Proof of ion-pair structures in ammonium-based protic ionic liquids using combined NMR and DFT/PCM-based chemical shift calculations.

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+-HN or N+-HO hydrogen bonding, whereas hydrophilic complexes with Cα-HO and Cα-HF 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.

An algorithm for an automatic NOE pathways analysis of 2D NMR spectra of RNA duplexes.

An algorithm is proposed to provide the tool for an automatic resonance assignment of 2D-NOESY spectra of RNA duplexes. The algorithm, based on a certain subproblem of the Hamiltonian path, reduces a number of possible connections between resonances within aromatic and anomeric region of 2D-NOESY spectra. Appropriate pathways between H6/H8 and H1' resonances were obtained by subsequent implementation of experimental data as limiting factors. Predictive power of the algorithm was tested on both experimental and simulated data for RNA and DNA duplexes.

Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A.

The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(Lys3)) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modified anticodon stem and loop domain of tRNA(Lys3)(UUU) (ASL(Lys3)(UUU)- t(6)A(37)) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL(Lys3)(UUU) is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 +/- 0.33 A (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL(Lys3)(UUU)-t(6)A(37) loop is significantly different than that of the unmodified ASL(Lys3)(UUU), although the five canonical base pairs of both ASL(Lys3)(UUU) stems are in the standard A-form of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U(33).A(37) base pair that is observed in the unmodified ASL(Lys3)(UUU). The anticodon wobble position U(34) nucleobase in ASL(Lys3)(UUU)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL(Lys3)(UUU) tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A(38) is protonated and positively charged in ASL(Lys3)(UUU)-t(6)A(37) and the unmodified ASL(Lys3)(UUU). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(+)(38). The protonated A(+)(38) can base pair with C(32), but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL(Lys3)(UUU) would bind as well as ASL(Lys3)(UUU)-t(6)A(37). t(6)A(37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.

Internal loop/bulge and hairpin loop of the iron-responsive element of ferritin mRNA contribute to maximal iron regulatory protein 2 binding and translational regulation in the iso-iron-responsive element/iso-iron regulatory protein family.

Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n = 28-30), and have a weaker signal response in vivo. Two trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505-1512]. IRE function (translation) and physical properties (T(m) and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substituting the HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen)(2) resistant] and more stable (T(m) increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T(m) decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The (1)H NMR spectrum of the Mg-IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/protein interactions that control protein synthesis rates with a range of signal sensitivities.

Does 29-mer RNA hairpin of the HIV-1 TAR RNA sequence bind magnesium? 19F-NMR and modelling studies.

5-Fluorouridine residues have been introduced into functionally important bulge and loop regions of 29-mer HIV-1 TAR RNA hairpins I and II to study Mg2+ and Ca2+ binding using 19F-NMR spectroscopy. There was no substantial binding detected up to 20-molar excess in case of both cations, whereas association of argininamide, used as a reference ligand, could be detected at less than 1-molar excess. The deltadelta 19F value of 1.93 ppm observed for (F)U23 upon argininamide binding is in agreement with former NMR studies of TAR RNA/argininamide complex. However, obtained results do not confirm U38 x A27 x U23 base-triple formation. The unmodified HIV-1 TAR RNA hairpin resulted from 600 ps in aqua molecular dynamics simulation was subjected to a molecular mechanics modelling of Mg+ binding.

Studies on synthesis and structure of O-beta-D-ribofuranosyl(1"-->2')ribonucleosides and oligonucleotides.

Minor nucleosides found in several eukaryotic initiator tRNASiMet, O-beta-D-ribofuranosyl(1"-->2')adenosine and -guanosine (Ar and Gr), as well as their pyrimidine analogues, were obtained from N-protected 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)ribonucle osides and 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in the presence of tin tetrachloride in 1,2-dichloroethane. A crystal structure has been solved for 2'-O-ribosyluridine. The 3'-phosphoramidites of protected 2'-O-ribosylribonucleosides were prepared as the reagents for 2'-O-ribofuranosyloligonucleotides synthesis. O-beta-D-Ribofuranosyl(1"-->2')adenylyl(3'-->5')guanosine (ArpG) was obtained and its structure was analysed by NMR spectroscopy.

