Conversion of L-arabinose to L-ribose by genetically engineered Candida tropicalis.

L-Ribose, a starting material for the synthesis of L-nucleoside, has attracted lots of attention since L-nucleoside is responsible for the antiviral activities of the racemic mixtures of nucleoside enantiomers. In this study, the L-ribulose-producing Candida tropicalis strain was engineered for the conversion of L-arabinose to L-ribose. For the construction of a uracil auxotroph, the URA3 gene was excised by homologous recombination. The expression cassette of codon-optimized L-ribose isomerase gene from Acinetobacter calcoaceticus DL-28 under the control of the GAPDH promoter was integrated to the uracil auxotroph. The resulting strain, K1 CoSTP2 LsaAraA AcLRI, was cultivated with the glucose/L-arabinose mixture. At 45.5 h of fermentation, 6.0 g/L of L-ribose and 3.2 g/L of L-ribulose were produced from 30 g/L of L-arabinose. The proportion between L-ribose and L-ribulose was approximately 2:1 and the conversion yield of L-arabinose to L-ribose was about 20% (w/w). The L-ribose-producing yeast strain was successfully constructed for the first time and could convert L-arabinose to L-ribose in one-pot fermentation using the mixture of glucose and L-arabinose.

The shift from early to late types of ribosomes in zebrafish development involves changes at a subset of rRNA 2'-O-Me sites.

During zebrafish development, an early type of rRNA is gradually replaced by a late type that is substantially different in sequence. We applied RiboMeth-seq to rRNA from developmental stages for profiling of 2'-O-Me, to learn if changes in methylation pattern were a component of the shift. We compiled a catalog of 2'-O-Me sites and cognate box C/D guide RNAs comprising 98 high-confidence sites, including 10 sites that were not known from other vertebrates, one of which was specific to late-type rRNA. We identified a subset of sites that changed in methylation status during development and found that some of these could be explained by availability of their cognate SNORDs. Sites that changed during development were enriched in the novel sites revealed in zebrafish. We propose that the early type of rRNA is a specialized form and that its structure and ribose methylation pattern may be an adaptation to features of development, including translation of specific maternal mRNAs.

Establishment of 5'-3' interactions in mRNA independent of a continuous ribose-phosphate backbone.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5'-3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'-3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.

Streptomyces acidicola sp. nov., isolated from a peat swamp forest in Thailand.

A novel actinobacterium, designated strain K1PN6T, was isolated from soil sample collected in Kantulee peat swamp forest, Surat Thani province, Thailand. The morphological, chemotaxonomic, and phylogenetic characteristics were consistent with its classification in the genus Streptomyces. Based on 16S rRNA gene sequence analysis, strain K1PN6T showed highest similarity to Streptomyces phyllanthi PA1-07T (98.6 %), Streptomyces spongiae Sp080513SC-24T (98.3%) and Streptomyces adustus WH-9T (98.3%). The G + C content of the genomic DNA was 70.3 mol%. Digital DNA-DNA hybridization and average nucleotide identity values between the genome sequence of strain K1PN6T with S. phyllanthi TISTR 2346T (33.7 and 89.1%), S. spongiae NBRC 106415T (38.6 and 90.6%) and S. adustus NBRC 109810T (26.0 and 86.2%) were below the thresholds of 70 and 95-96% for prokaryotic conspecific assignation. Chemotaxonomic data revealed that strain K1PN6T possessed MK-9(H8) (45%) and MK-9(H6) (34%) as the predominant menaquinones. It contained LL-diaminopimelic acid as the diagnostic diamino acid and galactose, glucose, mannose, and ribose as whole-cell sugars. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, two unidentified aminolipids, an unidentified phospholipid, and glycophospholipid. The predominant cellular fatty acids (>10%) were iso-C16:0, C16:0, anteiso-C15:0, and iso-C14:0. On the basis of these genotypic and phenotypic data, strain K1PN6T should be designated as a representative of a novel species of the genus Streptomyces, for which the name Streptomyces acidicola sp. nov. is proposed with the type strain K1PN6T (=TBRC 11341T=NBRC 114304T).

