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In this contribution we report the high-resolution NMR structure of a recently identified lanthanide-binding aptamer (LnA). We demonstrate that the rigid lanthanide binding by LnA allows for the measurement of anisotropic paramagnetic NMR restraints which to date remain largely inaccessible for nucleic acids. One type of such restraints - pseudocontact shifts (PCS) induced by four different paramagnetic lanthanides - was extensively used throughout the current structure determination study and the measured PCS turned out to be exceptionally well reproduced by the final aptamer structure. This finding opens the perspective for a broader application of paramagnetic effects in NMR studies of nucleic acids through the transplantation of the binding site found in LnA into other DNA/RNA systems.

RNA G-quadruplexes fold almost exclusively into parallel-stranded structures and thus display much less structural diversity than their DNA counterparts. However, also among RNA G-quadruplexes peculiar structural elements can be found which are capable of reshaping the physico-chemical properties of the folded structure. A striking example is provided by a uridine tetrad (U-tetrad) placed on the 3′-terminus of the tetramolecular G-quadruplex. In this context, the U-tetrad adopts a unique conformation involving chain reversal and is responsible for a tremendous stabilization of the G-quadruplex (ΔTm up to 30°C). In this report, we attempt to rationalize the origin of this stabilizing effect by concurrent structural, thermal stability, and molecular dynamics studies of a series of G-quadruplexes with subtle chemical modifications at their 3′-termini. Our results provide detailed insights into the energetics of the “reversed” U-tetrad motif and the requirements for its formation. They point to the importance of the 2′OH to phosphate hydrogen bond and preferential stacking interactions for the formation propensity and stability of the motif.

This work presents the synthesis and characterization of metal-free, zinc (II), and cobalt (II) porphyrins substituted with short PEG chains. The synthesized compounds were characterized by UV-Vis, 1H and 13C NMR spectroscopy, and MALDI-TOF mass spectrometry. The origin of the absorption bands for tested compounds in the UV-Vis range was determined using a computational model based on the electron density functional theory (DFT) and its time-dependent variant (TD-DFT). The photosensitizing activity was evaluated by measuring the ability to generate singlet oxygen (ΦΔ), which reached values up to 0.54. The photodynamic activity was tested using bladder (5637), prostate (LNCaP), and melanoma (A375) cancer cell lines. In vitro experiments clearly showed the structure–activity relationship regarding types of substituents, their positions in the phenyl ring, and the variety of central metal ions on the porphyrin core. Notably, the metal-free derivative 3 and its zinc derivative 6 exerted strong cytotoxic activity toward 5637 cells, with IC50 values of 8 and 15 nM, respectively. None of the tested compounds induced a cytotoxic effect without irradiation. In conclusion, these results highlight the potential value of the tested compounds for PDT application.

Triazoloacridinone C-1305, a potent antitumor agent recommended for Phase I clinical trials, exhibits high activity towards a wide range of experimental colon carcinomas, in many cases associated with complete tumor regression. C-1305 is a well-established dsDNA intercalator, yet no information on its mode of binding into DNA is available to date. Herein, we present the NMR-driven and MD-refined reconstruction of the 3D structures of the d(CGATATCG)2:C-1305 and d(CCCTAGGG)2:C-1305 non-covalent adducts. In both cases, the ligand intercalates at the TA/TA site, forming well-defined dsDNA:drug 1:1 mol/mol complexes. Orientation of the ligand within the binding site was unambiguously established by the DNA/ligand proton-proton NOE contacts. A subsequent, NMR-driven study of the sequence-specificity of C-1305 using a series of DNA duplexes, allowed us to confirm a strong preference towards TA/TA dinucleotide steps, followed by the TG/CA steps. Interestingly, no interaction at all was observed with duplexes containing exclusively the AT/AT, GG/CC and GA/TC steps.

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform–PAZ–Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03795-w.

The folding of tRNA fragments (tRFs) into G-quadruplex structures and the implications of G-quadruplexes in translational inhibition have been studied mainly in mammalian systems. To increase our knowledge of these phenomena, we determined the influence of human and plant tRFs and model G-quadruplexes on translation in rabbit reticulocyte lysate and wheat germ extract. The efficiency of translational inhibition in the mammalian system was strongly associated with the type of G-quadruplex topology. In the plant system, the ability of a small RNA to adopt the G-quadruplex conformation was not sufficient to repress translation, indicating the importance of other structural determinants.

Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.

The self-assembly of triethylammonium bis(trifluoromethylsulfonyl)imide, i.e. [(C2H5)3NH][TFSI], in chloroform and aqueous solutions has been investigated using 1H NMR spectroscopy and computational (DFT/PCM prediction) methods. We have examined a number of ion pairs formed between the [(C2H5)3NH]+ cation with different conformations of alkyl substituents as well as various dispositions of the multi-site [TFSI]− anion. Based on the agreement between the calculated (DFT) and observed 1H NMR chemical shifts, [(C2H5)3NH][TFSI] in chloroform formed lipophilic complexes with effective N+–H⋯N or N+–H⋯O hydrogen bonding, whereas hydrophilic complexes with Cα–H⋯O and Cα–H⋯F hydrogen bonding are found in aqueous solutions. This study provides a new insight into the self-aggregation of ammonium PILs incorporating the widely used [TFSI]− anion and demonstrates the importance of solvent effects on chemical shifts. The simulations with explicit and implicit dielectric continuum solvents are found to be the most realistic method, yielding a representative ensemble of structures.

RNA 25, 121 - 134 (2019). doi:10.1261/rna.068163.118, Uridine tetrads (U-tetrads) are a structural element encountered in RNA G-quadruplexes, for example, in the structures formed by the biologically relevant human telomeric repeat RNA. For these molecules, an unexpectedly strong stabilizing influence of a U-tetrad forming at the 3' terminus of a quadruplex was reported. Here we present the high resolution solution NMR structure of the r(UGGUGGU)4 quadruplex which, in our opinion, provides an explanation for this stabilization. Our structure features a distinctive, abrupt chain reversal just prior to the 3' uridine tetrad. Similar 'reversed U-tetrads' were already observed in the crystalline phase. However, our NMR structure coupled with extensive explicit solvent molecular dynamics (MD) simulations identifies some key features of this motif, that up to now remained overlooked. These include the presence of an exceptionally stable 2'OH to phosphate hydrogen bond, as well as, the formation of an additional K+ binding pocket in the quadruplex groove., Published by HighWire Press, Stanford, Calif.

S-adenosyl-l-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K+ ion and one Zn2+ ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K+ ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn2+ ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K+ or Rb+ ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a 23Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn2+ ion is bound in the area of a molecular gate that regulates access to the active site. Zn2+ coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.

The macrocyclization reactions led to higher-symmetry and lower-symmetry porphyrazines bearing peripheral 2,5-di(4′-chlorophenyl)pyrrol-1-yl and dimethylamino substituents, which were characterized using NMR and X-ray crystallography. The reactions were performed in the conditions of Linstead macrocyclization with magnesium n-butanolate in n-butanol with an addition of a malonitrile derivative in solid form or after dissolving it in DMF. It is interesting to note that the addition of DMF increases the total yield of the reaction from low to moderate, and forces the synthesis of the higher-symmetry over the lower-symmetry porphyrazine. The non-alternate order of peripheral substituents in lower-symmetry porphyrazine resulted in the non-equivalence of dimethylamino groups in 1H and 13C NMR spectra. The X-ray crystal structures of both porphyrazines revealed an almost perpendicular orientation of the bulky, 2,5-di(4′-chlorophenyl)pyrrol-1-yl substituents to the cores. Higher-symmetry porphyrazine was used in the Suzuki–Miyaura reaction with phenylboronic acid. This led with a high yield to the derivative possessing peripheral 2,5-di(biphenyl-4-yl)pyrrol-1-yl and dimethylamino substituents.

Diffusion and self-aggregation (micellization) processes of the neat 1-decyl-3-methyl-imidazolium chloride ([C10mim][Cl]) and its aqueous solutions have been studied using pulsed-field gradient 1H NMR. Different from many RTILs, whose temperature dependencies of the diffusion coefficient in log D vs. 1/T plot exhibit curved profiles and thus obey the Vogel–Fulcher–Tammann (VFT) equation, those in the neat [C10mim][Cl] have been found to be perfect Arrhenius-type. This finding has been discussed in terms of the classification of compounds into ‘fragile’-, ‘intermediate’- and ‘strong glass formers’. The Arrhenius-type behavior can be explained by the presence of the long and flexible alkyl chains, which soften the system making it more plastic and less ‘fragile’. The obtained activation energy values can be compared with those available in the literature for other RTILs and for some long-chain fatty acids (e.g. heptadecanoic (C17) and nonadecanoic (C19)) close to the melting point in the orientationally disordered solid phases. It can be assumed that the diffusion processes in the neat long-chain RTILs, similar to other long-chain molecular systems, go through the vacancies and the boundaries of highly dynamic mesoscopic structures. The critical aggregation concentration and the aggregation number have been determined from the dependence of diffusion coefficient on concentration in an aqueous solution.

