Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

Norovirus RNA‐dependent RNA polymerase: A computational study of metal‐binding preferences

Norovirus (NV) RNA‐dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer‐template duplex was investigated using X‐ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back‐tracked state of the enzyme, in which the ‐end of the primer occupies the position expected for the post‐incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435–1445. © 2017 Wiley Periodicals, Inc.     

Structural parameters affecting the kinetics of RNA hairpin formation

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program ‘Kinfold’. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem–loop structures.     

Potential anti‐bacterial drug target: Structural characterization of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase from Salmonella typhimurium LT2

3,4‐Dihydroxy‐2‐butanone‐4‐phosphate synthase (DHBPS) encoded by ribB gene is one of the first enzymes in riboflavin biosynthesis pathway and catalyzes the conversion of ribulose‐5‐phosphate (Ru5P) to 3,4‐dihydroxy‐2‐butanone‐4‐phosphate and formate. DHBPS is an attractive target for developing anti‐bacterial drugs as this enzyme is essential for pathogens, but absent in humans. The recombinant DHBPS enzyme of Salmonella requires magnesium ion for its activity and catalyzes the formation of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate from Ru5P at a rate of 199 nmol min^?1 mg^?1 with K_m value of 116 μM at 37°C. Further, we have determined the crystal structures of Salmonella DHBPS in complex with sulfate, Ru5P and sulfate‐zinc ion at a resolution of 2.80, 2.52, and 1.86 Å, respectively. Analysis of these crystal structures reveals that the acidic loop (residues 34–39) responsible for the acid‐base catalysis is disordered in the absence of substrate or metal ion at the active site. Upon binding either substrate or sulfate and metal ions, the acidic loop becomes stabilized, adopts a closed conformation and interacts with the substrate. Our structure for the first time reveals that binding of substrate Ru5P alone is sufficient for the stabilization of the acidic active site loop into a closed conformation. In addition, the Glu38 residue from the acidic active site loop undergoes a conformational change upon Ru5P binding, which helps in positioning the second metal ion that stabilizes the Ru5P and the reaction intermediates. This is the first structural report of DHBPS in complex with either substrate or metal ion from any eubacteria. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Preference for Ribose Over Deoxyribose in Loop-Closing Base Pairs of Extra Stable Nucleic Acid Hairpins

We have investigated the effect of switching ribose to deoxyribose at the closing base-pair of an extra-stable RNA hairpin. Specifically, we studied the sequence 5′-GGAC(UUCG)GUCC, a dodecanucleotide that folds into a well-characterized, “extra stable” RNA hairpin structure. Recently, we showed that hairpins containing a 2′,5′-linked (UUCG) loop instead of the native 3′,5′-linked loop also exhibit extra-stability (Hannoush and Damha, J. Am. Chem. Soc., 2001, 123, 12368–12374). In this article, we show that the ribose units located at the loop-closing positions (i.e., rC ₄ and rG ₉ ) contribute significantly to the stabilization of RNA hairpins, particularly those containing the 3′,5′-UUCG loop. Interestingly, the requirement of rC4 and rG9 is more relaxed for DNA hairpins containing the 2′,5′-UUCG loop and, in fact, they may be replaced altogether (ribose → deoxyribose) without affecting stability. The results broaden our understanding of the behavior of highly stable (UUCG) hairpin loops and how they respond to structural perturbation of the loop-closing base pairs.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

The Role of Structural Elements in Dimerization of RNA Fragments in Avian Retroviruses

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop–loop interaction of two hairpins, or kissing hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.     

Non-nearest-neighbor dependence of the stability for RNA group II single-nucleotide bulge loops

Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop.     

Analysis of the interacting partners eIF4F and 3′‐CITE required for Melon necrotic spot virus cap‐independent translation

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3′‐cap‐independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV‐Mα5 3′‐CITE and its translation initiation factor partner. We first mapped the minimal 3′‐CITE (Ma5TE) to a 45‐nucleotide sequence, which consists of a stem‐loop structure with two internal loops, similar to other I‐shaped 3′‐CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G_980–1159 (eIF4F_p20), but not with each subunit alone or with eIF4E + eIF4G_1003–1092, suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G_980–1159, or through a double interaction with eIF4E and eIF4G_980–1159. Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G_1003–1092 and cap‐independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE‐driven cap‐independent translation, showing that conserved amino acids in a positively charged RNA‐binding motif around amino acid position 228, implicated in eIF4E–eIF4G binding or belonging to the cap‐recognition pocket, are essential for cap‐independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.     

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype–phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction‐dependent destabilization of the underlying double helix, and a self‐structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self‐structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size‐dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1–12, 2014.     