Functional diversity of small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) are short non-protein-coding RNAs with a long-recognized role in tuning ribosomal and spliceosomal function by guiding ribose methylation and pseudouridylation at targeted nucleotide residues of ribosomal and small nuclear RNAs, respectively. SnoRNAs are increasingly being implicated in regulation of new types of post-transcriptional processes, for example rRNA acetylation, modulation of splicing patterns, control of mRNA abundance and translational efficiency, or they themselves are processed to shorter stable RNA species that seem to be the principal or alternative bioactive isoform. Intriguingly, some display unusual cellular localization under exogenous stimuli, or tissue-specific distribution. Here, we discuss the new and unforeseen roles attributed to snoRNAs, focusing on the presumed mechanisms of action. Furthermore, we review the experimental approaches to study snoRNA function, including high resolution RNA:protein and RNA:RNA interaction mapping, techniques for analyzing modifications on targeted RNAs, and cellular and animal models used in snoRNA biology research.

Lysosomal regulation of extracellular vesicle excretion during d-ribose-induced NLRP3 inflammasome activation in podocytes.

The NLRP3 inflammasome is activated in the cytoplasm of cells and its products such as IL-1ß are exported through a non-classical ER-Golgi pathway. Several mechanistically distinct models including exocytosis of secretory lysosomes, microvesicles (MVs) and extracellular vehicles (EVs) have been proposed for their release. In this study, we hypothesized that the NLRP3 inflammasome product, IL-1ß in response to exogenously administrated and endogenously produced d-ribose stimulation is released via extracellular vesicles including EVs via a sphingolipid-mediated molecular mechanisms controlling lysosome and multivesicular body (MVB) interaction. First, we demonstrated that both endogenous and exogenous d-ribose induced NLRP3 inflammasome activation to produce IL-1ß, which was released via EVs in podocytes. Then, we found that colocalization of marker MVB marker VPS16 with IL-1ß within podocytes increased upon d-ribose stimulation, which was accompanied by decreased colocalization of lysosome marker Lamp-1 and VPS16, suggesting decrease in MVB inclusion of IL-1ß due to reduced lysosome and MVB interaction. All these changes were mimicked and accelerated by lysosome v-ATPase inhibitor, bafilomycin. Moreover, ceramide in podocytes was found elevated upon d-ribose stimulation, and prior treatments of podocyte with acid sphingomyelinase (Asm) inhibitor, amitriptyline, acid ceramidase (AC) inducer, genistein, or AC CRISPR/cas9 activation plasmids were found to decrease d-ribose-induced ceramide accumulation, EVs release and IL-1ß secretion due to reduced interactions of lysosome with MVBs. These results suggest that inflammasome-derived products such as IL-1ß during d-ribose stimulation are released via EVs, in which lysosomal sphingolipid-mediated regulation of lysosome function plays an important role.

Ribose-Map: a bioinformatics toolkit to map ribonucleotides embedded in genomic DNA.

Recent advances in high-throughput sequencing techniques have made it possible to tag ribonucleoside monophosphates (rNMPs) embedded in genomic DNA for sequencing. rNMP sequencing experiments generate large, complex datasets that require efficient, scalable software that can accurately map embedded rNMPs independently of the particular sequencing technique used. Current computational pipelines designed to map rNMPs embedded in genomic DNA are customized for data generated using only one type of rNMP sequencing technique. To standardize the processing and analysis of rNMP sequencing experiments, we developed Ribose-Map. Through a series of analytical modules, Ribose-Map transforms raw sequencing data into summary datasets and publication-ready visualizations of results, allowing biologists to identify sites of embedded rNMPs, study the nucleotide sequence context of these rNMPs and explore their genome-wide distribution. By accommodating data from any of the available rNMP sequencing techniques, Ribose-Map can increase the reproducibility of rNMP sequencing experiments and enable a head-to-head comparison of these experiments.

