RNA Polynucleotide Research Articles

RNA degradation in human mitochondria: the journey is not finished.

Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.

Multiple Non-Canonical Base-Stacking Interactions as One of the Major Determinants of RNA Tertiary Structure Organization.

Stacking interactions of heterocyclic bases of ribonucleotides are one of the most important factors in the organization of RNA secondary and tertiary structure. Most of these (canonical) interactions are formed between adjacent residues in RNA polynucleotide chains. However, with the accumulation of data on the atomic tertiary structures of various RNAs and their complexes with proteins, it has become clear that nucleotide residues that are not adjacent in the polynucleotide chains and are sometimes separated in the RNA primary structure by tens or hundreds of nucleotides can interact via (non-canonical) base stacking. This paper presents an exhaustive database of such nonadjacent base-stacking elements (NA-BSEs) and their environment in the macromolecules of natural and synthetic RNAs. Analysis of these data showed that NA-BSE-forming nucleotides, on average, account for about a quarter of all nucleotides in a particular RNA and, therefore, should be considered as bona fide motifs of the RNA tertiary structure. We also classified NA-BSEs by their location in RNA macromolecules. It was shown that the structure-forming role of NA-BSEs involves compact folding of single-stranded RNA loops, transformation of double-stranded bulges into imperfect helices, and binding of RNA regions distant in the primary and secondary RNA structure.

Consequences of Genomic DNA Mono-Ribonucleotides for Chromosomal Stability

Abstract Mono-ribonucleotides are building blocks for polynucleotide RNA chains (e.g., messenger RNA), but if mis-incorporated into duplex DNA can cause mutagenesis and chromosomal instability. During DNA synthesis by Pol γ, remnants of unremoved RNA primers contribute to elevated mono-ribonucleotide triphosphates resulting in nucleotide pool imbalance, ultimately favoring mis-incorporated ribonucleotides during replication. Moreover, although polymerases generally replicate DNA with high fidelity, the steric gate occasionally allows a mis-incorporated ribonucleotide. Thus, a mono-ribonucleotide is one of the most abundant lesions in genomic DNA of eukaryotes. If unremoved from double-stranded DNA, the ribonucleotide exerts negative effects on replication, transcription, and genomic maintenance, with lasting effects on cellular homeostasis. Even a single ribonucleotide in telomeric DNA comprises shelterin binding and telomere capping causing vulnerability to spontaneous hydrolysis which potentiates telomere shortening. Consistent with this, a ribonucleotide positioned in double-helical DNA alters its structure by torsinally distorting the sugar-phosphate backbone. Fortunately, cellular response and repair pathways exist to help cells cope with mis-incorporated mono-ribonucleotides. The Ribonucleotide Excision Repair (RER) or a Topoisomerase 1 (Top1)-mediated pathway remove embedded ribonucleotides. For RER, RNase H2 incises 5’ of a mono-ribonucleotide, creating an access point for its removal. If cells are deficient in RNase H2, Top1 initiates removal of the ribonucleotide. However, Top1 is less accurate than RNase H2, which can lead to mutagenesis. Studying the mechanisms in which ribonucleotides are incorporated into DNA or further metabolized should provide insight to their negative consequences for chromosomal integrity, cancer, and auto-immune disease attributed to a genetic deficiency of RNase H2.

A conserved RNA degradation complex required for spreading and epigenetic inheritance of heterochromatin.

Heterochromatic domains containing histone H3 lysine 9 methylation (H3K9me) can be epigenetically inherited independently of underlying DNA sequence. To gain insight into the mechanisms that mediate epigenetic inheritance, we used a Schizosaccharomyces pombe inducible heterochromatin formation system to perform a genetic screen for mutations that abolish heterochromatin inheritance without affecting its establishment. We identified mutations in several pathways, including the conserved and essential Rix1-associated complex (henceforth the rixosome), which contains RNA endonuclease and polynucleotide kinase activities with known roles in ribosomal RNA processing. We show that the rixosome is required for spreading and epigenetic inheritance of heterochromatin in fission yeast. Viable rixosome mutations that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.

