Automated 3D RNA structure prediction using the RNAComposer method for riboswitches.

Understanding the numerous functions of RNAs depends critically on the knowledge of their three-dimensional (3D) structure. In contrast to the protein field, a much smaller number of RNA 3D structures have been assessed using X-ray crystallography, NMR spectroscopy, and cryomicroscopy. This has led to a great demand to obtain the RNA 3D structures using prediction methods. The 3D structure prediction, especially of large RNAs, still remains a significant challenge and there is still a great demand for high-resolution structure prediction methods. In this chapter, we describe RNAComposer, a method and server for the automated prediction of RNA 3D structures based on the knowledge of secondary structure. Its applications are supported by other automated servers: RNA FRABASE and RNApdbee, developed to search and analyze secondary and 3D structures. Another method, RNAlyzer, offers new way to analyze and visualize quality of RNA 3D models. Scope and limitations of RNAComposer in application for an automated prediction of riboswitches' 3D structure will be presented and discussed. Analysis of the cyclic di-GMP-II riboswitch from Clostridium acetobutylicum (PDB ID 3Q3Z) as an example allows for 3D structure prediction of related riboswitches from Clostridium difficile 4, Bacillus halodurans 1, and Thermus aquaticus Y5.1 of yet unknown 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.

The 1.19 A X-ray structure of 2'-O-Me(CGCGCG)(2) duplex shows dehydrated RNA with 2-methyl-2,4-pentanediol in the minor groove.

The crystal and molecular structure of 2'-O-Me(CGCGCG)(2) has been determined at 1.19 A resolution, at 100 K, using synchrotron radiation. The structure in space group P3(2)12 is a half-turn right-handed helix that includes two 2-methyl-2,4-pentanediol (MPD) molecules bound in the minor groove. The structure deviates from A-form RNA. The duplex is overwound with an average value of 9.7 bp per turn, characterised as having a C3'-endo sugar pucker, very low base pair rise and high helical twist and inclination angles. The structure includes 65 ordered water molecules. Only a single row of water molecules is observed in the minor groove due to the presence of hydrophobic 2'-O-methyl groups. As many as five magnesium ions are located in the structure. Two are in the major groove and interact with O(6) and N(7) of guanosine and N(4) of cytidine residues through their hydration spheres. This work provides the first example of molecular interactions of nucleic acids with MPD, which was used as a precipitant, cryo-solvent and resolution enhancing agent. The two MPD molecules intrude into the hydration network in the minor groove, each forming hydrogen bonds between their secondary hydroxyl group and exo-amino functions of guanosine residues. Comparison of the 2'-O-Me(CGCGCG)(2) structure in the P3(2)12 and P6(1)22 crystals delineates stability of the water network within the minor groove to dehydration by MPD and is of interest for evaluating factors governing small molecule binding to RNA. Intrusion of MPD into the minor groove of 2'-O-Me(CGCGCG)(2) is discussed with respect to RNA dehydration, a prerequisite of Z-RNA formation.

Structure and dynamics of a DNA duplex containing single alpha-anomeric deoxyadenosine residue.

Structure and dynamics of an undecamer DNA duplex containing a single alpha-anomeric deoxyadenosine residue placed in opposition to a thymidine unit have been studied using simulation of molecular dynamics in aqueous solution. Despite several noticeable deviations from the B-DNA duplex structure caused by the anomerisation, such as: West type puckering of the alpha-anomeric sugar, disrupted base stacking pattern and unstable duplex bending, the formation of a non-classical alpha-dA-T pair was observed. A novel way of visual presentation of trajectory data allowing high throughput screening of the conformational parameters is presented.

Structure and dynamics of adenosine loops in RNA bulge duplexes as revealed by linked application of thermodynamics, spectrofluorimetry and simulation of molecular dynamics.

Structure and dynamics of adenosine loops in RNA bulge duplexes was studied using time-resolved spectrofluorimetry and in aqua simulation of molecular dynamics. Thermodynamics revealed that 2-aminopurine riboside is an non-invasive fluorescent probe when built within the bulge region of chemically synthesized RNA duplexes.

Spatial distribution functions as a tool in the analysis of ribonucleic acids hydration--molecular dynamics studies.

The spatial distribution functions (SDFs) determined as three-dimensional density distribution of hydrogen and oxygen atoms of water in a local coordinate system linked with RNA molecule are used to study details of the spatial structure of aqueous solution around selected parts of RNA duplexes: r(CGCGCG)2 and 2'-O-Me(CGCGCG)2. The influence of the 2'-O-methylation on the hydration pattern of RNA helical fragments is visualized at the atomic level.

