Differentiate RNA Single‐Stranded Region of the Branched Structures and Hairpin Loops by an Octahedral Cobalt(II) Complex

Single‐stranded RNA molecules usually include secondary structural elements such as bulges, internal loops, and hairpin loops. These RNA secondary structural elements are often essential for the biological activity and functions of the RNA molecule. Chemical probe Co^II(HAPP)(TFA)₂ in the presence of H₂O₂ is found to differentiate single‐stranded RNA from branched structures and hairpin loops. This study uses Co^II(HAPP)(TFA)2 to analyze the structures of two RNA molecules: a fragment of HIV TAR RNA (TAR‐27) and the catalytic domain 5 of group II intron (D5‐29). The electrophoretic mobility of TAR‐27 does not shift in the presence of Co^II(HAPP)(TFA)₂, suggesting that the reagent does not change the conformation of RNA substrate. Cleavage of the RNA substrates by Co^II(HAPP)(TFA)₂ unambiguously differentiated RNA internal looped structures from hairpin loops. The results show that Co^II(HAPP)(TFA)2 is a sensitive, informative and convenient tool for analysis of RNA secondary structures.     

Automated RNA tertiary structure prediction from secondary structure and low‐resolution restraints

A novel protocol for all‐atom RNA tertiary structure prediction is presented that uses restrained molecular mechanics and simulated annealing. The restraints are from secondary structure, covariation analysis, coaxial stacking predictions for helices in junctions, and, when available, cross‐linking data. Results are demonstrated on the Alu domain of the mammalian signal recognition particle RNA, the Saccharomyces cerevisiae phenylalanine tRNA, the hammerhead ribozyme, the hepatitis C virus internal ribosomal entry site, and the P4–P6 domain of the Tetrahymena thermophila group I intron. The predicted structure is selected from a pool of decoy structures with a score that maximizes radius of gyration and base–base contacts, which was empirically found to select higher quality decoys. This simple ab initio approach is sufficient to make good predictions of the structure of RNAs compared to current crystal structures using both root mean square deviation and the accuracy of base–base contacts. © 2011 Wiley Periodicals, Inc. J Comput Chem, 2011     

A role for a single-stranded junction in RNA binding and specificity by the Tetrahymena group I ribozyme

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.     

Formation of a Ligand‐Assisted Complex of Two RNA Hairpin Loops

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non‐structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand‐assisted formation of loop–loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G–G mismatches in double‐stranded DNA, we successfully demonstrated the formation of both inter‐ and intra‐molecular NCT6‐assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)₃ in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly‐labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6‐assisted loop–loop complex. Förster resonance energy‐transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.     

Synthesis and Solution Conformation of β‐Hairpin Mimetics Utilizing a Template Derived from (2S,3R,4R)‐Diaminoproline

A novel template was synthesized for stabilizing β‐hairpin conformations in cyclic peptide mimetics. The template is a diketopiperazine derived formally from L ‐aspartic acid and (2S,3R,4R)‐diaminoproline, the latter being available by an efficient synthetic route from vitamin C. The template was incorporated by solid‐phase peptide synthesis into a cyclic loop mimetic containing the sequence (‐Ala‐Asn‐Pro‐Asn‐Ala‐Ala‐template‐). This mimetic was shown by NMR to adopt a stable β‐hairpin conformation in (D₆)DMSO solution. The template may prove to be generally useful for creating small‐molecule mimetics of hairpin loops on proteins of diverse function.     

Restoration of Ribozyme Tertiary Contact and Function by Using a Molecular Glue for RNA

Some RNA classes require folding into the proper higher‐order structures to exert their functions. Hammerhead ribozyme (HHR) requires a folding conformation stabilized by tertiary interaction for full activity. A rationally engineered HHR was developed that was inactive, but could be activated by a synthetic RNA‐binding ligand, naphthyridine carbamate tetramer with Z‐stilbene linker (Z‐NCTS). Binding of Z‐NCTS could induce the formation of an active folding structure and thereby restore ribozyme activity, where Z‐NCTS acts as a molecular glue to bring two isolated RNA loops into contact with each other. Next, we designed a Z‐NCTS‐responsive genetic switch using the HHR sequence inserted into the 3′ untranslated region as a cis‐acting element. We demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z‐NCTS and was functional in mammalian cells.     