Interaction of proflavine with the RNA polynucleotide polyriboadenylic acid–polyribouridylic acid: photophysical and calorimetric studies

The binding of proflavine, an acriflavine derivative, with the RNA polynucletodide polyadenylic acid–polyuridylic acid is investigated here to understand the structural and thermodynamic basis of the binding process. Such binding data are crucial for designing viable theraperutic agents. Spectroscopic studies clearly suggest a strong binding interaction between proflavine and polyadenylic acid–polyuridylic acid leading to efficient energy transfer between the poly AU base pairs and proflavine. The stoichiometry of proflavine polyadenylic acid–polyuridylic acid binding was independently estimated by continuous variation analysis of Job. An intercalative binding model is envisaged for the binding from hydrodynamic studies. Circular dichroism experiments revealed that the binding induced conformational changes in the RNA, and also led to induction of optical activity in the bound dye molecules. The binding affinity of the complex was deduced to be (6.57 ± 0.75) 105 M−1 at (298.15 ± 0.10) K from isothermal titration calorimetry experiment. Positive entropy and negative enthalpy changes characterized the complexation. The binding was observed to be weaker both at higher temperatures and increased [Na+]. The affinity of binding decreased with increasing [Na+]. When the Gibbs energy was parsed between polyelectrolytic and nonpolyelectropytic components, it surprisingly revealed a higher role for the non-polyelectrolytic forces. These results present new data for developing RNA targeted ligands.Communicated by Ramaswamy H. Sarma

Mitochondrial double-stranded RNA triggers antiviral signalling in humans

Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.

Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes

The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes.

Methods and compositions for amplification and detection of microRNAs (miRNAs) and noncoding RNAs (ncRNAs) using the signature sequence amplification method (SSAM).

The signature sequence amplification method (SSAM) described herein is an approach for amplifying noncoding RNA (ncRNA), microRNA (miRNA), and small polynucleotide sequences. A key point of the SSAM technology is the generation of signature sequences. The signature sequences include target sequences (miRNA, ncRNA, and/or any small polynucleotide sequence) flanked by two DNA fragments. Target sequences can be amplified through DNA synthesis, RNA synthesis, or the combination of DNA and RNA synthesis. The amplification of signature sequences provides an efficient and reproducible mechanism to determine the presence or absence of the target miRNAs/ncRNAs, to analyze the quantities of the miRNAs in biological samples, and for miRNA/ncRNA profiling.

Biological activities of the antiviral protein BE27 from sugar beet (Beta vulgaris L.).

The ribosome inactivating protein BE27 displays several biological activities in vitro that could result in a broad action against several types of pathogens. Beetin 27 (BE27), a ribosome-inactivating protein (RIP) from sugar beet (Beta vulgaris L.) leaves, is an antiviral protein induced by virus and signaling compounds such as hydrogen peroxide and salicylic acid. Its role as a defense protein has been attributed to its RNA polynucleotide:adenosine glycosidase activity. Here we tested other putative activities of BE27 that could have a defensive role against pathogens finding that BE27 displays rRNA N-glycosidase activity against yeast and Agrobacterium tumefaciens ribosomes, DNA polynucleotide:adenosine glycosidase activity against herring sperm DNA, and magnesium-dependent endonuclease activity against the supercoiled plasmid PUC19 (nicking activity). The nicking activity could be a consequence of an unusual conformation of the BE27 active site, similar to that of PD-L1, a RIP from Phytolacca dioica L. leaves. Additionally, BE27 possesses superoxide dismutase activity, thus being able to produce the signal compound hydrogen peroxide. BE27 is also toxic to COLO 320 cells, inducing apoptosis in these cells by either activating the caspase pathways and/or inhibiting protein synthesis. The combined effect of these biological activities could result in a broad action against several types of pathogens such as virus, bacteria, fungi or insects.

Entropy driven binding of the alkaloid chelerythrine to polyadenylic acid leads to spontaneous self-assembled structure formation

The binding thermodynamics and interaction of the putative anticancer alkaloid chelerythrine with polyadenylic acid were investigated by isothermal titration calorimetry, absorption and fluorescence spectroscopy, circular dichroism, differential scanning calorimetry and thermal melting experiments. The equilibrium binding constant was evaluated to be of the order of 107M−1. Strong positive entropic and favorable enthalpic contributions to the binding were revealed. The binding affinity was enhanced within (10 to 100)mM Na+ concentration. Circular dichroism spectra confirmed that the increase in entropy change was caused by a strong conformational change in the RNA polynucleotide. Absorption and circular dichroism melting studies revealed that chelerythrine binding induced self-assembled duplex structure formation in poly(A) molecules resulting in a cooperative melting profile. This was further confirmed from differential scanning calorimetry data. The intercalation binding of the alkaloid involved strong energy transfer from the polynucleotide bases to the bound alkaloid molecules. The remarkably high entropy driven binding of the alkaloid induced spontaneous self-assembled structure formation in poly(A) and the associated binding affinity is the highest so far reported for a small molecule binding to poly(A).