Iron regulatory element and internal loop/bulge structure for ferritin mRNA studied by cobalt(III) hexammine binding, molecular modeling, and NMR spectroscopy.

The ferritin IRE, a highly conserved (96-99% in vertebrates) mRNA translation regulatory element in animal mRNA, was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Cobalt(III) hexammine was used to model hydrated Mg2+. IRE isoforms in other mRNAs regulate mRNA translation or stability; all IREs bind IRPs (iron regulatory proteins). A G.C base pair, conserved in ferritin IREs, spans an internal loop/bulge in the middle of an A-helix and, combined with a dynamic G.U base pair, formed a pocket suitable for Co(III) hexammine binding. On the basis of the effects of Co(III) hexammine on the 1H NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III) hexammine at the pocket in the major groove; Mg2+ may bind to the IRE at the same site on the basis of an analogy to Co(III) hexammine and on the Mg2+ inhibition of Cu-(phen)2 cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP cross-linking site suggests a role for the pocket in ferritin IRE/IRP interactions.

Methoxyamine-induced mutagenesis of nucleic acids. A proton NMR study of oligonucleotides containing N4-methoxycytosine paired with adenine or guanine.

We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case paired with adenine on the opposite strand and the other with guanine. For the N4-methoxycytosine adenine pair, only the imino form of the N4-methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N4-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the cis conformation of the OCH3 group in the single strand, which inhibits base pair formation. For the N4-methoxycytosine that is base paired with guanine, we observe an amino form in Watson-Crick geometry in slow exchange with a base paired imino form in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 313 K. We have measured the exchange rate between the two forms at 303 K and observed a value of approximately 1 S-1. The relative ratio of amino and imino forms of N4-methoxycytosine is influenced by both the base that is in front and the temperature. Our results explain the preferential replacement of dTTP by N4-methoxycytosine in primer elongation.

Methoxyamine attack on cytosine produces ambivalent base pairing properties of the modified base.

We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case with adenine on the opposite strand and in the other with guanine. For the N4-methoxycytosine-adenine pair only the imino form of the N4-methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N4-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the syn conformation of the OCH3 group in the single strand which inhibits base pair formation. For N4-methoxycytosine base paired with guanosine we observe the N4-methoxycytosine base in the amino form in Watson-Crick geometry and a slow exchange of this species with an imino form base paired in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 40 degrees C. Our results point to preferential replacement of dTTP by N4-methoxycytosine in primer elongation.

Base-pair induced shifts in the tautomeric equilibrium of a modified DNA base.

We have examined the base-pairing properties of N4-methoxycytosine (mo4C), a mutagenic base analog, in DNA by nuclear magnetic resonance spectroscopy. Unlike standard bases, the tautomeric equilibrium of mo4C could be strongly influenced by base-pair formation. Paired with A, mo4C is found predominantly in the imino configuration in Watson-Crick geometry. However, when paired with G, two structurally distinct configurations are observed in equilibrium with one another. In one configuration, mo4C is in the amino form paired with G in Watson-Crick geometry. In the second species, mo4C is in the imino configuration paired with G in a wobble geometry. This is the first demonstration of basepair induced tautomeric shifts in DNA and supports the hypothesis that rare tautomeric forms may be involved in mutagenesis.

5' end fluorescent labelling of oligonucleotides with riboflavin-derived phosphitylating reagent.

Riboflavin was transformed within six steps into 3-isobutyryl-7,8-dimethyl-10-[2-O-(beta-cyanoethoxy-N,N- diisopropylaminophosphinyl)ethyl]isoalloxazine. This new fluorescent reagent was applied for direct phosphitylation of 5-OH function of protected oligonucleotide assembled on controlled-pore glass support by beta-cyanoethyl phosphoramidite chemistry. As the result of subsequent P(III)----P(V) oxidation and removal of protecting groups with concentrated ammonia, an oligonucleotide 5-labelled with fluorescent flavin moiety could be obtained. Using this procedure 15-mer oligonucleotide of a sequence corresponding to M13 hybridization primer was prepared.