Fluorescent alpha-anomeric 1,N(6)etheno-deoxyadenosine in DNA duplexes. The alpha-epsilondA / dG pair.

Structural properties of the fluorescent alpha-anomeric 1,N(6)ethenodeoxyadenosine residue placed in opposition to all four canonical deoxynucleotide units within 11-mer DNA duplexes have been studied. The duplex with alpha-epsilondA / dG pairing is most thermodynamically stable while the alpha-epsilondA / dC one is the least stable. Fluorescence measurements confirm the thermodynamic data and indicate base-pair dependent stacking properties of alpha-epsilondA within duplex structures. Results of molecular dynamics (MD) simulations in aqueous solution for the most stable duplex point to the presence of different conformational states of the alpha-1,N(6)etheno-deoxyadenosine residue, including formation of a hydrogen bonded pair with the dG and possible occurrence of severe kinking in the duplex.

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.

Acid promoted transformations of fluorescent luminarosine and its 2'-modified analogues.

The susceptibility of highly fluorescent luminarine nucleosides to acid promoted anomerization reactions has been studied in order to select a derivative with suitable properties for chemical synthesis of luminarine-labeled oligo(deoxy)ribonucleotides. Both O-acetylated derivatives Ia-c and parent luminarosine IIa, as well as 2'-O-methylluminarosine IIb, and 2'-deoxyluminarosine IIc undergo anomerization at pH = 4 however, at considerably different velocities. In the case of O-protected nucleosides (Ia-c), the anomerization leads to an equilibrium mixture of respective beta and alpha furanosides, the rate and extent of anomerization decreasing in the following order: Ic >> Ia > Ib. Parent nucleosides (IIa-c) bearing free hydroxyls are generally more susceptible to anomerization than the O-acetylated derivatives but a similar order of reactivity (IIc >> IIa > IIb) is observed. In each case, a complex mixture containing both beta and alpha ribopyranosyl and -furanosyl forms is formed. Their structure and anomeric configuration have been proved by 1H and 13C NMR spectroscopy. The results point to 2'-O-methylluminarosine as the fluorophore of choice for further derivatization and chemical introduction into oligo(deoxy)ribonucleotides.

Solution structure of RNA duplexes containing alternating CG base pairs: NMR study of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 under low salt conditions.

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.

Crystal structure of 2'-O-Me(CGCGCG)2, an RNA duplex at 1.30 A resolution. Hydration pattern of 2'-O-methylated RNA.

The molecular and crystal structure of 2'-O-Me (CGCGCG)2 has been determined using synchrotron radiation at near-atomic resolution (1.30 A), the highest resolution to date in the RNA field. The crystal structure is a half-turn A-type helix with some helical parameters deviating from canonical A-RNA, such as low base pair rise, elevated helical twist and inclination angles. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. This type of regularity is not seen in other RNA crystal structures. The structure includes 44 water molecules and two hydrated Mg2+ions one of which lies exactly on the crystallographic 2-fold axis. There are distinct patterns of hydration in the major and the minor grooves. The major groove is stabilised by water clusters consisting of fused five- and six-membered rings. Minor groove contains only a single row of water molecules; each water bridges either two self-parallel cytosines or two self-parallel guanines by a pair of hydrogen bonds. The structure provides the first view of the hydration scheme of 2'-O-methylated RNA duplex.

2-Aminopurine labelled RNA bulge loops. Synthesis and thermodynamics.

The chemical introduction of the blue-fluorescent 2-aminopurine riboside into oligoribonucleotides synthesized using the 2'-O-t-butyl-dimethylsilyl protection is reported. The proposed purification procedure led to the synthetic RNAs of high purity required for spectrofluorimetry studies. Thermodynamic parameters for the RNA bulge loops of type (A)n labelled with the 2-aminopurine (2AP) have been determined by optical melting. Results indicate that a bulge (B) in the RNA duplexes GUCG(B)GCUG + CAGCCGAC destabilize a regular helix structure and the destabilization increases monotonically as bulge length increases in the following order of (B) = A-2AP-A > 2AP-A; A-2AP > 2AP. The analysis of the free energy increments for bulged loops, delta G(o)37(bulge), allows to conclude that the structural properties of 2-aminopurine in RNA bulge loops are very similar to those of isomeric adenine. The fluorescent 2-aminopurine could therefore be used as non-invasive conformational probe.

Fluorescent nucleoside derivatives: Luminescence study of 4-dimethylaminopyridinium chloride derived from guanosine.