Identification of the Plant Ribokinase and Discovery of a Role for Arabidopsis Ribokinase in Nucleoside Metabolism.

Ribose can be used for energy or as a component of several important biomolecules, but for it to be used in either capacity it must first be phosphorylated by ribokinase (RBSK). RBSK proteins are part of the phosphofructokinase-B (pfkB) family of carbohydrate kinases. Sequence comparisons of pfkB proteins from the model plant Arabidopsis thaliana with the human and Escherichia coli RBSK identified a single candidate RBSK, At1g17160 (AtRBSK). AtRBSK is more similar to predicted RBSKs from other plant species and known mammalian and prokaryotic RBSK than to all other PfkB proteins in Arabidopsis AtRBSK contains a predicted chloroplast transit peptide, and we confirmed plastid localization using AtRBSK fused to YFP. Structure prediction software verified that the AtRBSK sequence mapped onto a known RBSK structure. Kinetic parameters of purified recombinant AtRBSK were determined to be Kmribose = 150 µm ± 17 µm, KmATP = 45 µm ± 5.6 µm, and kcat = 2.0 s-1 Substrate inhibition was observed for AtRBSK (KiATP = 2.44 mm ± 0.36 mm), as has been demonstrated for other RBSK proteins. Ribose accumulated in Arabidopsis plants lacking AtRBSK. Such plants grew normally unless media was supplemented with ribose, which led to chlorosis and growth inhibition. Both chlorosis and ribose accumulation were abolished upon the introduction of a transgene expressing AtRBSK-MYC, demonstrating that the loss of protein is responsible for ribose hypersensitivity. Ribose accumulation in plants lacking AtRBSK was reduced in plants also deficient in the nucleoside ribohydrolase NSH1, linking AtRBSK activity to nucleoside metabolism.

Identification and characterization of D-xylose reductase involved in pentose catabolism of the zygomycetous fungus Rhizomucor pusillus.

Rhizomucor pusillus NBRC 4578 efficiently produces ethanol from lignocellulosic biomass because of its ability to ferment not only d-glucose, but also d-xylose. When the strain was cultivated on d-xylose, ethanol was gradually formed in the culture medium with a decrease in d-xylose and the simultaneous accumulation of xylitol, which suggested that the strain catabolized d-xylose with d-xylose reductase (XR) and xylitol dehydrogenase (XDH). XR (RpXR) was purified to homogeneity from the crude extract prepared from the mycelia of the strain grown on d-xylose. The purified enzyme was found to be NADPH-dependent and prefer pentoses such as d-xylose, d-ribose, and l-arabinose as substrates. Isolation of the genomic DNA and cDNA of the xyl1 gene encoding RpXR revealed that the gene was interrupted by two introns and the exon of the gene encoded a protein composed of 322 amino acids with a Mr of 36,724. Phylogenetic analysis showed that RpXR is more related to 4-dihydromethyltrisporate dehydrogenases from Mucoraseae fungi rather than the previously reported fungal XRs. Quantitative real-time PCR indicated that transcription of the xyl1 gene was marked in the presence of d-xylose and l-arabinose, but was week in the presence of d-glucose. These biochemical and expression analyses suggest that RpXR is involved in the catabolism of l-arabinose as well as d-xylose. This is the first report of the purification, characterization, and gene cloning of XR from zygomycetous fungi.

Improvement of D-Ribose Production from Corn Starch Hydrolysate by a Transketolase-Deficient Strain Bacillus subtilis UJS0717.