A magnesium porphyrazine possessing isophthaloxybutylsulfanyl substituents in the periphery was synthesized and subjected to various photophysical studies, including optical absorption and emission measurements. Moreover, synchronous fluorescence spectra were recorded and a contour three-dimensional map of the excitation-emission of the studied porphyrazine was obtained. The porphyrazine macrocycle exhibited interesting solvatochromic effects in many different solvents. Upon excitation with visible light, it generated singlet oxygen with a low quantum yield, therefore when it was encapsulated in liposomes it exhibited no photocytotoxicity in the in vitro study on human carcinoma LNCaP cell line.

Chronic obstructive pulmonary disease, COPD, affects the condition of the entire human organism and causes multiple comorbidities. Pathological lung changes lead to quantitative changes in the composition of the metabolites in different body fluids. The obstructive sleep apnea syndrome, OSAS, occurs in conjunction with chronic obstructive pulmonary disease in about 10–20 % of individuals who have COPD. Both conditions share the same comorbidities and this makes differentiating them difficult. The aim of this study was to investigate whether it is possible to diagnose a patient with either COPD or the OSA syndrome using a set of selected metabolites and to determine whether the metabolites that are present in one type of biofluid (serum, exhaled breath condensate or urine) or whether a combination of metabolites that are present in two biofluids or whether a set of metabolites that are present in all three biofluids are necessary to correctly diagnose a patient. A quantitative analysis of the metabolites in all three biofluid samples was performed using 1H NMR spectroscopy. A multivariate bootstrap approach that combines partial least squares regression with the variable importance in projection score (VIP-score) and selectivity ratio (SR) was adopted in order to construct discriminant diagnostic models for the groups of individuals with COPD and OSAS. A comparison study of all of the discriminant models that were constructed and validated showed that the discriminant partial least squares model using only ten urine metabolites (selected with the SR approach) has a specificity of 100 % and a sensitivity of 86.67 %. This model (AUCtest = 0.95) presented the best prediction performance. The main conclusion of this study is that urine metabolites, among the others, present the highest probability for correctly identifying patents with COPD and the lowest probability for an incorrect identification of the OSA syndrome as developed COPD. Another important conclusion is that the changes in the metabolite levels of exhaled breath condensates do not appear to be specific enough to differentiate between patients with COPD and OSAS.

Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHP's backbone helical regions and turns have average order parameters of S2 = 0.87 ± 0.03 and S2 = 0.76 ± 0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τm is 9.49 ± 1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant Rex using the Lipari-Szabo model-free formalism. These include Lys3, Ile6, Leu13, Gln18, Arg32, Ser48, Met49, Thr56, Phe60, Arg69, Thr71 Cys73, Ala77, Asn81, Gly95, Arg109, Phe115, Leu127 and Met136, which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains >4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O2, H2O and H2O2 to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.

G-quadruplexes are non-canonical secondary structures which may be formed by guanine rich sequences, both in vitro and in living cells. The number of biological functions assigned to these structural motifs has grown rapidly since the discovery of their involvement in the telomere maintenance. Knowledge of the three-dimensional structures of G-quadruplexes plays an important role in understanding their conformational diversity, physiological functions, and in the design of novel drugs targeting G-quadruplexes. For the last decades, structural studies have been mainly focused on the DNA G-quadruplexes. Their RNA counterparts gained an increased interest along with still-emerging recognition of the central role of RNA in multiple cellular processes. In this review we focus on structural properties of RNA G-quadruplexes, based on high-resolution structures, available in RCSB PDB data base and on structural models. In addition, we point out to the current challenges in this field of research.