Photophysical properties of fluorescentN-[2-amino-9-(2',3',5'-tri-O-acetyl-ß-D-ribofuranosyl)-purin-6-yl]-4-dimethylaminopyridinium chloride (GDMAP) are determined in view of its possible use as a probe in DNA. The fluorescence intensity of GDMAP increases and exhibits doubleexponential decay in the presence of common nucleosides. The formation of ground-state complexes with nucleosides is inferred from absorption and emission measurements.

Z-RNA. The synthesis of 2'-O-[13C]methyl- and 5-methyl-analogs of ribo-CGCGCG.

Chemical synthesis of 2'-O-[13C]methyl-rCGCGCG and 5-methyl-rCGCGCG using support-aided phosphoramidite method is presented. 2'-O-Methyl guanosine derivative was separated from its 3'-O-methyl counterpart using transient 5',3'-O-silylation with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (Markiewicz reagent). The hexamers were obtained in a purity suitable for NMR studies.

Metal binding ability of hypermodified nucleosides of t-RNA. Potentiometric and spectroscopic studies on the metal complexes of N-[(9-beta-D-ribofuranosylpurin-6-yl)-carbamoyl] threonine.

Copper(II), nickel(II), zinc(II), manganese(II), and magnesium(II) complexes of t6A (N-[9-beta-D-ribofuranosylpurin-6-yl)carbamoyl] threonine and t6Ade (N6(threoninocarbonyl)adenine) were studied by potentiometric and spectroscopic methods. It was found that t6Ade has three dissociable protons in the accessible pH range (N1 and N9 of purine and carboxylate), while only two pK values are characteristic of t6A. Magnesium(II) and manganese(II) do not interact effectively with these ligands, but copper(II) and nickel(II) ions form very stable complexes with the coordination of purine N1, deprotonated amide nitrogen, and carboxylate oxygen donors.

Z-RNA: the solution NMR structure of r(CGCGCG).

Left-handed double-helical Z-RNA has been studied using the ribohexanucleotide pentaphosphate r(CpGpCpGpCpG). One-dimensional and two-dimensional proton nmr experiments were used to probe the structural details of the left-handed helix in concentrated sodium perchlorate solution. In 1M NaClO4 the RNA adopts the normal A-form double helix, and in 6M NaClO4 it is nearly all in the Z form. In 4M NaClO4 it exists as nearly equal parts of A form and Z form. Resonances corresponding to both A and Z form appear in the nmr spectrum, indicating that the duplex exchanges slowly between forms. Spin-spin coupling constants between protons in the ribose rings were used to determine the sugar-pucker conformations of the individual nucleotides. Quantitative nuclear Overhauser experiments were used to determine proton-proton distances within the nucleoside, and from these distances values for the glycosidic torsion angle were determined. The results show that the cytidines adopt C2'-endo sugar puckers (S type) with pseudo-rotation phase values (P) of approximately 165 degrees. The bases are in the anti conformation, with chi values of approximately -140 degrees. The internal guanosines adopt C3'-endo sugar puckers (N type) with P approximately 18 degrees, while the 3'-terminal guanosine ribose exists in an equilibrium between S- and N-type conformations. All three guanosine bases adopt the syn conformation, with chi approximately 70 degrees. The results indicate that the solution structure of Z-RNA is very similar to that of Z-DNA.

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.

Studies directed toward introduction of N6-isopentenyladenosine and its 2-methylthio analogue into oligoribonucleotides.

A new efficient route of i6A and ms2i6A synthesis, especially useful for the preparation of i6A-containing oligoribonucleotides eg. Api6A has been proposed. The method is based on aminolysis of a 6-methylsulfonylpurine system formed by oxidation of its 6-methylthio precursor.

New, ionic side-products in oligonucleotide synthesis: formation and reactivity of fluorescent N-/purin-6-yl/pyridinium salts.

Fluorescent N-/purin-6-yl/pyridinium salts are formed in pyridine assisted phosphorylations and arenesulphonations of the hypoxanthine lactam system under various conditions including those used in oligonucleotide synthesis. The N1-methyl-N3-/purin-6-yl/imidazolium salt is generated in phosphorylation with TPSCl/1-methylimidazole as a coupling system. Both salts are representatives of a new family of ionic side-products in oligonucleotide synthesis involving hypoxanthine residues. Their isolation procedure has been developed. High reactivity of N-/purin-6-yl/pyridinium salts towards some reagents used in oligonucleotide chemistry, e.g. pyridinium mediated conversion of hypoxanthine into 6-aminopurine, can result in point mutations in synthesized oligomer.