target a pathogenic point mutation in mitochondrial DNA was able to induce a decrease in the proportion of mutant mitochondrial genomes. This newly developed approach can be useful for a carrier-free delivery of therapeutic RNA into mitochondria of living human cells
Small Interfering RNA Targeted to IGF-IR Delays Tumor Growth and Induces Proinflammatory Cytokines in a Mouse Breast Cancer Model
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
Multivalent Aptamers: Versatile Tools for Diagnostic and Therapeutic Applications
Nucleic acid aptamers generated through an in vitro selection are currently extensively applied as very valuable biomolecular tools thanks to their prominent advantages. Diversity of spatial structures, ease of production through chemical synthesis and a large variety of chemical modifications make aptamers convenient building blocks for the generation of multifunctional constructs. An opportunity to combine different aptamer functionalities with other molecules of interest such as reporter groups, nanoparticles, chemotherapeutic agents, siRNA or antisense oligonucleotides provides a widest range of applications of multivalent aptamers. The present review summarizes approaches to the design of multivalent aptamers, various examples of multifunctional constructs and the prospects of employing them as components of biosensors, probes for affinity capture, tools for cell research and potential therapeutic candidates
Dendriplex-Impregnated Hydrogels With Programmed Release Rate
International audienceHydrogels are biocompatible matrices for local delivery of nucleic acids; however, functional dopants are required to provide efficient delivery into cells. In particular, dendrimers, known as robust nucleic acid carriers, can be used as dopants. Herein, we report the first example of impregnating neutral hydrogels with siRNA–dendrimer complexes. The surface chemistry of dendrimers allows adjusting the release rate of siRNA-containing complexes. This methodology can bring new materials for biomedical applications
Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides
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
Multipyrene Tandem Probes for Point Mutations Detection in DNA
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
Synthesis of dissymmetric phosphorus dendrimers using an unusual protecting group
International audienceThe presence of an azido group directly linked to phosphorus functionalized monomeric species allows the synthesis of neutral or polycationic original phosphorus dendrimers of reduced symmetry bearing branches of different generations on the core
Key Aspects of Nucleic Acid Library Design for in Vitro Selection
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
Novel Convenient Approach to the Solid-Phase Synthesis of Oligonucleotide Conjugates
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)