NMR Conformational Study Reveals that S-C-N Anomeric Effect in Thionucleosides Is Weaker than O-C-N Anomeric Effect in Natural Nucleosides

The comparative analysis by NMR and ab initio calculations of the energetics of North &#8644; South pseudorotational equilibrium in 4\u27-thio-nucleosides and in their natural 4\u27-oxo counterparts has shown that S-C-N anomeric effect in the former is weaker than O-C-N anomeric effect in the latter. The &Delta;&Delta;H° values between 2\u27-deoxy-4\u27-thio analogues and their 4\u27-oxo counterparts after accounting for the drive by 3\u27-OH group have been attributed to the weakening of the nucleobase-dependent S4\u27-C1\u27-N9/1 anomeric effect by 3.3, 6.5, 8.5 and 9.2 kJ mol-1 in adenine, guanine, cytosine and thymine, respectively. In addition, S-C-N anomeric effect is stronger in purine than in pyrimidine 4\u27-thionucleosides and increases in the following order: thymine < cytosine < guanine < adenine, which is in contrast to natural nucleosides

Identification of mixed di-cation forms of G-quadruplex in solution

Multinuclear NMR study has demonstrated that G-quadruplex adopted by d(G(3)T(4)G(4)) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K(+) ions into the solution of d(G(3)T(4)G(4))(2) folded in the presence of [Formula: see text] ions uncovered a mixed mono-K(+)-mono- [Formula: see text] form that represents intermediate in the conversion of [Formula: see text] into di-K(+) form. Analogously, [Formula: see text] ions were found to replace Na(+) ions inside d(G(3)T(4)G(4))(2) quadruplex. The preference of [Formula: see text] over Na(+) ions for the two binding sites is considerably smaller than the preference of K(+) over [Formula: see text] ions. The two cation binding sites within the G-quadruplex core differ to such a degree that [Formula: see text] ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K(+) ions. The second binding site is not taken up by K(+) ion until K(+) ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K(+) ion concentrations afforded equilibrium binding constants. K(+) ion binding to sites U and L within d(G(3)T(4)G(4))(2) is more favorable with respect to [Formula: see text] ions by Gibbs free energies of approximately −24 and −18 kJ mol(−1) which includes differences in cation dehydration energies, respectively

Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex

The isomerization of phosphodiester functionality of nucleic acids from 3′,5′- to a less common 2′,5′-linkage influences the complex interplay of stereoelectronic effects that drive pseudorotational equilibrium of sugar rings and thus affect the conformational propensities for compact or more extended structures. The present study highlights the subtle balance of non-covalent forces at play in structural equilibrium of 2′,5′-linked RNA analogue, 3′-O-(2-methoxyethyl) substituted dodecamer *CG*CGAA*U*U*CG*CG, 3′-MOE-2′,5′-RNA, where all cytosines and uracils are methylated at C5. The NMR and UV spectroscopic studies have shown that 3′-MOE-2′,5′-RNA adopts both hairpin and duplex secondary structures, which are involved in a dynamic exchange that is slow on the NMR timescale and exhibits strand and salt concentration as well as pH dependence. Unusual effect of pH over a narrow physiological range is observed for imino proton resonances with exchange broadening observed at lower pH and relatively sharp lines observed at higher pH. The solution structure of 3′-MOE-2′,5′-RNA hairpin displays a unique and well-defined loop, which is stabilized by Watson–Crick A5·*U8 base pair and by n → π* stacking interactions of O4′ lone-pair electrons of A6 and *U8 with aromatic rings of A5 and *U7, respectively. In contrast, the stem region of 3′-MOE-2′,5′-RNA hairpin is more flexible. Our data highlight the important feature of backbone modifications that can have pronounced effects on interstrand association of nucleic acids

Coexistence of two main folded G-quadruplexes within a single G-rich domain in the EGFR promoter

EGFR is an oncogene which codifies for a tyrosine kinase receptor that represents an important target for anticancer therapy. Indeed, several human cancers showed an upregulation of the activity of this protein. The promoter of this gene contains some G-rich domains, thus representing a yet unexplored point of intervention to potentially silence this gene. Here, we explore the conformational equilibria of a 30-nt long sequence located at position -272 (EGFR-272). By merging spectroscopic and electrophoretic analysis performed on the wild-type sequence as well as on a wide panel of related mutants, we were able to prove that in potassium ion containing solution this sequence folds into two main G-quadruplex structures, one parallel and one hybrid. They show comparable thermal stabilities and affinities for the metal ion and, indeed, they are always co-present in solution. The folding process is driven by a hairpin occurring in the domain corresponding to the terminal loop which works as an important stabilizing element for both the identified G-quadruplex arrangements

