Synthesis and properties of uniquely modified oligoribonucleotides: yeast tRNA(Phe) fragments with 6-methyluridine and 5,6-dimethyluridine at site-specific positions

The phosphoramidites of 6-methyluridine and 5,6-dimethyluridine were synthesized and the modified uridines site-selectively incorporated into heptadecamers corresponding in sequence to the yeast tRNA(Phe) anticodon and TpsiC domains. The oligoribonucleotides were characterized by NMR, MALDI-TOF MS and UV-monitored thermal denaturations. The 6-methylated uridines retained the syn conformation at the polymer level and in each sequence location destabilized the RNAs compared to that of the unmodified RNA. The decrease in RNA duplex stability is predictable. However, loss of stability when the modified uridine is in a loop is sequence context dependent, and can not, at this time, be predicted from the location in the loop.     

Simple statistical models predict C-to-U edited sites in plant mitochondrial RNA

Background  

RNA editing is the process whereby an RNA sequence is modified from the sequence of the corresponding DNA template. In the mitochondria of land plants, some cytidines are converted to uridines before translation. Despite substantial study, the molecular biological mechanism by which C-to-U RNA editing proceeds remains relatively obscure, although several experimental studies have implicated a role for cis-recognition. A highly non-random distribution of nucleotides is observed in the immediate vicinity of edited sites (within 20 nucleotides 5' and 3'), but no precise consensus motif has been identified.     

The Conformation of a Conserved Stem-Loop Structure in Ribosomal RNA

Abstract

The RNA of small ribosomal subunits contains a conserved stem-loop structure near the 3′ end. Characteristics for the hairpins are: (a) a nine-basepairs stem; (b) a conserved ^A-U _U-Gjunction in the stem; (c) a conserved sequence Gm⁶ ₂Am⁶ ₂A sequence in the loop (except yeast mitochondria and mutants from bacteria). We are using UV-optics, micro-calorimetry and 500 MHz-NMR to investigate fragments of about 50 nucleotides cleaved from the 3′ ends of small ribosomal subunit RNA's by bacteriocins. Our preliminary conclusions are: (1) Dimethylation of the adenines in the loop destabilizes the hairpin because of an increased stacking; (2) melting of the hairpin starts at the ends as well as in the middle at the 5-H junction; (3) basepair substitutions have an unexpectedly large effect on thermal stability.     

Mg2+ Dependence of the Structure and Thermodynamics of Wheat Germ and Lupin Seeds 5S rRNA

Abstract

The formation and stability of structural elements in two 5S rRNA molecules from wheat germ (WG) and lupin seeds (LS) as a function of Mg²⁺ concentration in solution was determined using the adiabatic differential scanning microcalorimetry (DSC). The experimentally determined thermodynamic parameters are compared with calculations using thermodynamic databases used for prediction of RNA structure. The 5S rRNA molecules which show minor differences in the nucleotide sequence display very different thermal unfolding profiles (DSC profiles). Numerical deconvolution of DSC profiles provided information about structural transformations that take place in both 5S rRNA molecules. A comparative analysis of DSC data and the theoretical thermodynamic models of the structure was used to establish a relationship between the constituting transitions found in the melting profiles and the unfolding of structural domains of the 5S rRNA and stability of its particular helical elements.

Increased concentration of Mg²⁺ ions induces additional internal interactions stabilising 5S rRNA structures found at low Na⁺ concentrations. Observed conformational transitions suggest a structural model in which the extension of helical region E dominates over the postulated tertiary interaction between hairpin loops. We propose that helix E is stabilised by a sequence of non-standard pairings extending this helix by the formation of tetra loop e and an almost total reduction of loop d between helices E and D. Two hairpin structures in both 5S rRNA molecules: the extended C-C' and the extended E-E'-E” hairpins appear as the most stable elements of the structure. The cooperativity of the unfolding of helixes in these 5S rRNA molecules changes already at 2 mM Mg²⁺.     

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.     