Three-dimensional structure of glycyrrhizic acid is determined by the conformation of the disaccharide unit and its relative orientation to a virtually rigid aglycone. X-ray crystallography, NMR spectroscopy, and density functional theory (DFT) calculations were used to study the conformational preferences of the glycosidic linkages in solid state, solution and vacuo, respectively. Experimental data have revealed that conformation of glycosidic bonds, that is adopted in the solid state, is also favored in solution. The molecular geometry optimizations have shown that a strongly twisted orientation of the two glucopyranose units, characteristic of the solid state, is stabilized by the bridging interaction of water molecules and/or cations with disaccharide COOH/COO¯ groups. Our results illustrate the impact of environment on the preferred conformation of disaccharide unit in the studied glycoside and point to a possible reason for the observed rigid conformation of the glycosidic bonds in solution and in t...

Acyclovir (ACV) belongs to a class of drugs with low bioavailability. Selected ACV esters including acetyl (Ac-), isobutyryl (iBut-), pivaloyl (Piv-), ethoxycarbonyl (Etc-) and nicotinoyl (Nic-) were synthesized, and their lipophilicity was determined by the high-performance liquid chromatography (HPLC) RP method. Statistical analyses of the comparative values of log P and clog P were carried out using computational methods. It was proved that the AC log P algorithm can be useful for the analysis of these compounds and has a statistically justified application in the assessment of the quantitative structure–activity relationship. Moreover, the lipophilicity determined by the HPLC method appears as follows: ACV

Background The nature of the polyamine–DNA interactions at a molecular level is not clearly understood. Methods In order to shed light on the binding preferences of polyamine with nucleic acids, the NMR solution structure of the DNA duplex containing covalently bound spermine was determined. Results The structure of 4-N-[4,9,13-triazatridecan-1-yl]-2′-deoxycytidine (dCSp) modified duplex was compared to the structure of the reference duplex. Both duplexes are regular right-handed helices with all attributes of the B-DNA form. The spermine chain which is located in a major groove and points toward the 3′ end of the modified strand does not perturb the DNA structure. Conclusion In our study the charged polyamine alkyl chain was found to interact with the DNA surface. In the majority of converged structures we identified the presumed hydrogen bonding interactions between O6 and N7 atoms of G4 and the first internal –NH2+− amino group. Additional interaction was found between the second internal –NH2+− amino group and the oxygen atom of the phosphate of C3 residue. General significance The knowledge of the location and nature of a structure-specific binding site for spermine in DNA should be valuable in understanding gene expression and in the design of new therapeutic drugs.

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.

N6-Threonylcarbamoyladenosine (t6A) and its derivatives are universally conserved modified nucleosides found at position 37, 3΄ adjacent to the anticodon in tRNAs responsible for ANN codons. These modifications have pleiotropic functions of tRNAs in decoding and protein synthesis. In certain species of bacteria, fungi, plants and protists, t6A is further modified to the cyclic t6A (ct6A) via dehydration catalyzed by TcdA. This additional modification is involved in efficient decoding of tRNALys. Previous work indicated that the chemical structure of ct6A is a cyclic active ester with an oxazolone ring. In this study, we solved the crystal structure of chemically synthesized ct6A nucleoside. Unexpectedly, we found that the ct6A adopted a hydantoin isoform rather than an oxazolone isoform, and further showed that the hydantoin isoform of ct6A was actually present in Escherichia coli tRNAs. In addition, we observed that hydantoin ct6A is susceptible to epimerization under mild alkaline conditions, warning us to avoid conventional deacylation of tRNAs. A hallmark structural feature of this isoform is the twisted arrangement of the hydantoin and adenine rings. Functional roles of ct6A37 in tRNAs should be reconsidered.

5-Bromouracil ((Br)U) modified di- and hexanucleotides having (Br)U flanked on the 5' or the 3' side by uracil (U) have been synthesized, and their photochemical reactivity was examined under the conditions of irradiation with near UV light. The results indicate that the primary photochemical process in all of these compounds involves the formation of an intermediate cyclobutane phodoadduct composed of (Br)U and U, which undergoes further photochemically and thermally induced transformations to 5,5'-bipyrimidine type adducts.

All guanine-rich DNA and RNA molecules tend to fold into inter- and intra-molecular structures called G-quadruplexes. The core of a G-quadruplex is composed of at least two adjacent G-tetrads stabilized by monovalent cations. While it has been suggested that there could potentially be over 375 000 quadruplex forming sequences in the human genome alone, only a small number of three-dimensional G-quadruplex structures are known. More structural data are needed to explain their presence and function in vivo. We offer here a short review of experimental methods commonly used to determine G-quadruplex topologies.

