D. S. Novopashina

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action

This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed.

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (A). Mechanisms of interaction.

This paper examines a perspective on the use of newly engineered nanomaterials as effective and safe carriers of genes for the therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus and carbosilane) were complexed with anticancer siRNA and their biophysical properties of the dendriplexes analyzed. The potential of the dendrimers as nanocarriers for anticancer siBcl-xl, siBcl-2, siMcl-1 siRNAs and a siScrambled sequence was explored. Dendrimer/siRNA complexes were characterized by methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. Some of the experiments were done with heparin to check if siRNA can be easily disassociated from the complexes, and whether released siRNA maintains its structure after interaction with the dendrimer. The results indicate that siRNAs form complexes with all the dendrimers tested. Oligoribonucleotide duplexes can be released from dendriplexes after heparin treatment and the structure of siRNA is maintained in the case of PAMAM or carbosilane dendrimers. The dendrimers were also effective in protecting siRNA from RNase A activity. The selection of the best siRNA carrier will be made based on cell culture studies (Part B).

[Oligo(2'-O-methylribonucleotides) and their derivatives: III. 5'-mono- and 5'-bispyrenyl derivatives of oligo(2'-O-methylribonucleotides) and their 3'-modified analogues: synthesis and properties].

Abstract5′-Pyrenylmethylphosphamide and 5′-bispyrenylmethylphosphordiamide derivatives of oligo(2′-O-methylribonucleotides) and their analogues with thymidine attached at their 3′-termini by a 3′-3′-phosphodiester internucleotide bond (“inverted” thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5′-mono- and 5′-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5′-Pyrenylphosphamide derivatives of oligo(2′-O-methylribonucleotides) and their inverted analogues were shown to be used as sensitive probes for the detection of single nucleotide polymorphisms in RNA and DNA by the method of thermal duplex denaturation with fluorescence change registration.

Conjugates of Oligo(2′-O-Methylribonucleotides) with Minor Groove Binders as New Sequence-Specific Agents Recognizing Both Grooves of Double-Stranded DNA

Abstract Design, synthesis and physico-chemical studies of new pyrimidine oligo(2′-O-methylribonucleotide) conjugates with one or two oligo(pyrrolecarboxamide) minor groove binders (MGB) are described.

Fluorescently labeled single-walled carbon nanotubes and their hybrids with oligonucleotides

The approach to the preparation of fluorescein-labeled single-walled carbon nanotubes was proposed. The investigation of physico-chemical properties of obtained modified nanotubes was performed. The method of the design of non-covalent hybrids of fluorescein-labeled carbon nanotubes with nucleic acid fragments containing pyrene residues at 5′-termini was developed. This method is based on stacking interactions of pyrene residues with carbon nanotubes surface. The effect of carbon nanotubes functionalization type on the efficacy of sorption of pyrene conjugates of oligonucleotides was examined.

3′-MODIFIED OLIGO(2′-O-METHYLRIBONUCLEOTIDES) AS IMPROVED PROBES FOR HYBRIDIZATION WITH RNA

A series of octa(2′-O-methylribonucleotides) with an additional 3′-terminal deoxynucleoside (T, dC, dA or dG) linked by the 3′-3′ (“inverted”) bond was synthesized. The exceptional stability of these oligomers to a 3′-exonuclease (SVP) and nucleases in culture medium containing 10% heat-inactivated fetal calf serum was demonstrated. It was shown that the addition of the 3′-dangling inverted deoxynucleoside increases substantially the thermal stability of the duplexes of oligo(2′-O-methylribonucleotides) with complementary RNA and DNA in the case of a relatively weak terminal AmU(T) pair and enhances the mismatch sensitivity.

Stabilization of DNA double and triple helices by conjugation of minor groove binders to oligonucleotides.

Abstract New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis, Fluorescent Properties, and Duplex Stability

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.

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation

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.

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.