Zofia Gdaniec

The contribution of pseudouridine to stabilities and structure of RNAs

Thermodynamic data are reported revealing that pseudouridine (Ψ) can stabilize RNA duplexes when replacing U and forming Ψ-A, Ψ-G, Ψ-U and Ψ-C pairs. Stabilization is dependent on type of base pair, position of Ψ within the RNA duplex, and type and orientation of adjacent Watson–Crick pairs. NMR spectra demonstrate that for internal Ψ-A, Ψ-G and Ψ-U pairs, the N3 imino proton is hydrogen bonded to the opposite strand nucleotide and the N1 imino proton may also be hydrogen bonded. CD spectra show that general A-helix structure is preserved, but there is some shifting of peaks and changing of intensities. Ψ has two hydrogen donors (N1 and N3 imino protons) and two hydrogen bond acceptors because the glycosidic bond is C-C rather than C-N as in uridine. This greater structural potential may allow Ψ to behave as a kind of structurally driven universal base because it can enhance stability relative to U when paired with A, G, U or C inside a double helix. These structural and thermodynamic properties may contribute to the biological functions of Ψ.

NMR and Quantum Chemistry Study of Mesoscopic Effects in Ionic Liquids

(1)H, (13)C, and (81)Br NMR spectra of the neat room-temperature ionic liquid (RTIL), namely, 1-decyl-3-methyl-imidazolium bromide ([C(10)mim][Br]) as well as its solutions in acetonitrile, dichloromethane, methanol, and water have been investigated. The most important observation of the present work is the significant broadening of (81)Br NMR signal in the solutions of [C(10)mim][Br] in organic solvents, which molecules tend to associate into hydrogen bond networks and the appearance of the complex contour of (81)Br NMR signal in the neat RTIL as well as in the liquid crystalline (LC) ionogel formed in RTIL/water solution. The complex structure of (81)Br signal changes upon heating and dilution in water. It disappears at ca. 353 K and in the aqueous solution below ca. 0.1 mol fraction of RTIL. Several new (1)H NMR signals appear at the [C(10)mim][Br]/water compositions just before the solidification of the sample (approximately 0.3 mol fraction of [C(10)mim][Br]). These additional peaks can be attributed to the H(2)O protons placed in inhomogeneous regions of the sample or due to the appearance of nonequivalent water sites in LC ionogel, the exchange between which is highly restricted or even frozen. The complex shape of (81)Br NMR signal can originate from the presence of supra-molecular structures (mesoscopic domains) that live over the period of the NMR time-scale due to a very high viscosity of [C(10)mim][Br]. These domains exhibit some features of partially disordered solids (liquid- or plastic crystals). To evaluate the static and dynamic contributions into the relaxation rate of (81)Br nuclei, the quantum chemistry calculations of the electronic structure, magnetic shielding, and electric field gradient (EFG) tensors of [C(10)mim][Br] and related model systems (Br(-).6H(2)O cluster, with addition of the dipoles (hydrogen fluoride) and charged particles - cations: H(+) or C(1)mim(+)) were performed.

Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats

Trinucleotide repeats are microsatellite sequences that are polymorphic in length. Their expansion in specific genes underlies a number of neurodegenerative disorders. Using ultraviolet-visible, circular dichroism, nuclear magnetic resonance (NMR) spectroscopies and electrospray ionization mass spectrometry, the structural preferences of RNA molecules composed of two and four repeats of AGG, CGG and UGG in the presence of K+, Na+ and NH4+ were analysed. (AGG)2A, (AGG)4A, p(UGG)2U and p(UGG)4U strongly prefer folding into G-quadruplexes, whereas CGG-containing sequences can adopt different types of structure depending on the cation and on the number of repeats. In particular, the two-repeat CGG sequence folds into a G-quadruplex in potassium buffer. We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG)2A, whereas mixed G:C:G:C tetrads and U-tetrads were observed in the NMR spectra of G(CGG)2C and p(UGG)2U, respectively. Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellular environment on the folding. Importantly, we highlight that although potassium ions are prevalent in cells, the structures observed in the HeLa cell extract are not always the same as those prevailing in biophysical studies in the presence of K+ ions.

On the Application of t-Butyldimethylsilyl Group in Chemical RNA Synthesis. Part I. 31P NMR Study of 2′-O-t-BDMSi Group Migration During Nucleoside 3′-OH Phosphorylation and Phosphitylation Reactions

Abstract 31P NMR spectroscopy has been used for evaluation of 2′-O-t-BDMSi group migration during reactions of suitably protected 3′-OH ribonucleosides with P(V) and P(III) reagents used in major methodologies for oligoribonucleotide synthesis.