Heating‐induced conformational change of a novel β‐(1→3)‐D‐glucan from Pleurotus geestanus

Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical–chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel β‐(1→3)‐D ‐glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical β‐(1→3)‐D ‐glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8–12.5°C and 25–60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro‐differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating‐induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating‐induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121–131, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Sequence‐dependent folding and unfolding of ligand‐bound purine riboswitches

Riboswitch regulation of gene expression requires ligand‐mediated RNA folding. From the fluorescence lifetime distribution of bound 2‐aminopurine ligand, we resolve three RNA conformers (C_o, C_i, C_c) of the liganded G‐ and A‐sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg²⁺, together with results from mutagenesis, suggest that C_o and C_i are partially unfolded species compromised in key loop‐loop interactions present in the fully folded C_c. These data verify that the ligand‐bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand‐mediated folding of the G‐sensing riboswitch is more effective, less dependent on Mg²⁺, and less debilitated by mutation, than the A‐sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence‐dependent RNA dynamics, which adjust the balance of ligand‐mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine‐tuning of gene regulatory mechanisms. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 953–965, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.     

Trinucleotide repeat DNA alters structure to minimize the thermodynamic impact of 8-oxo-7,8-dihydroguanine

In the phenomenon of trinucleotide repeat (TNR) expansion, an important interplay exists between DNA damage repair of 8-oxo-7,8-dihydroguanine (8-oxoG) and noncanonical structure formation. We show that TNR DNA adapts its structure to accommodate 8-oxoG. Using chemical probe analysis, we find that CAG repeats composing the stem-loop arm of a three-way junction alter the population of structures in response to 8-oxoG by positioning the lesion at or near the loop. Furthermore, we find that oligonucleotides composed of odd-numbered repeat sequences, which form populations of two structures, will also alter their structure to place 8-oxoG in the loop. However, sequences with an even number of repeats do not display this behavior. Analysis by differential scanning calorimetry indicates that when the lesion is located within the loop, there are no significant changes to the thermodynamic parameters as compared to the DNA lacking 8-oxoG. This contrasts with the enthalpic destabilization observed when 8-oxoG is base-paired to C and indicates that positioning 8-oxoG in the loop avoids the thermodynamic penalty associated with 8-oxoG base-pairing. Since formation of stem-loop hairpins is proposed to facilitate TNR expansion, these results highlight the importance of defining the structural consequences of DNA damage.     

Diversity of DIS, SD and psi hairpins in HIV-1 isolates of group M: in silico study

The primary sequence and secondary structure of the region encompassing DIS, SD and psi hairpins in HIV-1 genomic RNAs have been analyzed for 731 group M isolates from NCBI database. The secondary structures have been predicted by the m fold program (M. Zuker). Though the primary sequence of the region studied was found to be highly heterogeneous, this region is folded into DIS, SD and psi hairpins (DIS-, SD- and psi-like hairpins) in 96% of the isolates studied. The phylogenetic analysis showed that the most frequent variants of DIS hairpin (DIS(Lai), DIS(Mal) and DIS(C)) tolerate certain base changes. Particularly, base changes at stem position 23 occur 5 and 33 times more frequently in DIS(Lai) than in DIS(Mal) and DIS(C), respectively, while A insertion at the 5'end of apical loop is tolerated in DIS(Mal) and DIS(C) but not in DIS(Lai). We have revealed that the bottom base pair substitution G-C --> A-U in SD hairpin is highly specific for subtype D isolates. All variants of DIS, SD and psi hairpins found in our database are discussed, systematized and presented in schemes of hypothetical transitions between variants via a single base change. Most variants of DIS and psi hairpins were found to adopt several conformations.     

Deformability in the cleavage site of primary microRNA is not sensed by the double‐stranded RNA binding domains in the microprocessor component DGCR8

The prevalence of double‐stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA‐binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD‐containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8‐Core, consists of its two dsRBDs and a C‐terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N‐terminal dsRBD of DGCR8 in isolation nor the DGCR8‐Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single‐stranded segments flanking the stem. Extending DGCR8‐Core to include an N‐terminal heme‐binding region does not change our conclusions. Thus, our data suggest that although the DGCR8‐Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. Proteins 2015; 83:1165–1179. © 2015 Wiley Periodicals, Inc.     

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

Structure of the RNA‐directed RNA Polymerase from the cystovirus ϕ12

We have determined the structure of P2, the self‐priming RdRp from cystovirus ?12 in two crystal forms (A, B) at resolutions of 1.7 Å and 2.1 Å. Form A contains Mg²⁺ bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts‐mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo. Proteins 2013; 81:1479–1484. © 2013 Wiley Periodicals, Inc.