NMR evaluation of ammonium ion movement within a unimolecular G-quadruplex in solution

d[G4(T4G4)3] has been folded into a unimolecular G-quadruplex in the presence of 15NH4+ ions. NMR spectroscopy confirmed that its topology is the same as the solution state structure determined earlier by Wang and Patel (J. Mol. Biol., 1995; 251: 76–94) in the presence of Na+ ions. The d[G4(T4G4)3] G-quadruplex exhibits four G-quartets with three 15NH4+-ion-binding sites (O1, I and O2). Quantitative analysis utilizing 15NH4+ ions as a NMR probe clearly demonstrates that there is no unidirectional 15NH4+ ion movement through the central cavity of the G-quadruplex. 15NH4+ ions move back and forth between the binding sites within the G-quadruplex and exchange with ions in bulk solution. 15NH4+ ion movement is controlled by the thermodynamic preferences of individual binding sites, steric restraints of the G-quartets for 15NH4+ ion passage and diagonal versus edge-type arrangement of the T4 loops. The movement of 15NH4+ ions from the interior of the G-quadruplex to bulk solution is faster than exchange within the G-quadruplex. The structural details of the G-quadruplex define stiffness of individual G-quartets that intimately affects 15NH4+ ion movement. The stiffness of G-quartets and steric hindrance imposed by thymine residues in the loops contribute to the 5-fold difference in the exchange rate constants through the outer G-quartets

Strand directionality affects cation binding and movement within tetramolecular G-quadruplexes

Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG3T) and its modified analogs containing a 50-50 or 30-30 inversion of polarity sites, namely d(30TG50-50G2T30), d(30T50- 50G3T30) and d(50TG30-30G2T5’) demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 50-30 backbone. Exchange of 15NH+ 4 ions between G-quadruplex and bulk solution is faster at the 30-end in comparison to the 50-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG3T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures

Solution Structure of a Prion Protein Aptamer Analogue

It has previously been shown that r(GGA)4 folds into a G-quadruplex structure, which binds to the normal cellular form of the prion protein (PrPC) with high affinity. The current study utilizes CD and NMR spectroscopy to show that a dimeric parallel G-quadruplex structure is formed by r(GGA)2 in a KCl solution. Each r[(GGA)2]2 G-quadruplex unit exhibits two G-quartets, one of which is hydro- gen bonded to two additional adenines forming a hexade. Through stacking of hexade planes, two G-quadruplex units interact with each other and form a symmetric dimer, r[(GGA)2]4. The topolo¬gy of r[(GGA)2]4 is in agreement with the fold of r[(GGA)4]2, however, subtle differences are found in the region responsible for PrPC binding

Solubilization of ibuprofen for freeze dried parenteral dosage forms

Ibuprofen, a weakly acidic non-steroidal anti-inflammatory drug having poor aqueous solubility, is a challenging drug for the development of pharmaceutical formulations, resulting in numerous research attempts focusing on improvement of its solubility and consequently bioavailability. Most studies have been done for solid dosage forms, with very little attention paid to parenterals. Hence, the main purpose of the present study was to enhance ibuprofen solubility as a result of formulation composition and the freeze drying process. Moreover, the purpose was to prepare a freeze dried dosage form with improved ibuprofen solubility that could, after simple reconstitution with water for injection, result in an isotonic parenteral solution. Solubility of ibuprofen was modified by various excipients suitable for parenteral application. Drug interactions with selected excipients in the final product/lyophilisate were studied by a combined use of XRPD, DSC, Raman and ssNMR. Analyses of lyophilized samples showed solubility enhancement of ibuprofen and in situ formation of an ibuprofen salt with the alkaline excipients used

Non-canonical Structures in Promoter Modulate Gene Expression in Escherichia coli

Herein we show how sequences that can form different non-canonical structures affect gene expression levels when inserted in the core of σ70-dependent promoter, between the −35 and −10 elements recognized by RNA polymerase, in E. coli. We note that influence on level of GFP expression varies considerably depending on introduction of non-canonical structural elements in the antisense and sense strands as well as with their propensities to form G-triplex, G-hairpin, hairpin or G-quadruplex structures. Moreover, the extent of repression of expression does not relate to the in vitro thermal stability in a simple manner. Repression is most likely caused by steric interference rather than improper distance between the −35 and −10 elements. Although properties like thermal stability and topology can be somewhat different under in vivo and in vitro conditions, our results suggest that the extent of expression suppression cannot be dependent solely on thermal stabilities of G-rich structures alone. This work is licensed under a Creative Commons Attribution 4.0 International License