A fully enzymatic method for site-directed spin labeling of long RNA.

Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5'-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies.

A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage.

Oligoribonucleotides containing a 5'-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5'-phosphorothiolate linkage, with the adjacent 2'-hydroxyl group protected as the photolabile 2'-O-o-nitrobenzyl or 2'-O-α-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2'-protected dinucleotide to transcribed or chemically synthesized 5' and 3' flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.

RNA analysis by biosynthetic tagging using 4-thiouracil and uracil phosphoribosyltransferase.

RNA analysis by biosynthetic tagging (RABT) enables sensitive and specific queries of (a) how gene expression is regulated on a genome-wide scale and (b) transcriptional profiling of a single cell or tissue type in vivo. RABT can be achieved by exploiting unique properties of Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT), a pyrimidine salvage enzyme that couples ribose-5-phosphate to the N1 nitrogen of uracil to yield uridine monophosphate (UMP). When 4-thiouracil is provided as a TgUPRT substrate, the resultant product is 4-thiouridine monophosphate which can, ultimately, be incorporated into RNA. Thio-substituted nucleotides are not a natural component of nucleic acids and are readily tagged, detected, and purified with commercially available reagents. Thus, one can do pulse/chase experiments to measure synthesis and decay rates and/or use cell-specific expression of the TgUPRT to tag only RNA synthesized in a given cell type. This chapter updates the original RABT protocol (1) and addresses methodological details associated with RABT that were beyond the scope or space allotment of the initial report.

Amplification of 4'-thioDNA and its utility as a shRNA expression system.

In this presentation, we discuss PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside triphosphate (sdNTPs). The amplified 4'-thioDNA acted as templates for not only in vitro transcription by T7 RNA polymerase, but also transcription in cells by RNA polymerase III. Accordingly, we succeeded to inhibit gene expression in cells by transfection of 4'-thioDNA that expresses a short-hairpin RNA (shRNA).

Microarray analysis of newly synthesized RNA in cells and animals.

Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.

Bcl-2 antisense oligonucleotide overcomes resistance to E1A gene therapy in a low HER2-expressing ovarian cancer xenograft model.

We are currently conducting clinical trials of E1A gene therapy for patients with ovarian cancer. The adenovirus type 5 E1A gene suppresses growth of ovarian cancer cells that overexpress HER-2/neu (HER2) and growth of some--but not all--that express low HER2. In HER2-overexpressing cells, suppression by E1A is predominantly by down-regulation of HER2, but the mechanism in low HER2-expressing cells is not fully understood. The adenoviral E1B protein has sequential and functional homology to Bcl-2 and prolongs the viability of adenovirus host cells by inhibiting E1A-induced apoptosis. Bcl-2 is overexpressed in ovarian cancer and participates in chemoresistance; we hypothesized that Bcl-2 inhibits E1A-induced apoptosis leading to resistance to E1A gene therapy. E1A suppressed colony formation of ovarian cancer cells that express low levels of Bcl-2 and HER2 (OVCAR-3 and OVCA 433), but enhanced colony formation in low HER2-, high Bcl-2-expressing ovarian cancer cells (2774 and HEY). Treating 2774 or HEY cells with antisense oligonucleotide Bcl-2 (Bcl-2-ASO) did not reduce cell viability. E1A combined with Bcl-2-ASO led to significant decreases in cell viability resulting from increased apoptosis relative to cells treated with E1A alone (P < 0.05). The increase in apoptosis was partly due to cytochrome c release and subsequently caspase-9 activation by Bcl-2-ASO. Finally, in an ovarian cancer xenograft model, treatment with Bcl-2-ASO did not prolong survival, but E1A plus Bcl-2-ASO did (P < 0.001). In conclusion, ovarian tumors overexpressing Bcl-2 may not respond well to E1A gene therapy, but treatment with a combination of E1A and Bcl-2-ASO may overcome this resistance.

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo.

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium.

Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations. In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene. This site may be suppressed by peptidyl-tRNAProcmo5UGG (cmo(5)U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNAGlnmnm5s2UUG (mnm(5)s(2)U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site. We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS. Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm(5)s(2)U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S. enterica were reduced in the iscS51 mutant. In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level. After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm(5)s(2)U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNAGlnmnm5s2UUG due to mnm(5)s(2)U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mnmA) lacking only mnm(5)s(2)U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm(5)s(2)U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant.