D-Ribose is a five-carbon sugar and generally used as an energy source to improve athletic performance and the ability. The culture conditions for maximum D-ribose production performance from cheap raw material corn starch hydrolysate were improved by using one-factor-at-a-time experiments and a three-level Box-Behnken factorial design. The optimal fermentation parameters were obtained as 36°C culture temperature, 10% inoculum volume, and 7.0 initial pH. The mathematical model was then developed to show the effect of each medium composition and their interactions on the production of D-ribose and estimated that the optimized D-ribose production performance with the concentration of 62.13 g/L, yield of 0.40 g/g, and volumetric productivity of 0.86 g/L·h could be obtained when the medium compositions were set as 157 g/L glucose, 21 g/L corn steep liquor, 3.2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.05 g/L MnSO4·H2O, and 20 g/L CaCO3. These findings indicated the D-ribose production performance was significantly improved compared to that under original conditions.

Cloning and characterization of the l-ribose isomerase gene from Cellulomonas parahominis MB426.

A newly isolated bacterium, Cellulomonas parahominis MB426, produced l-ribose isomerase (CeLRI) on a medium containing l-ribose as a sole carbon source. A 32 kDa protein isomerizing l-ribose to l-ribulose was purified to homogeneity from this bacterium. A set of degenerated primers were synthesized based on amino acid sequences of the purified CeLRI, and a 747 bp gene encoding CeLRI was cloned, sequenced and overexpressed in Escherichia coli. This gene encoded a 249 amino acid protein with a calculated molecular mass of 27,435. The deduced amino acid sequence of this gene showed the highest identity with l-ribose isomerase from Acinetobacter calcoaceticus DL-28 (71%). The recombinant l-ribose isomerase (rCeLRI) was optimally active at pH 9.0 and 40°C, and was stable up to 40°C for 1 h and not dependent for metallic ions for its activity. The rCeLRI showed widely substrate specificity for the rare sugar which involved l-erythro form such as l-ribose, d-lyxose, d-talose, d-mannose, l-gulose, and l-allose.

Unique O-methoxyethyl ribose-DNA chimeric oligonucleotide induces an atypical melanoma differentiation-associated gene 5-dependent induction of type I interferon response.

Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Characterization of hMTr1, a human Cap1 2'-O-ribose methyltransferase.

Cellular eukaryotic mRNAs are capped at their 5' ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2'-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2'-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo.

Co-factor engineering in lactobacilli: effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei.

The hydrolytic F(1)-part of the F(1)F(0)-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity resulted in a decrease in intracellular energy level (ATP/ADP ratio), biomass yield and growth rate. Interestingly, the glycolytic and ribolytic flux increased in L. plantarum with uncoupled ATPase activity compared to the reference strain by up to 20% and 50%, respectively. The ATP demand was estimated to have approximately 80% control on both the glycolytic and ribolytic flux in L. plantarum under these conditions. In contrast, the glycolytic and ribolytic flux decreased in L. sakei with uncoupled ATPase activity.

The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs.

Piwi-interacting RNAs (piRNAs) are a novel class of small regulatory RNAs that are expressed specifically and abundantly in germ cells. Mammalian piRNAs are 26-31 nucleotides in length and bind to Piwi proteins, but their function and biogenesis remain elusive. We previously showed that mammalian piRNAs are 2'-O-methylated at their 3' termini. The biosynthetic mechanism and function of this modification is unknown. Here, we report that the mouse homolog (mHEN1) of HEN1, a plant microRNA (miRNA) 2'-O-methyltransferase, is expressed specifically in testis and methylates 3' termini of piRNAs in vitro. These findings provide insight into the biogenesis of piRNAs.

No induction of anti-viral responses in human cell lines HeLa and MCF-7 when transfecting with siRNA or siLNA.

