Magdalena Janicka

Histidine triad nucleotide-binding protein 1 (HINT-1) phosphoramidase transforms nucleoside 5'-O-phosphorothioates to nucleoside 5'-O-phosphates.

Nucleoside 5′-O-phosphorothioates are formed in vivo as primary products of hydrolysis of oligo(nucleoside phosphorothioate)s (PS-oligos) that are applied as antisense therapeutic molecules. The biodistribution of PS-oligos and their pharmacokinetics have been widely reported, but little is known about their subsequent decay inside the organism. We suggest that the enzyme responsible for nucleoside 5′-O-monophosphorothioate ((d)NMPS) metabolism could be histidine triad nucleotide-binding protein 1 (Hint-1), a phosphoramidase belonging to the histidine triad (HIT) superfamily that is present in all forms of life. An additional, but usually ignored, activity of Hint-1 is its ability to catalyze the conversion of adenosine 5′-O-monophosphorothioate (AMPS) to 5′-O-monophosphate (AMP). By mutagenetic and biochemical studies, we defined the active site of Hint-1 and the kinetic parameters of the desulfuration reaction (P-S bond cleavage). Additionally, crystallographic analysis (resolution from 1.08 to 1.37 Å) of three engineered cysteine mutants showed the high similarity of their structures, which were not very different from the structure of WT Hint-1. Moreover, we found that not only AMPS but also other ribonucleoside and 2′-deoxyribonucleoside phosphorothioates are desulfurated by Hint-1 at the following relative rates: GMPS > AMPS > dGMPS ≥ CMPS > UMPS > dAMPS ≫ dCMPS > TMPS, and during the reaction, hydrogen sulfide, which is thought to be the third gaseous mediator, was released.

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.

Synthesis of a Fluorescent Cationic Phosphorus Dendrimer and Preliminary Biological Studies of Its Interaction with DNA

The synthesis of a water-soluble phosphorus-containing dendrimer possessing a fluorophore (maleimide-type) linked to the core is described. This dendrimer is found brightly fluorescent in CH2Cl2, but poorly fluorescent in water. The cytotoxicity of this compound is relatively low towards HeLa and A549 cells, and less toxic after 48 hours than after 24 hours. Association of this dendrimer with plasmid DNA (BACE-GFP) analyzed with circular dichroism (CD) indicates a possible disturbing of the helical B-type structure of DNA. The strength of this association (a “dendriplex”) with BACE-GFP (also with HygEGFP) was measured by electrophoresis.

Crystal structure, stability and Ago2 affinity of phosphorodithioate-modified RNAs

Small interfering RNAs (siRNAs) with phosphorodithioate modifications (PS2-RNA) possess favourable properties for use as RNAi therapeutics. Beneficial here is the combining of PS2 and 2′-O-methyl modifications (MePS2). SiRNAs with MePS2 moieties in the sense strand show promising efficacies in vitro and in vivo. Crystal structures of PS2- and MePS2- modified RNAs reveal subtle changes in geometry and hydration compared with natural RNA. A model of an MePS2-RNA–PAZ domain complex points to a hydrophobic effect as the source of the higher affinity of MePS2-RNA for Ago2.

Chelating ability and biological activity of hesperetin Schiff base

Hydrazone hesperetin Schiff base (HHSB) - N-[(±)-[5,7-dihydroxy-2-(3-hydroxy-4-methoxy-phenyl)chroman-4-ylidene]amino]benzamide has been synthesized and its crystal structure was determined. This compound was used for the formation of Cu(II) complexes in solid state and in solution which were characterized using different spectroscopic methods. The analyses of potentiometric titration curves revealed that monomeric and dimeric complexes of Cu(II) are formed above pH7. The ESI-MS (electrospray ionization-mass spectrometry) spectra confirmed their formation. The EPR and UV-visible spectra evidenced the involvement of oxygen and nitrogen atoms in Cu(II) coordination. Hydrazone hesperetin Schiff base can show keto-enol tautomerism and coordinate Cu(II) in the keto (O(-), N, Oket) and in the enolate form (O(-), N, O(-)enol). The semi-empirical molecular orbital method PM6 and DFT (density functional theory) calculations have revealed that the more stable form of the dimeric complex is that one in which the ligand is present in the enol form. The CuHHSB complex has shown high efficiency in the cleavage of plasmid DNA in aqueous solution, indicating its potential as chemical nuclease. Studies on DNA interactions, antimicrobial and cytotoxic activities have been undertaken to gain more information on the biological significance of HHSB and copper(II)-HHSB chelate species.

Biological and physicochemical characterization of siRNAs modified with 2′,2′-difluoro-2′-deoxycytidine (gemcitabine)

The use of synthetic short interfering RNAs (siRNAs) is currently a method of choice to manipulate gene expression in mammalian cells. Efforts aimed at improving siRNA biological activity, including increased silencing properties, higher substrate specificity and cellular stability, lower cytotoxicity, and improved target delivery, have been made through the introduction of various chemical modifications into the siRNA strands. In these studies, we present the synthesis of oligoribonucleotides with the single replacement of a cytidine unit for 2′,2′-difluoro-2′-deoxycytidine (gemcitabine, dFdC) and the use of them in a series of siRNAs for gene silencing experiments. The dFdC modifications are located in six different positions of the antisense strand, which are crucial for siRNA silencing activity. The results indicate a position-dependent tolerance for the dFdC modification. Gemcitabine units present in the “seed region”, at positions 1 or 8, resulted in only a ∼15% silencing activity in the corresponding duplexes. The dFdC unit at position 10 virtually switched off the silencing activity (below 10%), while the dFdC unit at the positions 2, 4 or 5 produced duplexes of silencing potential comparable to that of the non-modified duplex (70% silencing). The dFdC modification had little impact on the structure of the siRNA duplexes, as determined by circular dichroism analysis, while melting experiments showed their lower thermal stability.

RNA fragments mimicking tRNA analogs interact with cytochrome c.

In times, when drug seeking assays focus on the natural molecular triggers and their analogs, a deeper insight into molecular mechanisms governing the initial step of intrinsic apoptosis (cytochrome c release) is essential to suppress the immortality of pathologically changed cells. In this study, we examined RNA molecules mimicking mitochondrial tRNAs interacting with cytochrome c and possibly affecting its cellular function. tRNA analogs were designed and synthesized prior to the conformational analysis and gel assays clearly stating the nucleic acid–protein complex formation. The circular dichroism spectroscopic (CD) and microscale thermophoresis examination revealed the structural and conformational differences between four tRNA analogs in their interactions with cytochrome c. Obtained CD spectra and gel studies resulted in the complex ratio estimation and conclusion that not only the complex formation may be preferential towards specific tRNAs present in the cell, but nucleobase modifications are not essential for such interaction.

A stereogenic heteroatom-containing substituent as an inducer of chirality in the derivatives of thiophenes (mono, oligo, and poly), fullerenes C60, and multiwalled nanotubes

GRAPHICAL ABSTRACT Abstract Synthetic procedures applied recently in our laboratory for the preparation of a new thiophene, fullerene C60 and multiwalled carbon nanotube (MWCNT) derivatives functionalized with a substituent containing an achiral and/or a stereogenic heteroatom are reviewed. Polymerization reactions of selected optically active 2-(3′-thienyl)ethyl aryl(alkyl) sulfoxides and tert-butyl-[2-(3′-thienyl)ethyl]phenylphosphine oxide are also mentioned. Mechanistic and stereochemical aspects of the presented protocols are discussed.