Structural Studies of 2-Thiouridine in RNA

Abstract

A pentamer RNA sequence, Gs²UUUC, and a s²U containing 14-mer RNA tetraloop hairpin were synthesized and characterized by NMR and by UV melting studies. These oligonucleotides were used as models to understand the effect of 2-thiouridine substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through sU-34 modification. The magnitude of the effect of sU in our model system is comparable to the 20° C stabilization reported for 2-thiolation in a codonanticodon model system composed of two tRNAs with complementary anticodon sequences.     

Transfer ribonucleic acids from radish seedlings germinated with 4-thiouridine

Transfer RNAs containing 4-thiouridine residues were prepared from etiolated cotyledons or isolated chloroplasts of radish seedlings germinated with the analog. They possess a higher melting temperature and tend to have a reduced hyperchromicity. Incorporation of 4-thiouridine into tRNA reduces the ability to accept a mixture of 14 amino acids or phenylalanine alone. After desulphurization of 4-thiouridine to uridine in the tRNA, aminoacylation was restored almost completely to the level of control tRNA prepared from untreated seedlings. These results indicate that a tRNA containing 4-thiouridine in the anticodon and/or in another part of molecule has low or no ability to accept amino acids.     

Carbon-13 NMR studies on [4-13C] uracil labelled E. coli transfer RNA1(Val1).

In this paper we describe carbon-13 nuclear magnetic resonance results on 13C-enriched purified transfer RNAI(VAL) from from E. coli SO-187, a uracil requiring auxotroph. The organism was grown on uracil 90% 13C-enriched at the carbonyl C4 position. Transfer RNAI(Val) was purified from bulk tRNA by sequential chromatography on columns of BD cellulose, DEAE-Sephadex A-50 and reverse gradient sepharose 4B. Dihydrouridine, 4-thiouridine, and uridine 5-oxyacetic acid located at discrete positions in the polymer backbone were tentatively assigned in the highly resolved 25 MHz 13C-spectra. Chemical shift versus temperature plots reveal differential thermal perturbation of the ordered solution structure, evident in the large dispersion (ca 3-4 ppm) of the uridine C4 resonances. Over the range 26-68 degrees C, V in the anticodon displays the largest downfield shift. Whereas several uridine residues rapidly shift downfield between 50-68 degrees, one moves upfield beginning at 37 degrees. The results are qualitatively compared with proton NMR analysis of the three dimensional structure.     

Desulfuration of 2-thiouridine with hydrogen peroxide in the physiological pH range 6.6–7.6 is pH-dependent and results in two distinct products

The 2-thiomodified nucleosides, located at first position of tRNAs anticodon, may constitute a primary target for oxidative attack under conditions of oxidative stress. Desulfuration of 2-thiouridine (S2U) was investigated in the ¹H NMR scale in the presence of 100 mM H₂O₂ and phosphate buffer in the physiological pH range, from pH 6.6 to 7.6. The obtained data demonstrate an intriguing result that within one unit of the pH range uridine is the major product of the S2U desulfuration in the pH 7.6, while the 4-pyrimidinone nucleoside (H2U) is dominant in pH 6.6. The possible desulfuration pathway and the biological importance of the transformation of S2U either to U or H2U are discussed in the context of the tRNA oxidative damage.     

Isolation and characterization of 5-carbamoylmethyluridine and 5-carbamoylmethyl-2-thiouridine from human urine