Discrimination of RNA from DNA by Polynucleotide Phosphorylase

Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also thought to participate in bacterial DNA transactions. Here we used chimeric polynucleotides, composed of alternating RNA and DNA tracts, to analyze whether and how Mycobacterium smegmatis PNPase discriminates RNA from DNA during the 3'-phosphorolysis reaction. We find that a kinetic block to 3'-phosphorolysis of a DNA tract within an RNA polynucleotide is exerted when resection has progressed to the point that a 3'-monoribonucleotide flanks the impeding DNA segment. The position of the pause one nucleotide before the first deoxynucleotide encountered is independent of DNA tract length. However, the duration of the pause is affected by DNA tract length, being transient for a single deoxynucleotide and durable when two or more consecutive deoxynucleotides are encountered. Substituting manganese for magnesium as the metal cofactor allows PNPase to "nibble" into the DNA tract. A 3'-phosphate group prevents RNA phosphorolysis when the metal cofactor is magnesium. With manganese, PNPase can resect an RNA 3'-phosphate end, albeit 80-fold slower than a 3'-OH. We discuss the findings in light of the available structures of PNPase and the archaeal exosome·RNA·phosphate complex and propose a model for catalysis whereby the metal cofactor interacts with the scissile phosphodiester and the penultimate ribose.

Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of Mycobacterium smegmatis Polynucleotide Phosphorylase

Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also suggested to participate in bacterial DNA transactions. Here we characterize and compare the RNA and DNA modifying activities of Mycobacterium smegmatis PNPase. The full-length (763-aa) M. smegmatis PNPase is a homotrimeric enzyme with Mg(2+)•PO(4)-dependent RNA 3'-phosphorylase and Mg(2+)•ADP-dependent RNA polymerase activities. We find that the enzyme is also a Mn(2+)•dADP-dependent DNA polymerase and a Mn(2+)•PO(4)-dependent DNA 3'-phosphorylase. The Mn(2+)•DNA and Mg(2+)•RNA end modifying activities of mycobacterial PNPase are coordinately ablated by mutating the putative manganese ligand Asp526, signifying that both metals likely bind to the same site on PNPase. Deletions of the C-terminal S1 and KH domains of mycobacterial PNPase exert opposite effects on the RNA and DNA modifying activities. Subtracting the S1 domain diminishes RNA phosphorylase and polymerase activity; simultaneous deletion of the S1 and KH domains further cripples the enzyme with respect to RNA substrates. By contrast, the S1 and KH domain deletions enhance the DNA polymerase and phosphorylase activity of mycobacterial PNPase. We observe two distinct modes of nucleic acid binding by mycobacterial PNPase: (i) metal-independent RNA-specific binding via the S1 domain, and (ii) metal-dependent binding to RNA or DNA that is optimal when the S1 domain is deleted. These findings add a new dimension to our understanding of PNPase specificity, whereby the C-terminal modules serve a dual purpose: (i) to help capture an RNA polynucleotide substrate for processive 3' end additions or resections, and (ii) to provide a specificity filter that selects against a DNA polynucleotide substrate.

S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting autoregulation and apparent KD values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase.

Surface-enhanced Raman Scattering Study of the Binding Modes of a Dibenzotetraaza[14]annulene Derivative with DNA/RNA Polynucleotides

Binding modes of a dibenzotetraaza14annulene (DBTAA) derivative with synthetic nucleic ac- ids were studied using surface-enhanced Raman spectroscopy (SERS). Changes in SERS intensity and appearance of new bands in spectra were attributed to different complexes formed between the DBTAA molecules and DNA/RNA polynucleotides. A decrease in intensity pointed to intercalation as the domi- nant binding mode of the annulene derivative with poly dGdC-poly dGdC and poly rA-poly rU, whereas new bands in the spectra at 735 cm −1 and 1345 cm −1 revealed binding within the minor groove of poly dAdT-poly dAdT. When all the dominant binding sites were occupied, SERS spectra implied that small molecules bind on the outside of the DNA analogues, while exist mainly as free molecules in equimolar ratio with the synthetic RNA polynucleotide, thereby indicating higher affinity for DNA than for RNA.(doi: 10.5562/cca2106)

Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.