International audience; The NMR spectral densities of a complex motion consisting of combination of anisotropic overall motion and internal motion have been derived. Two approximations of the equations derived for the cases of slow, Jslow(), and fast, Jfast(), internal motions are presented. These equations imply that reduction in spectral density of overall motion can be observed if the maxima of internal and overall motions spectral densities versus temperature are well separated, so for the fast internal motion. Slow intramolecular motion influences the values of spectral densities of the overall motion if one of the two spins performs a motion as for example a proton in double minimum of the 15N-H.....N hydrogen bond. The analysis presented reveals small differences between the temperature dependencies of spectral densities of the isotropic and anisotropic overall motions. The theory is illustrated by the 13C protonated carbon spin-lattice relaxation of -cyclodextrin macromolecule, using the expected motional parameters: DII/D  5 at room temperature and for a fast or slow internal motion.

The complex shaped 1H NMR signal of [C10mim][Br] has been observed after it was mixed with water. Within minutes the contour transformed into Voigt (V) profile with Gauss (G) contribution as dominant and started to evolve towards Lorentz-shaped (L) contour. Relative G/L contribution in V-profile was proposed as the measure of nonequilibrium aggregation in mesoscopic/supramolecular scale. The equilibration kinetics was monitored by time dependence of the half-widths G/L with waiting time up to 2–4 days. The ‘nonequilibrium’ covers only certain degrees of freedom of the guest (ionic liquid) molecules, whereas the matrix, i.e. the hydrogen-bonded network of water, is equilibrated.

Nanosecond transient absorption and steady-state photochemical studies showed that interactions of the nucleic acid bases: uracil, thymine (5-methyluracil), 5-ethyluracil and adenine with triplet excited 9-substituted 6-thiopurine chromophore (TP) is influenced by the mutual orientation of the heterocyclic rings. In an aqueous solution containing a mixture of monomers, where no restrictions are imposed on ring-to-ring orientation, all the nucleobases quench the T1 state of 9-propyl-6-thiopurine (PTP) with similarly large rate constants ( k q inter ∼ 10 9 M − 1 s − 1 ) . Steady-state irradiation of TP in the presence of uridine and adenosine led to adducts formed via [2 + 2] cycloaddition of C S to the olefinic fragments of the nucleobases. In newly synthesized dyads composed of trimethylene-linked TP and nucleobase pairs, only thymine and 5-ethyluracil reduced the TP-like T1 state lifetime ( k q intra ∼ 5 × 10 6 s − 1 ) . The relative orientations of the 6-thiopurine and nucleobase rings in the dyads are limited by the spacer. The length of the trimethylene chain does not allow for a close approach of the reactive centers for [2 + 2] photocycloadditions. In steady-state irradiation only the dyads containing thymine or 5-ethyluracil are photoreactive, and they form intramolecular cyclophane-type products albeit with low quantum yields (ϕ ≤ 6 × 10−3). The structure of the photoproducts can be rationalized by assuming an initial H atom abstraction from the 5-alkyl group at the C(5) position of the uracil ring by an excited thiocarbonyl group. The preferential reversal of biradicals formed from [2 + 2] photocycladditon and from H atom abstraction has been suggested as mechanisms responsible for quenching of the TP T1 state by nucleobases.

In the cytidine and adenosine derivatives an isomerization of a 2,4-dinitrophenyl group between the 2'- and 3'-positions of the ribose was observed under neutral conditions. Moreover, it was shown that isomerization of the 2,4-dinitrophenyl group in conditions required to synthesize phosphoramidites and lability in aqueous ammonia make chemical synthesis of 2'-O-(2,4-dinitrophenyl) oligonucleotides impossible.

Two highly fluorescent, thermally stable diastereomeric photoadducts, 3a,b, are formed when either 5-chloro-4-thiouridine, 1, or 5-fluoro-4-thiouridine, 2, are photoexcited with 366 nm UV light in the presence of thymidine (T). 5-Fluoro-4-thiouridine, 2, exhibits photoreactivity much higher than that of the 5-chloro derivative 1. In both cases the photoreaction is very clean, leading to highly eficient conversion of the 5-halogeno-4-thiouridine (1, 2) and T to photoadducts 3a,b. The identity and structure of 3a was confirmed using mass spectrometry and 2-D NMR. The epimeric relationship of 3a,b was established by UV circular dichroism spectroscopy. The geometry of the fluorescent photoadduct is consistent with formation of an interstrand cross-link in a DNA duplex if 1 or 2 is flanked by T in an opposite strand.