Two new modified uracil nucleosides, 5-carbamoylmethyuridine (ncm5U, I) and 5-carbamoylmethyl-2-thiouridine (ncm5s2U, II) were isolated from a 24 hr collection of a normal human urine. The structures were assigned on the basis of UV, NMR and mass spectral data and confirmed by comparison of the spectral data and HPLC mobilities with those of authentic samples. On the basis of experimental data it appears possible that 5-carbamoylmethyl-2-thio-uridine (ncm5s2U, II) may be a degradation product produced from a labile precursor by the chemical treatments during the isolation procedure. However, the other nucleoside (ncm5U,I) certainly appears to be of metabolic origin and was also found in the urines of one chronic myelogenous leukemia and one lung carcinoma patient. Abbreviations used are: tRNA-transfer ribonucleic acid, TMS-trimethylsilyl, RP-HPLC--reverse phase high performance liquid chromatography, EI--electron impact, cm5U-5-carboxymethyluridine, mcm5U-5-methoxycarbonylmethyluridine, cm5s2U-5-carboxymethyl-2-thiouridine, mcm5s2U-5-methoxycarbonylmethyl-2-thiouridine, t6A-9-beta-D-ribofuranosyl-[N(purin-6-yl)carbamoyl]-1-threonine, C-cytidine, acp3u-3-(3-amino-3-carboxypropyl)uridine, AICR-aminoimidazole carboxamide riboside, alpha-4-PCNR & beta-4-PCNR-9-alpha-D-(or beta-D)-ribofuranosyl-pyridin-4-one-3-carboxamide, H x 7R-7-beta-D-ribofuranosyl hypoxanthine, m3U-3-methyluridine, m1I-1-methylinosine, m1G-1-methylguanosine, DI-5'-deoxyinosine, dms5OA-5'-deoxy-5'-methylthioadenosine sulfoxide, m2(2)G-N2-dimethylguanosine, psi-psi-uridine, A-adenosine, I-inosine, CML-chronic myelogenous leukemia mam5s2U-5-methylaminomethyl-2-thiouridine, ncm5U-5-carbamoylmethyluridine, ncm5s2U-5-carbamoylmethyl-2-thiouridine, UV-ultraviolet, NMR-nuclear magnetic resonance, HPLC-high performance liquid chromatography, GC-MS-gas chromatography-mass spectrometry.     

MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli

Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

5-(carboxymethylaminomethyl)-2-thiouridine, a new modified nucleoside found at the first letter position of the anticodon.

The structure of a modified uridine derivative which was detected at the first letter position of the anticodon of Bacillus subtilis tRNA1Lys was determined to be 5-(carboxymethylaminomethyl)-2-thiouridine. The determination was mainly based in this ultraviolet absorption spectra and mass spectrometric analysis of the trimethylsilyl derivative.     

Synthesis and properties of oligonucleotides containing 2'-O-methyl-2-thiouridine.

New methods to synthesize 2'-O-methyl-2-thiouridine and its phosphoramidite building block for incorporation into oligonucleotides were developed. Oligonucleotides containing 2'-O-methyl-2-thiouridine were expected to be favorable as antisense agents in several respects, i.e., nuclease resistance, stable RNA duplex formation, and exact base recognition. Therefore, to make them clear, we synthesized oligonucleotides having 2'-O-methyl-2-thiouridine and analyzed their properties in detail.     

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

A series of nucleobase-modified siRNA duplexes containing "rare" nucleosides, 2-thiouridine (s(2)U), pseudouridine (Psi), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s(2)U or Psi units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (DeltaTm 3.9 and 6.6 degrees C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s(2)U and Psi units at their 3'-ends and with a D unit at their 5'-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s(2)U and Psi units at their 5'-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 3'-adjacent to the mutation site. Thermally stable siRNA molecules containing several s(2)U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.     

Synthesis of oligoribonucleotides containing 4-thiouridine using the convertible nucleoside approach and the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group

Oligoribonucleotides containing 4-thiouridine were prepared using the Fpmp group for protection of the 2'-OH. Two uridine derivatives with the 1,2,4-triazolyl and the 2-nitrophenyl groups at position 4 were used to obtain 4-thiouridine by postsynthetic substitution with sodium hydrogen sulfide. Both uridine derivatives allow the preparation of the desired oligonucleotides in good yields.     

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.     

Synthesis and Properties of Uniquely Modified Oligoribonucleotides: Yeast TrnaPhe Fragments with 6-Methyluridine and 5,6-Dimethyluridine at Site-Specific Positions

Abstract

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 TΦC 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.     

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture.

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.     

In vivo incorporation of the intrinsic photolabel 4-thiouridine into Escherichia coli RNAs.

The in vivo incorporation of the photoactivable uridine analogue 4-thiouridine into the RNAs of an Escherichia coli K12 pyrD strain has been optimised. s4Urd uptake in RNAs appears to be strikingly dependent upon the age of the preculture, i.e. the number of generations the cells have undergone immediately before dilution in the thiolation medium. Conditions have been set up where efficient RNA thiolation occurs in cells growing exponentially at 50 to 70% the rate of the control. The substitution level s4U/U is maximal after growth for 9 to 10 generations in the thiolation medium and reaches 17 +/- 3% in tRNA and bulk RNA. Most of ribosomal derived ribonucleoproteins, 65 +/- 5%, sediment as 70S ribosomes (s4U/U = 7 +/- 2%) on a high Mg2+ sucrose gradient. The thiolated RNAs were characterized by their migration on a thiol-specific affinity electrophoretic gel.