Gene silencing by RNAi and siRNAs has become a well-used tool for researchers. Because of its relatively small size, siRNA was originally thought to avoid activation of anti-viral responses. Recent reports demonstrating so-called "off-target effects" are therefore alarming. One issue raised is that siRNA induces interferon-regulated genes at the transcriptional level. We characterize the anti-viral responses of synthetic siRNA and in vitro-transcribed siRNA by measuring the mRNA levels of IFN-beta and OAS2 in HeLa cells. Transfections with both traditional and LNA-modified synthetic siRNA cause no anti-viral responses, whereas transfection with either long dsRNA or in vitro-transcribed siRNA leads to greater than 1000-fold induction of these genes. The lack of response was also demonstrated at the level of phosphorylated eIF2alpha, and measuring of IFN-beta by ELISA in cell culture media from the human cell line MCF-7. Altogether, transfection with synthetic siRNA does not induce anti-viral responses in these two cell lines. Our results reinforce the role of siRNA as an effective tool for reverse genetics and strengthen siLNA as a tool for future therapeutic applications.

Crystal structure of an RNA duplex r(gugucgcac)(2) with uridine bulges.

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gugucgcac)(2), was determined at 1.4 A resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R(work)/R(free) of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4(1)22 with two independent molecules in the asymmetric unit. The unit cell dimensions are a=b=47.18 A and c=80.04 A. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (approximately +50 degrees) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2'-endo conformation while the rest, including the bulges, are in the C3'-endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (approximately 4.4 A) are significantly shorter than the average P-P distance in the duplex (6.0 A). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.

Multiple snoRNA gene clusters from Arabidopsis.

Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modification (2'-O-ribose methylation and pseudouridylation). In all eukaryotes, certain snoRNAs (e.g., U3) are transcribed from classical promoters. In vertebrates, the majority are encoded in introns of protein-coding genes, and are released by exonucleolytic cleavage of linearized intron lariats. In contrast, in maize and yeast, nonintronic snoRNA gene clusters are transcribed as polycistronic pre-snoRNA transcripts from which individual snoRNAs are processed. In this article, 43 clusters of snoRNA genes, an intronic snoRNA, and 10 single genes have been identified by cloning and by computer searches, giving a total of 136 snoRNA gene copies of 71 different snoRNA genes. Of these, 31 represent snoRNA genes novel to plants. A cluster of four U14 snoRNA genes and two clusters containing five different snoRNA genes (U31, snoR4, U33, U51, and snoR5) from Arabidopsis have been isolated and characterized. Of these genes, snoR4 is a novel box C/D snoRNA that has the potential to base pair with the 3' end of 5.8S rRNA and snoR5 is a box H/ACA snoRNA gene. In addition, 42 putative sites of 2'-O-ribose methylation in plant 5.8S, 18S, and 25S rRNAs have been mapped by primer extension analysis, including eight sites novel to plant rRNAs. The results clearly show that, in plants, the most common gene organization is polycistronic and that over a third of predicted and mapped methylation sites are novel to plant rRNAs. The variation in this organization among gene clusters highlights mechanisms of snoRNA evolution.

Experimental and theoretical studies of the effects of deoxyribose substitutions on the stability of the UUCG tetraloop.

Experimental and theoretical thermodynamic studies of the consequences of 2'-hydroxyl substitution in the RNA UUCG tetraloop show distinct position dependence consistent with the diverse structural contexts of the four-loop ribose hydroxyls in this motif. The results suggest that even for simple substitutions, such as the replacement of the ribose hydroxyl (2'-OH) with hydrogen (2'-H), the free energy change reflects a complex interplay of hydrogen bonding and solvation effects and is influenced by the intrinsic pucker preferences of the nucleotides. Furthermore, theoretical studies suggest that the effect of these mutations in the single-strand state is sequence dependent, in contrast to what is commonly assumed. Free energy perturbation simulations of ribose-deoxyribose mutations in a single-strand dodecamer and in trinucleotide models suggest that in the denatured state, the magnitude of the free energy change for deoxyribose substitutions is determined to a larger extent by the identity of the nucleotide (A, C, G or U) rather than its structural context. Single-strand mutational effects must be considered when interpreting mutational studies in molecular terms.