Effect of methotrexate polyglutamates on thioguanine nucleotide concentrations during continuation therapy of acute lymphoblastic leukemia with mercaptopurine.

Methotrexate is widely administered with mercaptopurine, a prodrug requiring activation into thioguanine nucleotides (TGN) to exert antileukemic effects. In vitro, methotrexate enhances TGN formation, but in vivo, such enhancement has yet to be demonstrated. We investigated whether TGN concentrations were related to methotrexate concentrations in children with acute lymphoblastic leukemia who received a weekly intravenous methotrexate (40 mg/m(2)) dose combined with daily mercaptopurine (75 mg/m(2)). A total of 141 erythrocyte TGN concentrations were measured with erythrocyte methotrexate polyglutamates (MTX-PG) concentrations in 87 patients. Average TGN concentrations ranged from 137 to 958 pmol/8 x 10(8) cells (median 389), average total MTX-PG concentrations (MTX- PG(1-7)) from 0.60 to 97.7 pmol/10(9)cells (median 29), and average long chain polyglutamate concentrations (MTX-PG(5-7)) from 0 to 8.35 pmol/10(9) cells (median 2.43). Higher TGN concentrations correlated with higher MTX-PG(5-7) concentrations (P = 0.002). These data support the practice of administering methotrexate with mercaptopurine during continuation therapy of acute lymphoblastic leukemia.

[In vitro antiviral activity of antisense oligonucleotides against influenza virus].

For developing of antisense oligonucleotides as potential antiviral therapeutic agents against influenza A virus, phosphorothioate oligodeoxynucleotides(PS-ODN) targeted to part to the 3' and 5' end sequences which are common to the eight RNAs of type A influenza virus were synthesized. The in vitro cytotoxicity of these PS-ODNs was assayed and then antiviral activity of these PS-ODNs was evaluated by using cultured MDCK cells infected by A/JingFang/86-1(H1N1). It was found that(1) No in vitro cytotoxicity was seen when concentration of PS-ODN was up to 50 mumol/L. (2) Oligonucleotides(IV4 #) complementary to 5' terminal conserved sequences and ODN(IV6 #, IV7 #)composed by 3'/5' terminal conserved sense sequences or antisense sequences all exhibited the most potent antiviral activity. IV4 # ODN exhibited significant antiviral activity with reduced hemegglutination titer about 50% at concentration of 1 mumol/L, and over 70% at 10 mumol/L. The antiviral activity of IV4 # was in a dose- and sequence-dependent manner. (3) IV4 # ODN showed significant antiviral activity not only against H1N1 subtype strains of influenza virus, but also against H3N2 subtype strains. (4) The multiplicity of infection(MOI) of influenza virus had effects on antiviral activity of IV4 # ODN. IV4 # ODN showed better dose-dependent antiviral activity when there was lower MOI. These results suggested the the 3'/5' terminal sequences of influenza A virus could be used as a target for designing antiviral oligonucleotides against influenza A virus.

Biochemical basis of the prevention of 6-thiopurine toxicity by the nucleobases, hypoxanthine and adenine.

Co-incubation of human leukemia cell lines with naturally occurring nucleobases (hypoxanthine or adenine) significantly prevented the cytotoxic activity of 6-thiopurines. Extracellular hypoxanthine decreased the transport of 6-mercaptopurine into cells, but adenine had no significant effect. However, intracellular thioinosine monophosphate accumulation in the presence of 10 microM, 6-mercaptopurine was reduced to below 1% or 10% of that of the controls when 50 microM hypoxanthine or adenine was added, respectively. Finally, in adenine phosphoribosyl transferase deficient mutants, adenine provided no protective effect against 6-thiopurines, whereas hypoxanthine retained its modulating activity. These data suggest that the nucleobases compete with 6-thiopurines for the ribose-phosphate donor, 5'-phosphoribosyl-1-pyrophosphate, thus preventing the formation of active metabolites of 6-thiopurines.

2',5'-Oligoadenylates chiral at phosphorus: enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioate trimer and tetramer analogues synthesized from (SP)-ATP alpha S.