Novel Activities of Glycolytic Enzymes in Bacillus subtilis

Glycolysis is one of the most important metabolic pathways in heterotrophic organisms. Several genes encoding glycolytic enzymes are essential in many bacteria even under conditions when neither glycolytic nor gluconeogenic activities are required. In this study, a screening for in vivo interaction partners of glycolytic enzymes of the soil bacterium Bacillus subtilis was used to provide a rationale for essentiality of glycolytic enzymes. Glycolytic enzymes proved to be in close contact with several other proteins, among them a high proportion of essential proteins. Among these essential interaction partners, other glycolytic enzymes were most prominent. Two-hybrid studies confirmed interactions of phosphofructokinase with phosphoglyceromutase and enolase. Such a complex of glycolytic enzymes might allow direct substrate channeling of glycolytic intermediates. Moreover we found associations of glycolytic enzymes with several proteins known or suspected to be involved in RNA processing and degradation. One of these proteins, Rny (YmdA), which has so far not been functionally characterized, is required for the processing of the mRNA of the glycolytic gapA operon. Two-hybrid analyses confirmed the interactions between the glycolytic enzymes phosphofructokinase and enolase and the enzymes involved in RNA processing, RNase J1, Rny, and polynucleotide phosphorylase. Moreover RNase J1 interacts with its homologue RNase J2. We suggest that this complex of mRNA processing and glycolytic enzymes is the B. subtilis equivalent of the RNA degradosome. Our findings suggest that the functional interaction of glycolytic enzymes with essential proteins may be the reason why they are indispensable.

Ribosome-inactivating proteins from plants: more than RNA N-glycosidases?

Many plants contain proteins that are capable of inactivating ribosomes and accordingly are called ribosome-inactivating proteins or RIPs. These typical plant proteins receive a lot of attention in biological and biomedical research because of their unique biological activities toward animal and human cells. In addition, evidence is accumulating that some RIPs play a role in plant defense and hence can be exploited in plant protection. To understand the mode of action of RIPs and to optimize their medical and therapeutical applications and their use as antiviral compounds in plant protection, intensive efforts have been made to unravel the enzymatic activities of RIPs and provide a structural basis for these activities. Though marked progress has been made during the last decade, the enzymatic activity of RIPs has become a controversial issue because of the concept that RIPs possess, in addition to their classical RNA N-glycosidase and polynucleotide:adenosine glycosidase activity, other unrelated enzymatic activities. Moreover, the presumed novel enzymatic activities, especially those related to diverse nuclease activities, are believed to play an important role in various biological activities of RIPs. However, both the novel enzymatic activities and their presumed involvement in the biological activities of RIPs have been questioned because there is evidence that the activities observed are due to contaminating enzymes. We offer a critical review of the pros and cons of the putative novel enzymatic activities of RIPs. Based on the available data, it is suggested that there is little conclusive evidence in support of the presumed activities and that in the past too little attention has been given to the purity of the RIP preparation. The antiviral activity and mode of action of RIPs in plants are discussed in view of their classical and presumed novel enzymatic activities.

Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.

Solid-phase synthesis of oligoribonucleotides using T4 RNA ligase and T4 polynucleotide kinase

The concept of solid-phase synthesis of oligoribonucleotide using T4 RNA l ipase and T4 polynucleotide kinase has been proposed and tested with model homo-oligoribonucleotides. The method consists of the immobilization of the first oligomer block at the 3′-terminus on a solid support followed by a chain elongation in the 5′-direction with trinucleoside diphosphates using T4 RNA-ligase and phosphorylation using polynucleotide kinase. Hydrazides of Biogel P-300, Sepharose 4B and cellulose were tested as solid supports for immobilization of initial oligomers. The properties of supports were rated on reactivities of immobilized 5′-phosphorylated oligomers as phosphate donors in the solid phase reactions, hydrodynamical properties and capacity to eliminate donor molecules spontaneously during reactions. Hydrazide of Sepharose 4B appeared to be a more suitable support because of better hydrodynamic properties and highest reactivities of immobilized donors. Saturated concentrations of RNA ligase and polynucleotide kinase and optimal time of joining reaction were determined. In a model experiment ApApA was twice attached to the immobilized hydrazide of Sepharose 4B donor (pA) 6pA ox. The yield of (Ap) 12 was 25%.

A highly sensitive probe for guanine N7 in folded structures of RNA: application to tRNAPhe and Tetrahymena group I intron

A nickel complex has been shown to promote conformation-specific oxidation of guanosine in polynucleotide RNA. In all cases, reaction was strictly dependent on the solvent exposure and surface properties of guanine N7. Modification of native tRNA(Phe) (yeast) was detected at G18, G19, G20, and Gm34 and concurred with predictions based on its crystal structure. Additional guanine derivatives became exposed to oxidation only after the tRNA unfolded in the absence of Mg2+. Reaction of the Tetrahymena group I intron RNA (L-21 ScaI) also compared favorably to its three-dimensional model by appropriately identifying guanosine residues in hairpin loops, duplex termini, and the essential cofactor binding site. These results complemented prior data generated by hydroxyl radical, and in combination they served to distinguish the solvent accessibility of sugar backbone and base positions in guanosine residues. Most importantly, this nickel complex exhibited greater selectivity than either dimethyl sulfate or RNase T1 for characterizing tRNA(Phe) and intron RNA.