Specificity of Phage Display Selected Peptides for Modified Anticodon Stem and Loop Domains of tRNA

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA^Lys3 having 2-thiouridine (s²U₃₄) and pseudouridine (Ψ₃₉), bound the modified human ASL^Lys3(s²U₃₄;Ψ₃₉) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL^Lys3(s²U₃₄;Ψ₃₉), followed by the hypermodified ASL^Lys3 (mcm⁵s²U₃₄;ms²t⁶A₃₇) and the unmodified ASL^Lys3, but bound poorly to singly modified ASL^Lys3 constructs (Ψ₃₉, ms²t⁶A_37, s²U₃₄), ASL^Lys1,2 (t⁶A₃₇) and Escherichia coli ASL^Glu (s²U₃₄). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.     

Mass Spectrometric Study of Modified Uridines and Their N3—Isomers

Abstract

The mass spectral fragmentations of modified uridines and their N³—isomers are discussed in context of the b+41 ion formation.     

Base Pairing at the Stem-loop Junction in the SL1 Kissing Complex of HIV-1 RNA: A Thermodynamic Study Probed by Molecular Dynamics Simulation

Abstract

The thermodynamics of the opening/closure process of a GC base pair located at the stem-loop junction of the SL1 sequence from HIV-1_Lai genomic RNA was investigated in the context of a loop-loop homodimer (or kissing complex) using molecular dynamics simulation. The free energy, enthalpy and entropy changes for the closing reaction are 0 kcal·mol^?1, ?11 kcal·mol^?1and ?0.037 kcal·mol^?1-K^?1 at 300° K respectively. Furthermore it is found that the free energy change is the same for the formation of a 11 nucleotide loop closed with UG and for the formation of a 9 nucleotide loop closed with GC. Our study evidences the high flexibility of the nucleotides at the stem-loop junction explaining the weak stability of this structure.     

Solution Structure of a GAAG Tetraloop in Helix 6 of SRP RNA from Pyrococcus furiosus

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U:G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif, the conformational stabilities are significantly different to each other. It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.     

A role for hydrophobicity in a Diels–Alder reaction catalyzed by pyridyl-modified RNA

New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels–Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu²⁺, as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modification''s role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism.     

The Stability of DNA and RNA G-Quartets

Abstract

We have investigated the thermodynamic stability of DNA and RNA G-quartet by circular dichroism spectroscopy, gel electrophoresis, and melting analysis. The free energy (δG°37) for G-quartet formation of d(TTGGGG)⁴ and r(UUGGGG)⁴ at 100 mM NaCl and 37°C were 1.4 kcal mol-¹ and ?3.0 kcal mol-¹, respectively. On the other hand, at 100 mh4 KCI, δG°₃₇ of the DNA and RNA were ?10.0 kcal mol-1 and ?8.2 kcal mol-¹. This result indicates that the dependence of DNA G-quartet stability on these ions is larger than that of RNA.

     

The stability of RNA hairpin loops containing A-U-G: An-U-G-Um

RNA oligomers with the sequence A_n-U-G-U_m, n = 7–9, m = 5–10, have been synthesized and found to form hairpin loops in 21 mM or 101 mM Na⁺. The hairpin loops displayed melting temperatures 13°–29°C greater than that of the hairpin loop A₆-C₈-U₆ in the same solvent. The increased stability of these hairpin loops is attributed to the presence of the trinucleotide A-U-G in the loop. Circular dichroism (CD) spectra were taken of the hairpin loops A₇-U-G-U_6,7,8, A₈-U-G-U_6,7,8, and A₉-U-G-U₆ in 21 mM Na⁺, and compared with circular dichroism spectra of A₆-U₆ in 1 M Na⁺. Difference spectra were calculated between each A_n-U-G-U_m and 11.5 mM (nucleotide) A₆-U₆ at similar temperatures and identical singlestrand fractions to give the “experimental” CD spectrum of the unbase-paired nucleotides in the loop, assuming, five, six, or seven base pairs. CD spectra were calculated for each of the assumed unbase-paired sequences using the measured CD spectra of ApA, A-U-G-, A, and U, and compared with the experimental spectra. The best agreement was found for hairpin-loop models containing five base pairs and five to eight unbase-paired nucleotides in the loop.     