Novel photoadducts were obtained by irradiation of thioinosine (6-thiopurine riboside, TI) in deaerated aqueous solution without and in the presence of uridine and adenosine. Excitation (lambda300 nm) of TI to its excited S2 state yields a single bimolecular photoproduct. It is a purine-pyrimidine diriboside in which the purine ring is attached to the amide nitrogen of 6-amino-4-thioxo-5-formamidopyrimidine. When TI was irradiated in the presence of an excess of adenosine, two photoproducts were isolated: diribosides of N-(4,6-diaminopirymidin-5-yl)-N-formyl-6-aminopurine and N-(4-amino-6-formylamino-pyrimidin-5-yl)-6-aminopurine, both containing a purine and a formylaminopyrimidine (Fapy) fragment. The photoreaction of TI with uridine gave two regioisomeric photoproducts identified as diribosides containing either 5- or 6-(purin-6-yl)uracil as aglycones. A multistep mechanism leading to the stable photoproducts is proposed. In the first step of the mechanism, the C=S group of the excited TI undergoes a [2 + 2] cycloaddition regioselectively to the N(7)=C(8) bond of the purine ring or adds in a non-regioselective manner to the C(5)=C(6) bond of uracil. The unstable photoproducts thus formed undergo a series of dark reactions at room temperature. The photocycloaddition reactions originate from the excited T1 state of TI. This conclusion is supported by a combination of evidence from reaction quenching studies using both steady-state quantum yield determinations and kinetics results from nanosecond laser flash photolysis. The T1 state of TI is quenched by other TI molecules in their S0 state (self-quenching) and also by uridine and adenosine, all with large rate constants (0.8-5) x 10(9) M(-1) s(-1). The quantum yields of the reactions are in general very low (phi(R)or = 8 x 10(-3)). The sources of the inefficiency in the photocycloaddition of TI to uridine and adenosine are discussed. The photoproducts containing the Fapy residue undergo deformylation and isomerization of the ribosyl moiety (anomerization, furanose/pyranose transformation) upon heating in aqueous solution. Products of the transformations were identified.

The RNA single bulge motif is an unpaired residue within a strand of several complementary base pairs. Structure and dynamics of RNA duplexes containing single adenosine (5′GUCGAGCUG-3′/5′-CAGCCGAC-3′) and uridine bulge (5′-GUCGUGCUG-3′/5′CAGCCGAC-3′) have been studied using NMR spectroscopy and in aqua simulation of molecular dynamics. In both NMR structures, the bulged residues are oriented toward the minor groove. The bulged adenosine participates in the base triple formation being coplanar with the 5′neighboring residue G4 which pairs with cytidine C14 from the opposite strand. The uridine bulge adopts a looped-out conformation pointing toward the residue G15 and bridges the minor groove to 5′ side of the helix. NMR-derived structures have been used to formulate the initial RNA duplex models for the MD simulation. Results of molecular dynamics simulations in explicit solvent support the conclusion concerning the formation of a dinucleotide platform within the C:G-A triple. As suggested by NMR results, the uridine bulge shows considerable dynamics.

The RNA single bulge motif is an unpaired residue within a strand of several complementary base pairs. To gain insight into structural changes induced by the presence of the adenosine bulge on RNA duplex, the solution structures of RNA duplex containing a single adenine bulge (5'-GCAGAAGAGCG-3'/5'-CGCUCUCUGC-3') and a reference duplex with all Watson-Crick base pairs (5'-GCAGAGAGCG-3'/5'-CGCUCUCUGC-3') have been determined by NMR spectroscopy. The reference duplex structure is a regular right-handed helix with all of the attributes of an A-type helix. In the bulged duplex, single adenine bulge stacks into the helix, and the bulge region forms a well-defined structure. Both structures were analyzed by the use of calculated helical parameters. Distortions induced by the accommodation of unpaired residue into the helical structure propagate over the entire structure and are manifested as the reduced base pairs inclination and x-displacement. Intrahelical position of bulged adenine A5 is stabilized by efficient stacking with 5'-neighboring residues G4.