Transalkylation of N-methyl tertiary amines with 3, 4-dibromobutenolides

The selective transalkylation of N-methyl tertiary amines with 3,4-dibromobutenolides is described.The N-methyl group of the parent tertiary amines was replaced by alkenyl units of the butenolides;and a series of butenolide-containing tertiary enamines were obtained in moderate to good yields.Interestingly,the product 2b has shown a promising anticancer activity against HeLa cell lines(IC50=0.19 mmol/L).     

Palladium-catalyzed acylation of activated alkenes with bridgehead acid chlorides

Bridgehead acid chlorides 1a and 1b react with activated alkenes 2 in the presence of a catalytic amount of palladium and 1 equiv of a tertiary amine. The reaction proceeds regio- and stereoselectively at the terminal carbon atoms to yield acylated alkenes 3 with E-configuration.     

In vitro selection of a viomycin-binding RNA pseudoknot

Background: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection.Results: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb²⁺-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot.Conclusions: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.     

Highly efficient and diastereoselective gold(I)-catalyzed synthesis of tertiary amines from secondary amines and alkynes: substrate scope and mechanistic insights

An efficient method for the synthesis of tertiary amines through a gold(I)‐catalyzed tandem reaction of alkynes with secondary amines has been developed. In the presence of ethyl Hantzsch ester and [{(tBu)₂(o‐biphenyl)P}AuCl]/AgBF₄ (2 mol %), a variety of secondary amines bearing electron‐deficient and electron‐rich substituents and a wide range of alkynes, including terminal and internal aryl alkynes, aliphatic alkynes, and electron‐deficient alkynes, underwent a tandem reaction to afford the corresponding tertiary amines in up to 99 % yield. For indolines bearing a preexisting chiral center, their reactions with alkynes in the presence of ethyl Hantzsch ester catalyzed by [{(tBu)₂(o‐biphenyl)P}AuCl]/AgBF₄ (2 mol %) afforded tertiary amines in excellent yields and with good to excellent diastereoselectivity. All of these organic transformations can be conducted as a one‐pot reaction from simple and readily available starting materials without the need of isolation of air/moisture‐sensitive enamine intermediates, and under mild reaction conditions (mostly room temperature and mild reducing agents). Mechanistic studies by NMR spectroscopy, ESI‐MS, isotope labeling studies, and DFT calculations on this gold(I)‐catalyzed tandem reaction reveal that the first step involving a monomeric cationic gold(I)–alkyne intermediate is more likely than a gold(I)–amine intermediate, a three‐coordinate gold(I) intermediate, or a dinuclear gold(I)–alkyne intermediate. These studies also support the proposed reaction pathway, which involves a gold(I)‐coordinated enamine complex as a key intermediate for the subsequent transfer hydrogenation with a hydride source, and reveal the intrinsic stereospecific nature of these transformations observed in the experiments.     

Stability and Unfolding Mechanism of the N‐terminal β‐Hairpin from [2Fe‐2S] Ferredoxin I by Molecular Dynamics Simulations

The stability and unfolding mechanism of the N‐terminal β‐hairpin of the [2Fe‐2S] ferredoxin I from the blue‐green alga Aphanothece sacrum in pure methanol, 40% (v/v) methanol‐water, and pure water systems were investigated by 10 ns molecular dynamics simulations under periodic boundary conditions. The β‐hairpin was mostly in its native‐like state in pure methanol, whereas it unfolds dramatically following the ‘zip‐up’ mechanism when it was placed in pure water. Both interstrand and inside‐turn hydrogen bonds account for the stability of the β‐hairpin in its native‐like conformation, whereas hydrophobic interactions among nonpolar side chains are responsible for maintaining its stable loop‐like intermediate structures in 40% (v/v) methanol‐water. Reducing solvent polarity seems to increase the stability of the β‐hairpin in its native‐like structure. Methanol is likely to mimic the partially hydrophobic environment around the N‐terminal β‐hairpin by the subsequent α‐helix.     