NMR and Raman Spectroscopy Monitoring of Proton/Deuteron Exchange in Aqueous Solutions of Ionic Liquids Forming Hydrogen Bond: A Role of Anions, Self-Aggregation, and Mesophase Formation

The H/D exchange process in the imidazolium-based room temperature ionic liquids (RTILs) 1-decyl-3-methyl-imidazolium bromide- and chloride ([C10mim][Br] and [C10mim][Cl]) in D2O solutions of various concentrations was studied applying (1)H, (13)C NMR, and Raman spectroscopy. The time dependencies of integral intensities in NMR spectra indicate that the H/D exchange in [C10mim][Br] at very high dilution (10(-4) mole fraction of RTIL) runs only slightly faster than in [C10mim][Cl]. The kinetics of this process drastically changes above critical aggregation concentration (CAC). The time required to reach the apparent reaction saturation regime in the solutions of 0.01 mole fraction of RTIL was less 10 h for [C10mim][Br], whereas no such features were seen for [C10mim][Cl] even tens of days after the sample was prepared. The H/D exchange was not observed in the liquid crystalline gel mesophase. The role of anions, self-aggregation (micellization), and mesophase formation has been discussed. Crucial influence of Br(-) and Cl(-) anions on the H/D exchange rates above CAC could be related to the short-range ordering and molecular microdynamics, in particular that of water molecules. The concept of the conformational changes coupled with the H/D exchange in imidazolium-based ionic liquids with longer hydrocarbon chains can be rejected in the light of (13)C NMR experiment. The revealed changes in (13)C NMR spectra are caused by the secondary ((13)C) isotope effects not being the signal shifts due to the conformational trans-gauche transition.

Mature MiRNAs Form Secondary Structure, which Suggests Their Function beyond RISC

The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics.

Diazepinoporphyrazines Containing Peripheral Styryl Substituents and Their Promising Nanomolar Photodynamic Activity against Oral Cancer Cells in Liposomal Formulations

The photochemical properties and photodynamic activity of three porphyrazines (Pzs) containing annulated diazepine rings, including novel demetalated porphyrazine‐possessing bis(styryl)diazepine moieties were investigated. The porphyrazines were evaluated in terms of their electronic absorption and emission properties, their tendency to undergo aggregation and photodegradation, as well as their singlet oxygen generation efficiency. The in vitro photodynamic activity of the porphyrazines and their liposomal formulations were examined by using two oral squamous cell carcinoma cell lines. Magnesium(II) tribenzodiazepinoporphyrazine (1) revealed the highest phototoxic effect in both cell lines used, H413 and HSC‐3. Encapsulation of Pz 1 into L‐α‐phosphatidyl‐d,l‐glycerol:1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine liposomes resulted in a nearly threefold increase in photocytotoxicity relative to that of the solution of Pz 1 (IC50 values of 45 and 129 nM, respectively).

General Conception of the Virtual Laboratory

In the paper some theoretical considerations about virtual laboratory (VL) aspects are discussed. Possibilities of the VL system creation to control many laboratory apparatus in a remote way are considered by the authors. The main research goals on which the authors want to focus their attention are: laboratory framework and dynamic measurement scenarios.

19F NMR of RNA. The Structural and Chemical Aspects of 5-Fluoro-cytidine and-uridine Labelling of Oligoribonucleotides #

Abstract Results of PM3 semiempirical calculation revealed that energy and hydrogen bonds geometry of 1-methyl-5-fluoro-uracil and -cytosine base-pairs with 9-methyl-adenine and -guanine respectively are virtually the same as for the natural bases. Analysis of proton coupling constants proved that the sugar puckering of 5-fluorouridine and 5-fluorocytidine is analogous to non-modified ribonucleosides. 5-Fluorocytidine was regioselectively introduced to oligoribonucleotides, prepared using 2′-O-tert-dimethylsilyl protection, via post-synthetic quantitative ammonolysis of 4-O-methyl-5-fluorouridine derived precursor. #Dedicated to Professor Yoshihisa Mizuno on the occasion of his 75th birthday.

Photochemical studies and nanomolar photodynamic activities of phthalocyanines functionalized with 1,4,7-trioxanonyl moieties at their non-peripheral positions

Manganese(III), cobalt(II), copper(II), magnesium(II), zinc(II) and metal-free phthalocyanines, possessing 1,4,7-trioxanonyl substituents, at their non-peripheral positions, were subjected to photochemical, photodynamic and biological activity studies. Demetallated phthalocyanine and its metallated d-block analogues, with copper(II), cobalt(II), manganese(III) chloride, were found to be less efficient singlet oxygen generators in comparison to the zinc(II) analogue and zinc(II) phthalocyanine reference. Irradiation of several phthalocyanines for short time periods resulted in a substantially increased cytostatic activity against both suspension (leukemic/lymphoma at 85nM) and solid (cervix carcinoma at 72nM and melanoma at 81nM) tumour cell lines (up to 200-fold). Noteworthy is that enveloped viruses, such as for herpesvirus and influenza A virus, but not, non-enveloped virus strains, such as Coxsackie B4 virus and reovirus-1, exposed to irradiation in the presence of the phthalocyanines, markedly lost their infectivity potential.