23Na and 81Br NMR spin-lattice relaxation times and signal half widths (Δ1/2) have been measured in 3-methylpyridine (3MP)/H2O/NaBr mixture along T = 294 and 301 K isotherms gradually increasing the mass fractions of salt (X) up to the phase separation boundary. The extreme narrowing condition and thus 1/T 1 = 1/T 2 = πΔ 1/2 was found to be valid in all cases. Discontinuous changes in slope of 1/T 1,2 = f(X) were detected, and then corresponding points on the phase diagram (X, T) were attributed to the borderline between the molecular-ionic solution and the area of enhanced mesoscopic structuring. A very strong relaxation effect was observed for 81Br nuclei reaching relaxation rates of 14,000 s–1. 23Na and 81Br NMR relaxation data together with calculations of quantum chemistry model of electrical field gradient tensor evidence the migration of 3-methylpyridinium at increasing X from the anions hydration shells towards cations. An interchange from migration to steady distribution regimes is observed for a...

Nucleic acids symposium series 44(5), 2409 - 2416(2016). doi:10.1093/nar/gkv1534, Fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS) are neurodegenerative disorders caused by the pathogenic expansion of CGG triplet repeats in the FMR1 gene. FXTAS is likely to be caused by a ‘toxic’ gain-of-function of the FMR1 mRNA. We provide evidence for the existence of a novel quadruplex architecture comprising CGG repeats. The 8-bromoguanosine ($^{Br}$G)-modified molecule GC$^{Br}$GGCGGC forms a duplex in solution and self-associates via the major groove to form a four-stranded, antiparallel (GC$^{Br}$GGCGGC)4 RNA quadruplex with $^{Br}$G3:G6:$^{Br}$G3:G6 tetrads sandwiched between mixed G:C:G:C tetrads. Self-association of Watson–Crick duplexes to form a four-stranded structure has previously been predicted; however, no experimental evidence was provided. This novel four-stranded RNA structure was characterized using a variety of experimental methods, such as native gel electrophoresis, NMR spectroscopy, small-angle X-ray scattering and electrospray ionization mass spectrometry., Published by Oxford Univ. Press8619, Oxford

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in β-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2′-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by 1H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.

High hydrostatic pressure (HHP) is an emerging tool for studying conformational changes in lipids, proteins and nucleic acids. Although many thermodynamic parameters describing those processes have been determined, a molecular mechanism of HHP action is poorly recognized. To get insight into that, we have studied quinolizidine alkaloid, lupanine. It consists of two quinolizidine moieties, one of which contains lactam group. Using Fourier-transform infrared (FT-IR) spectroscopy, we have showed that at 6 kbar, the intensity of amide band at 1589 cm−1 decreased and a new band at 1556 cm−1 appeared. These changes are due to the hydrolysis of lupanine to lupanic acid. That reaction was confirmed with 13C NMR spectra of lupanine exposed to HHP. The NMR signals at 176.6 and 184.3 ppm were assigned to lactam group of lupanine and carboxylic group of lupanic acid, respectively. The ring opening reaction of lupanine under HHP is reversible at ambient pressure, as evidenced by CD measurements. A slightly acidic condition induced by HHP causes protonation of lactam group and carbocation is formed, while on the other hand, water molecule as nucleophile attacks electrophilic carbon of lactam and electrons move towards oxonium ion. Finally, C N bond breaks down and carboxyl group is formed.

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.

The photochemical behavior of 2-azidopurine 2',3',5'-tri-O-acetylribonucleoside has been investigated in aqueous solution under aerobic and anaerobic conditions. The two major processes under anaerobic irradiation of 2-azidopurine 2',3',5'-tri-O-acetylribonucleoside involve unprecedented transformation into 1-(5'-O-acetyl-β-D-ribofuranosyl)-5-[(2-oxo-1,3,5-oxadiazocan-4-ylidene)amino]-1H-imidazole-4-carbaldehyde and photoreduction to respective 2-aminopurine derivative, whereas under aerobic conditions these two processes occur to a much lesser extent and photooxidation to respective 2-nitropurine derivative dominates. The structures of photoproducts formed were confirmed by NMR and high-resolution electrospray ionization mass spectral data.

The mechanism of chemical synthesis of adenosine has been reinvestigated. Depending on the reaction conditions and the presence of N6-protecting groups, ribosylation of adenine proceeds via different kinetic products: 3-riboadenine in strongly acidic media, 7-ribosylated derivative in the silyl method, and 1-(β- d -ribofuranosyl)adenine when applying N6-acyladenine and silylating conditions.