Synthesis of Carbo- and Heterocyclic Aldehydes Bearing an Adjacent Donor Group – Ozonolysis versus OsO4/KIO4-Oxidation

Summary. The synthesis of carbo- and heterocyclic aldehydes bearing an ipso-methoxy group is investigated. The synthetic sequence is based on an initial Grignard addition of an olefin to a cyclic ketone followed by methylation of the resulting tertiary alcohol. The terminal olefin serves as precursor for the aldehyde functionality. Oxidation by ozonolysis turned out to depend significantly on the distance of the donor methoxy group. The observed side reactions could be circumvented by applying a one-pot OsO₄ mediated diol formation followed by Malaprade oxidation using KIO₄. A series of carbo- and heterocyclic precursors were successfully converted to the title products.     

Interactions of acyclic and cyclic bis-phenanthridinium derivatives with ss- and ds-polynucleotides

The acyclic bis-phenanthridinium ligands 1, 2 and the cyclic analogue 3 bind to ss-RNA by bis-intercalation. Due to it's shorter linker 1 exhibits mono-intercalative binding to ds-polynucleotides, while a mixed mode of binding with 2 is shown to strongly dependent on the base composition and tertiary structure of ds-DNA and RNA. The cyclic analogue 3 binds to ds-polynucleotides by non-intercalative mode. Comparing the ss-/ds-polynucleotide selectivity obtained for 3 and previously reported for 3,8-linked bis-phenanthridinium analogues, it is clear that the more rigid structure and sterically more restricted cleft of the latter could better distinguish ss- from ds-polynucleotide regions.     

Facile strategies for the synthesis and crystallization of linear trinuclear nickel(II)-Schiff base complexes with carboxylate bridges: Tuning of coordination geometry and magnetic properties

Three new linear trinuclear nickel(II) complexes, [Ni₃(salpen)₂(OAc)₂(H₂O)₂]·4H₂O (1) (OAc = acetate, CH₃COO⁻), [Ni₃(salpen)₂(OBz)₂] (2) (OBz = benzoate, PhCOO⁻) and [Ni₃(salpen)₂(OCn)₂(CH₃CN)₂] (4) (OCn = cinnamate, PhCHCHCOO⁻), H₂salpen = tetradentate ligand, N,N′-bis(salicylidene)-1,3-pentanediamine have been synthesized and characterized structurally and magnetically. The choice of solvent for growing single crystal was made by inspecting the morphology of the initially obtained solids with the help of SEM study. The magnetic properties of a closely related complex, [Ni₃(salpen)₂(OPh)₂(EtOH)] (3) (OPh = phenyl acetate, PhCH₂COO⁻) whose structure and solution properties have been reported recently, has also been studied here. The structural analyses reveal that both phenoxo and carboxylate bridging are present in all the complexes and the three Ni(II) atoms remain in linear disposition. Although the Schiff base ligand and the syn–syn bridging bidentate mode of the carboxylate group remain the same in complexes 1–4, the change of alkyl/aryl group of the carboxylates brings about systematic variations between six- and five-coordination in the geometry of the terminal Ni(II) centres of the trinuclear units. The steric demand as well as hydrophobic nature of the alkyl/aryl group of the carboxylate is found to play a crucial role in the tuning of the geometry. Variable-temperature (2–300 K) magnetic susceptibility measurements show that complexes 1–4 are antiferromagnetically coupled (J = −3.2(1), −4.6(1), −3.2(1) and −2.8(1) cm⁻¹ in 1–4, respectively). Calculations of the zero-field splitting parameter indicate that the values of D for complexes 1–4 are in the high range (D = +9.1(2), +14.2(2), +9.8(2) and +8.6(1) cm⁻¹ for 1–4, respectively). The highest D value of +14.2(2) and +9.8(2) cm⁻¹ for complexes 2 and 3, respectively, are consistent with the pentacoordinated geometry of the two terminal nickel(II) ions in 2 and one terminal nickel(II) ion in 3.     