The enzymatic synthesis and characterization of (RP)-2',5'-AMPS trimer and tetramer (SP)-5'-O-(1-thiotriphosphates) from chirally substituted (SP)-[alpha-35S]ATP alpha S by 2',5'-oligoadenylate synthetase from interferon-treated L cell extracts are described. The (RP)-ATP alpha S isomer is not a substrate for the synthetase. The identification of the trimer and tetramer analogues (molar ratio 70:30) was accomplished by high-performance liquid chromatography and subsequent separation by charge using DEAE-cellulose thin-layer chromatography. The digestion of the analogue by snake venom phosphodiesterase I (SVPD) to [alpha-35S]ATP alpha S and [35S]AMPS but not by T2 RNase demonstrated the presence of the 2',5' linkage. The assignment of RP configuration of the 2',5'-phosphorothiodiester linkage was based on the highly specific stereoselectivity of SVPD for RP diastereomers [Burgers, P. M. J., & Eckstein, F. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4978-4800; Bryant, F. R., & Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Nelson, P. S., Bach, C. T., & Verheyden, J. P. H. (1984) J. Org. Chem. 49, 2314-2317]. This suggests that the synthesis of the phosphorothioate analogues proceeded via inversion of configuration at the chiral phosphorus of (SP)-ATP alpha S. The putative (RP)-2',5'-AMPS tetramer (SP)-5'-O-(1-thiotriphosphate) displaced the 2',5'-p3A4[32P]pCp analogue from 2',5'-oligoadenylate-dependent endonuclease 5 times more efficiently than did equimolar concentrations of authentic 2',5'-adenylate tetramer triphosphate. Furthermore, in studies using the calcium phosphate coprecipitation technique, the 2',5'-phosphorothioate trimer and tetramer analogues inhibited protein synthesis better than did 2',5'-adenylate trimer and tetramer triphosphates.(ABSTRACT TRUNCATED AT 250 WORDS)

Assay of thioinosinic acid, an active metabolite of azathioprine, in human lymphocytes.

1 A specific assay for the measurement of thioinosinic acid, in human lymphocytes, has been developed with a sensitivity of 50 ng of thioinosinic acid per 5 x 10(6) lymphocytes. 2 Thioinosinic acid is precipitated from purified lymphocytes as the lanthanum salt. Acid hydrolysis results in the formation of 6-mercaptopurine which, when converted into its phenyl mercury derivative, can be easily extracted into toluene. Back-extraction of the toluene layer with 0.1N HCl regenerates 6-mercaptopurine which is then assayed fluorometrically. 3 Blood samples were taken from renal transplant recipients 3 h after an oral dose of 50 mg azathioprine. The results from 5 patients gave a range of 54 to 173 ng of thioinosinic acid per 5 x 10(6) lymphocytes, with a mean of 110 ng. 4 In an in vitro incubation of azathioprine, 1mM with fresh human blood, 160 and 180 ng of thioinosinic acid per 5 x 10(6) lymphocytes was formed after 0.5 h and 5 h respectively. 5 The assay is suitable for the study of the kinetics of thioinosinic acid formation in lymphocytes of patients with kidney transplants. It could also prove useful in the study of thioinosinic acid formation in leukaemia patients undergoing 6-mercaptopurine treatment.

Polyribonucleotides containing thiopurines. Synthesis and properties of poly(1-methyl-6-thioguanylic acid).

The synthesis of 1-methyl-6-thioguanosine 5'-diphosphate and its conversion to poly(1-methyl-6-thioguanylic acid) by means of polynucleotide phosphorylase are described. The polymer exhibited cooperative behavior (Tm = 294 K in the absence of added NaCl) characteristic of a highly stacked single-stranded helical array. In a high salt environment (0.5 M NaCl) the melting was much less cooperative and gave a higher Tm (313 K); this is suggestive of interstrand aggregation involving hydrogen bonding. The polynucleotide exhibited a remarkably high pKa (6.2) compared to that of the mononucleotide (2.6), and formed a very stable acid structure (Tm = 356 K in 50% ethylene glycol). Comparisons with poly(1-methyl-6-thioinosinic acid) and poly(6-thioguanylic acid) establish that both the 2-amino group and the 1-methyl group are required for the formation of the stable acid structure.