Barbara Nawrot

Water-soluble polycationic dendrimers with a phosphoramidothioate backbone: preliminary studies of cytotoxicity and oligonucleotide/plasmid delivery in human cell culture.

A series of water-soluble polycationic dendrimers with a phosphoramidothioate backbone (P-dendrimers) was studied in human cell culture. Preliminary studies have shown that P-dendrimers of series 1 and 2, possessing N,N-diethyl-ethylenediamine hydrochloride functions at the surface, show rather moderate cytotoxicity toward HeLa, HEK 293, and HUVEC cells in a standard MTT assay in serum-containing medium, generally lower than lipofectin. The experiments of cellular uptake have shown the necessity for the presence of serum for transfection with P-dendrimers of series 1 and 2. These compounds efficiently delivered fluorescein-labeled oligodeoxyribonucleotide into HeLa cells in serum-containing medium, but they failed to do so in HUVEC cell culture. The dendrimers were found to be successful mediators of transfection of the HeLa cells with a DNA plasmid containing the functional gene of enhanced green fluorescent protein (EGFP).

Polycationic phosphorus dendrimers: synthesis, characterization, study of cytotoxicity, complexation of DNA, and transfection experiments

Four series of phosphorus dendrimers (generations 1 and 4) having various types of amine terminal groups (pyrrolidine, morpholine, methyl piperazine, or phenyl piperazine) are synthesized. After protonation, the fourth generations of three of them are found water-soluble, and used for several biological experiments. First, the cytotoxicity of these polycationic dendrimers towards three cell strains (one healthy: HUVEC, and two cancerous: HEK 293 and HeLa) is assayed and found low. Second, their ability to interact with DNA is tested by electrophoresis: the dendrimer terminated by pyrrolidinium groups is found efficient to form dendriplexes. Finally, the polycationic dendrimers are used as transfection agents to deliver single- and double-stranded DNA into the three above-mentioned cell strains. Here also the dendrimer having pyrrolidinium groups is found the most efficient.

Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10-23

The RNA phosphodiester bond cleavage activity of a series of 16 thio‐deoxyribozymes 10–23, containing a P‐stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single‐turnover conditions in the presence of either 3 mm Mg2+ or 3 mm Mn2+. A metal‐specificity switch approach permitted the identification of nonbridging phosphate oxygens (proRP or proSP) located at seven positions of the core (P2, P4 and P9–13) involved in direct coordination with a divalent metal ion(s). By contrast, phosphorothioates at positions P3, P6, P7 and P14–16 displayed no functional relevance in the deoxyribozyme‐mediated catalysis. Interestingly, phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes, thio‐substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg2+, and this was reversed in the presence of Mn2+. Further experiments with thio‐deoxyribozymes of stereodefined P‐chirality suggested direct involvement of both oxygens of the P5 phosphate and the proRP oxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G6 contributes to metal ion binding and that this interaction is essential for 10–23 deoxyribozyme catalytic activity.

Nucleoside modifications in the regulation of gene expression: focus on tRNA.

Both, DNA and RNA nucleoside modifications contribute to the complex multi-level regulation of gene expression. Modified bases in tRNAs modulate protein translation rates in a highly dynamic manner. Synonymous codons, which differ by the third nucleoside in the triplet but code for the same amino acid, may be utilized at different rates according to codon–anticodon affinity. Nucleoside modifications in the tRNA anticodon loop can favor the interaction with selected codons by stabilizing specific base pairs. Similarly, weakening of base pairing can discriminate against binding to near-cognate codons. mRNAs enriched in favored codons are translated in higher rates constituting a fine-tuning mechanism for protein synthesis. This so-called codon bias establishes a basic protein level, but sometimes it is necessary to further adjust the production rate of a particular protein to actual requirements, brought by, e.g., stages in circadian rhythms, cell cycle progression or exposure to stress. Such an adjustment is realized by the dynamic change of tRNA modifications resulting in the preferential translation of mRNAs coding for example for stress proteins to facilitate cell survival. Furthermore, tRNAs contribute in an entirely different way to another, less specific stress response consisting in modification-dependent tRNA cleavage that contributes to the general down-regulation of protein synthesis. In this review, we summarize control functions of nucleoside modifications in gene regulation with a focus on recent findings on protein synthesis control by tRNA base modifications.

tRNA structural and functional changes induced by oxidative stress

Oxidatively damaged biomolecules impair cellular functions and contribute to the pathology of a variety of diseases. RNA is also attacked by reactive oxygen species, and oxidized RNA is increasingly recognized as an important contributor to neurodegenerative complications in humans. Recently, evidence has accumulated supporting the notion that tRNA is involved in cellular responses to various stress conditions. This review focuses on the intriguing consequences of oxidative modification of tRNA at the structural and functional level.

Evoking picomolar binding in RNA by a single phosphorodithioate linkage

RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by ∼1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference.

RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimers disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

5-Ethynyl-1-β-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide (ETCAR) and its analogues: synthesis and cytotoxic properties.

Efficient Pd(0)-catalysed synthesis of 5-alkynyl-1-β-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide depends on the presence of different protecting groups of the ribose moiety. Peracetylated 5-iodo substrate (15) couples with terminal alkynes or trimethyl-[(tributylstannyl)ethynyl]silane in 50-71% and 72% yield (ETCAR), respectively, although its hydrodehalogenation to 19 is noticeable. On the other hand, hydrodehalogenation of acetonide (16) predominates over coupling with terminal alkyne and slightly decreases a yield of cross-coupling reaction with trimethyl[(tributylstannyl)ethynyl]silane. Alternative conditions of reaction with terminal alkynes, to exclude so far identified hydride sources to produce hydridopalladium species, have been established for acetonide 16 and allowed to achieve 72% of coupling. Fluoromethyl derivative (42) was prepared from its 5-hydroxymethyl precursor by fluorination with DAST. Additionally, X-ray structural analysis of 42 was performed. All 1,2,3-triazolonucleosides and two synthesized cycloSal-pronucleotides were evaluated for cytotoxic activity against K562, HeLa and HUVEC cells.

2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes

2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon–anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3′-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif.

siRNAs modified with boron cluster and their physicochemical and biological characterization.

RNA interference (RNAi) technology provides a powerful, yet selective, molecular tool to reduce the expression of genes in eukaryotic cells. Despite the success associated with the effective use of siRNA duplexes for gene silencing, there is a need to improve their properties. These properties, related mainly to migration through the cell membranes, stability of siRNA in vivo, and specificity of their silencing activity, can be improved by chemical modifications of siRNA backbone. In this study, we examined the physicochemical and biological properties of siRNA duplexes targeted against BACE1 gene modified at various positions with a lipophilic boron cluster (C2B10H11, CB). The lipophilicity and resistance to enzymatic degradation of the modified oligomers was higher than the unmodified counterparts. As measured in a dual fluorescence assay (BACE1-GFP/RFP), the carboranyl siRNAs (CB-siRNAs) were as active as the parent nonmodified duplexes and their toxicity toward HeLa cells was also similar. The helical structure of CB-siRNAs remained unchanged upon boron cluster introduction, as determined by CD and UV melting experiments.