The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines

The N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines make up over half of the population of all naturally modified adenosines and they are present in the transfer ribonucleic acids (tRNA) at position 37. We measured effects of N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines on the thermodynamic stability of RNA duplexes containing a U-A^Mod base pair at internal and terminal duplex positions, as well as containing modified adenosines as a 3′-terminal unpaired nucleotide. Beside naturally modified adenosines such as N⁶-isopentenyladenosine (i⁶A), N⁶-methyladenosine (m⁶A), 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A) and 2-methylthio-N⁶-methyladenosine (ms²m⁶A), we studied several artificial modifications to evaluate the steric and electronic effects of N⁶-alkyl substituents. Moreover, some N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines were placed in hairpins at positions corresponding to nucleotide 37 of the tRNA anticodon arm, and the thermodynamic stability of those hairpins was studied. The stability of the modified RNA hairpins was measured in standard melting buffer containing 1 M sodium chloride as well as in physiological buffer containing 10 mM magnesium chloride and 150 mM potassium chloride. The results obtained indicate that the nature of the adenosine modification and the position of U-A^Mod base pairs within the duplex influence the thermodynamic stability of RNA duplexes. For most of the modification, the destabilization of duplexes was observed. Moreover, we found that the buffer composition and the structure of the modified adenosine very significantly affect the thermodynamic stability of RNA.     

The Chemical Synthesis of E. coli tRNALys Anticodon Loop Fragment and Its Analogues

Abstract

E. coli tRNA^Lys anticodon loop fragment (Umnm⁵sUUUt⁶A) 1 and its analogues 2–6 were synthesized by the classical phosphotriester approach in solution. The preparation of suitably protected derivatives of N⁶-threonylcarbamoyladenosine 18 is also described.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

Free energy of imperfect nucleic acid helices. II. Small hairpin loops

Physical studies of enzymically synthesized oligonucleotides of defined sequence are used to evaluate quantitatively the stability of small RNA hairpin loops and helices. The series (Ap)₄G(pC) _N(pU)₄, N = 4, 5 or 6, exists as monomolecular hairpin helices when N ≥ 5, and as imperfect dimer helices when N ≤ 4. In this size range, hairpin loops become more favorable (less destabilizing thermodynamically) as they increase in size from 3 to 4 to 5 unbonded nucleotides. Very small hairpin loops are particularly destabilizing; molecules whose base sequence would imply a hairpin loop of three nucleotides will generally exist with a loop of five, including a broken terminal base pair.Thermodynamic parameters for base pair and loop formation are calculated by a method which makes unnecessary the use of measured enthalpies of polynucleotide melting. Literature data on oligonucleotide double helices yield estimates of the free energy contribution from each of the six types of stacking interactions between three possible neighboring base pairs. The advantage of this approach is that the properties of oligonucleotides are used in predicting the stability of small RNA helices, avoiding the long extrapolation from the properties of high polymers.We provide Tables of temperature-dependent free energies that allow one to predict the stability and thermal transition temperature of many simple RNA secondary structures (applicable to ~1 m-Na⁺ concentration). As an example, we apply the rules to an isolated fragment of tRNA^Ser (yeast) (Coutts, 1971), whose properties were not used in calculating the free-energy parameters. The experimental melting temperature of 88 °C is predicted with an error margin of 5 deg. C.     

Archaeal Tuc1/Ncs6 Homolog Required for Wobble Uridine tRNA Thiolation Is Associated with Ubiquitin-Proteasome,Translation, and RNA Processing System Homologs

While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA^Lys_UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA^Lys_UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.     

NMR Studies of a Lead Ribozyme and Its Non-Cleavable Analogue

Abstract

The structure of a lead ribozyme, which consists of two RNA strands, at neutral pH has been studied by NMR. Nearly all resonances of imino protons, base protons (H2, H5, H6 and H8) and sugar protons (H1′ and H2′) were assigned sequentially. Interesting structural features which deviate from the standard structure were found for the residues at an active site which consists of an internal loop. No indication of stable G:A base pairs was found in the loop. The effect of addition of Pb²⁺ was studied by the use of a non-cleavable analogue in which the cytidine at a cleavage site is replaced by 2′-O-methylcytidine. It was suggested that Pb²⁺ binds close to the cleavage site and that the structural change induced by Pb²⁺ is moderate and localized.