The origin of the high stability of 3'-terminal uridine tetrads: contributions of hydrogen bonding, stacking interactions, and steric factors evaluated using modified oligonucleotide analogs.

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.

Solution Structure of a Lanthanide-binding DNA Aptamer Determined Using High Quality pseudocontact shift restraints.

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 and DNA G-quadruplexes bind to human dicer and inhibit its activity.

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.

Novel Short PEG Chain-Substituted Porphyrins: Synthesis, Photochemistry, and In Vitro Photodynamic Activity against Cancer Cells.

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.

Unraveling the structural basis for the exceptional stability of RNA G-quadruplexes capped by a uridine tetrad at the 3' terminus.

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.

A hydantoin isoform of cyclic N6-threonylcarbamoyladenosine (ct6A) is present in tRNAs.

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.

Structural characterization of a dimer of RNA duplexes composed of 8-bromoguanosine modified CGG trinucleotide repeats: a novel architecture of RNA quadruplexes.

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.

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.

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.

Polyaminooligonucleotide: NMR structure of duplex DNA containing a nucleoside with spermine residue, N-[4,9,13-triazatridecan-1-yl]-2'-deoxycytidine.

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.

Calculation procedures and HPLC method for analysis of the lipophilicity of acyclovir esters.

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 < Ac- < Nic- < Etc- < iBut- < Piv-.

Photochemistry of 6-amino-2-azido, 2-amino-6-azido and 2,6-diazido analogues of purine ribonucleosides in aqueous solutions.

The photochemistry of 6-amino-2-azidopurine, 2-amino-6-azidopurine and 2,6-diazidopurine ribonucleosides has been investigated in aqueous solutions under aerobic and anaerobic conditions. Near UV irradiation of 6-amino-2-azido-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine and 2-amino-6-azido-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine in the presence of oxygen leads to efficient formation of 6-amino-2-nitro-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine and 2-amino-6-nitro-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine. Under anaerobic conditions, both azidopurine ribonucleosides preferentially undergo photoreduction to 2,6-diamino-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)purine. The structures of the photoproducts formed were confirmed by UV, NMR and HR ESI-TOF MS spectral data. The photoproducts observed in this study for the aminoazidopurines are distinctly different from those observed previously for 6-azidopurine. When no amino group is present, the photochemistry of 6-azidopurine leads to the formation of a 1,3,5-triazepinone nucleoside. The energetics of the 6-nitreno moiety along both oxidation and ring expansion pathways was calculated using the nudged elastic band (NEB) method based on density functional theory (DFT) using DMol3. The role of the 2-amino group in regulating the competition between these pathways was elucidated in order to explain how the striking difference in reactivity under irradiation arises from the greater spin density on the 6-nitreno-9-methyl-9H-purin-2-amine, which essentially eliminates the barrier to oxidation observed in 6-nitreno-9-methyl-9H-purine. Finally, the importance of tetrazolyl intermediates for the photochemical activation of azide bond cleavage to release N2 and form the 6-nitreno group was also corroborated using the DFT methods.

The 8-17 DNAzyme can operate in a single active structure regardless of metal ion cofactor.

DNAzymes - synthetic enzymes made of DNA - have long attracted attention as RNA-targeting therapeutic agents. Yet, as of now, no DNAzyme-based drug has been approved, partially due to our lacking understanding of their molecular mode of action. In this work we report the solution structure of 8-17 DNAzyme bound to a Zn2+ ion solved through NMR spectroscopy. Surprisingly, it turned out to be very similar to the previously solved Pb2+-bound form (catalytic domain RMSD = 1.28 Å), despite a long-standing literature consensus that Pb2+ recruits a different DNAzyme fold than other metal ion cofactors. Our follow-up NMR investigations in the presence of other ions - Mg2+, Na+, and Pb2+ - suggest that at DNAzyme concentrations used in NMR all these ions induce a similar tertiary fold. Based on these findings, we propose a model for 8-17 DNAzyme interactions with metal ions postulating the existence of only a single catalytically-active structure, yet populated to a different extent depending on the metal ion cofactor. Our results provide structural information on the 8-17 DNAzyme in presence of non-Pb2+ cofactors, including the biologically relevant Mg2+ ion.

Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Photoaddition of 5-bromouracil to uracil in oligonucleotides leading to 5,5'-bipyrimidine-type adducts: mechanism of the photoreaction.

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.

1H, 13C NMR and DFT Study of Hydrogen Bonding in Imidazolium-based Ionic Liquids.

The ionic liquid 1-decyl-3-methyl-imidazolium bromide [C10mim][Br], the neat material, and also dissolved (~0.01 mole fraction) in various dielectric media (acetonitrile, benzene, chloroform, dichloromethane, methanol, 2-butanol and H2O) was studied using 1H and 13C NMR spectroscopy. The most important interaction in this compound is considered to be the Br-...H-C2+ hydrogen bond, which is formed between the anions and cations. The obtained results show that dielectric medium influence mostly the behavior of the Br-...H-C2+ bridge proton. The changes observed in 1H and 13C NMR spectra of [C10mim][Br] with increasing solvents polarity and temperature can be explained applying the model of the lengthening of the H2...Br- bond with the accompanying thickening of the solvation shell of bromine anion and C2-H bond contraction. The short-range order effects related to the configuration of neighboring dipoles of solvent molecules are more important for the solvation ability of small anions than the bulk solvent field effect. However, the solvents, molecules of which tend to associate via hydrogen bonding, can significantly affect the dynamics of anions.

Impact of a Single Nucleotide Change or Non-Nucleoside Modifications in G-Rich Region on the Quadruplex-Duplex Hybrid Formation.

In this paper, a method to discriminate between two target RNA sequences that differ by one nucleotide only is presented. The method relies on the formation of alternative structures, i.e., quadruplex-duplex hybrid (QDH) and duplex with dangling ends (Dss), after hybridization of DNA or RNA G-rich oligonucleotides with target sequences containing 5'-GGGCUGG-3' or 5'-GGGCGGG-3' fragments. Using biophysical methods, we studied the effect of oligonucleotide types (DNA, RNA), non-nucleotide modifications (aliphatic linkers or abasic), and covalently attached G4 ligand on the ability of G-rich oligonucleotides to assemble a G-quadruplex motif. We demonstrated that all examined non-nucleotide modifications could mimic the external loops in the G-quadruplex domain of QDH structures without affecting their stability. Additionally, some modifications, in particular the presence of two abasic residues in the G-rich oligonucleotide, can induce the formation of non-canonical QDH instead of the Dss structure upon hybridization to a target sequence containing the GGGCUGG motif. Our results offer new insight into the sequential requirements for the formation of G-quadruplexes and provide important data on the effects of non-nucleotide modifications on G-quadruplex formation.

Photoinduced fluorescent cross-linking of 5-chloro- and 5-fluoro-4-thiouridines with thymidine.

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.

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.

Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide.

Antisense DNA oligonucleotides, short interfering RNAs (siRNAs), and CRISPR/Cas9 genetic tools are the most useful therapeutic nucleic acids regulating gene expression based on the antisense specificity towards messenger RNA. Here, we present an effective novel strategy for inhibiting translation based on the antisense-controlled formation of an RNA quadruplex-duplex hybrid (QDH) between a G-rich RNA antisense oligoribonucleotide (Q-ASO) and specific mRNA, comprising two distant G-tracts. We selected epidermal growth factor receptor (EGFR) as a well-established target protein in anticancer therapy. The chemically modified, bi-functional anti-EGFR Q-ASO and a 56-nt long EGFR mRNA fragment, in the presence of potassium ions, were shown to form in vitro very stable parallel G-quadruplex containing a 28-nt long external loop folding to two duplex-stem structure. Besides, the Q-ASOs effectively reduced EGFR mRNA levels compared to the non-modified RNA and DNA antisense oligonucleotides (rASO, dASO). In addition, the hybridization specificity of Q-ASO comprising a covalently attached fluorescent tag was confirmed in living cells by visualization of the G4 green fluorescent species in the presence of other antisense inhibitors under competitive conditions. The results presented here offer novel insights into the potential application of Q-ASOs for the detection and/or alteration of (patho)biological processes through RNA:RNA quadruplex-duplex formation in cellular systems.