Origin and evolution of exon/intron junctions

In nuclear mRNA genes, exon/intron junctions (both exon/intron and intron/exon junctions in this paper) possess the specific duplex pattern with the corresponding ends (3′ to 3′, 5′ to 5′) of exons and introns more or less identical. In genes with group I or group II introns, overall analyses indicate there are also related patterns in their exon/intron junctions. From the analysis of these specific regions of split genes and the study of the composition of primitive genomes, it is proposed that the sequences of primitive exons and introns are identical at least in their corresponding boundary regions. And more fundamentally, it may be concluded that exon/intron junctions were originally related to tandem repeated sequences in the earliest genomes. Results from a preliminary analysis of specific motifs in modern repeated sequences support such a view on the origin of exon/intron junctions. As for the evolution of exon/intron junctions, there have been multiple rather than single paths.     

Protonation‐Dependent Base Flipping at Neutral pH in the Catalytic Triad of a Self‐Splicing Bacterial Group II Intron

NMR spectroscopy has revealed pH‐dependent structural changes in the highly conserved catalytic domain 5 of a bacterial group II intron. Two adenines with pK_a values close to neutral pH were identified in the catalytic triad and the bulge. Protonation of the adenine opposite to the catalytic triad is stabilized within a G(syn)–AH⁺(anti) base pair. The pH‐dependent anti‐to‐syn flipping of this G in the catalytic triad modulates the known interaction with the linker region between domains 2 and 3 (J23) and simultaneously the binding of the catalytic Mg²⁺ ion to its backbone. Hence, this here identified shifted pK_a value controls the conformational change between the two steps of splicing.     

Hydrolysis of Phosphodiester Bonds within RNA Hairpin Loops in Buffer Solutions: the Effect of Secondary Structure on the Inherent Reactivity of RNA Phosphodiester Bonds

The hydrolysis of phosphodiester bonds of chimeric 2′-O-methyloligoribonucleotides was studied in buffer solutions. Pseudo-first-order rate constants for cleavage of phosphodiester bonds within hairpin loops were calculated and compared with those for cleavage of phosphodiester bonds within double-stranded stems and linear single-stranded oligonucleotides. No large differences in reactivity were observed: some of the hairpin structures studied were slightly less and others slightly more reactive than the linear reference. These results suggest that phosphodiester bonds within small hairpin loops are conformationally free to cleave by an in-line mechanism, but also that the secondary structure may influence the reactivity of phosphodiester bonds.     

Combinatorial Biomimetic Chemistry: Parallel Synthesis of a Small Library of β‐Hairpin Mimetics Based on Loop III from Human Platelet‐Derived Growth Factor B

Combinatorial diversity in hypervariable β‐hairpin loops is exploited by the immune system to select binding sites on antibodies for a wide variety of different protein antigens. In a first step towards mimicking this strategy in vitro, for the selection of novel protein ligands, an approach is described here for the parallel synthesis of small libraries of conformationally defined β‐hairpin protein epitope mimetics. Starting from a protruding hairpin loop in platelet‐derived growth factor B (PDGF‐B), 8 and 12 residues were first transplanted from the protein to a D ‐Pro‐L ‐Pro template, to afford the cyclic peptide‐loop mimetics 1 and 2 , respectively. NMR and MD studies in aqueous solution show that both mimetics populate conformations which closely mimic the β‐hairpin in the crystal structure of the native protein (Fig. 5). Based on 1 as a scaffold, a library of 24 mimetics was synthesized in which the four residues at the tip of the loop (VRKK) were held constant, and flanking residues at positions 1, 2, 7, and 8 in the hairpin were varied (Fig. 7). The library was prepared by parallel synthesis in a two‐stage solid‐phase assembly/solution‐phase cyclization process. The products were analyzed by MS, NMR, and CD. 2D‐NOESY revealed for most library members characteristic long‐range NOEs that show that the hairpin conformation is stably maintained. The results suggest that this approach may be useful for the synthesis of much larger libraries of peptide and protein mimetics based on a β‐hairpin scaffold.