Quantitative gas chromatography mass spectrometric analysis of 2'-deoxyinosine in tissue DNA

Several studies examining DNA deamination have published levels of 2'-deoxyinosine that illustrated a large variation between studies. Most of them are the result of artifactual DNA deamination that occurs during the process of sample preparation, particularly acid hydrolysis. This protocol for measurement of 2'-deoxyinosine describes the use of nuclease P1 and alkaline phosphatase to achieve release of nucleosides from DNA, followed by HPLC prepurification with subsequent gas chromatography-mass spectrometry analysis of the nucleosides. It has been used in the measurement of the levels of 2'-deoxyinosine in DNA of commercial sources and DNA from cells and animal tissues, and gives values ranging from 3 to 7 2'-deoxyinosine per 10(6) 2-deoxyadenosine. This protocol should take approximately 7 days to complete.     

A one-pot preparation of 1-benzyl-2'-deoxyinosine from ionized 2'-deoxyinosine

A simple and one step synthetic method for the formation of 1-benzyl-2'-deoxyinosine was developed by direct benzylation of ionized 2'-deoxyinosine. Treatment of 2'-deoxyinosine, in the presence of NaOH, with benzyl bromide in 2,2,2-trifluoroethanol (TFE) or N,N-dimethylaetamide (DMA) gave 1-benzyl-2'-deoxyinosine in 35% and 80% yields, respectively.     

Phosphoramidites of base-modified 2''-deoxyinosine isosteres and solid-phase synthesis of d(GCI*CGC) oligomers containing an ambiguous base.

Novel phosphoramidites (1,2) of appropriately protected 2'-deoxyinosine isosteres (I*) such as allopurinol 2'-deoxyribofuranoside (4a) and 7-deaza-2'-deoxyinosine (4b) have been synthesized. They were employed together with the phosphoramidite of 2'-deoxyinosine in solid-phase synthesis of d(GCI*CGC) hexamers (12a-d). From thermodynamic data of these alternating hexamers it was shown that allopurinol 2'-deoxyribofuranoside destabilizes such duplexes less strongly than 2'-deoxyinosine. Additionally, the phosphoramidite of 7-deaza-2'-deoxyinosine (2) exhibits an extraordinary stability of the N-glycosylic bond. Since the new phosphoramidites are structurally related to 2'-deoxyinosine, they can be used in the construction of hybridization probes containing an ambiguous base.     

Synthesis and enzymatic polymerisation of 5-amino-1-(2''-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide-5''- triphosphate.

The chemical synthesis of 5-amino-1-(2'-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide, referred to as dZ, and of its 5'-triphosphate derivative (dZTP), from 2'-deoxyinosine is described. The polymerisation of dZTP using terminal deoxynucleotidyltransferase to give a homopolymer is also presented.     

Potential artifacts in the measurement of DNA deamination

Attack on DNA by some reactive nitrogen species results in deamination of adenine and guanine, leading to the formation of hypoxanthine and xanthine, respectively. Published levels of these products in cellular DNA have varied widely. Although these two deamination products are often measured by GC-MS analysis, the procedure of acid hydrolysis to release DNA bases for derivatization poses a risk of artifactual deamination of the DNA. In this study, we demonstrated the artifactual formation of these two deamination products during acid hydrolysis and hence developed a method for detecting and measuring 2'-deoxyinosine, the nucleoside of hypoxanthine. Our assay for 2'-deoxyinosine employs nuclease P1 and alkaline phosphatase to achieve release of the nucleosides from DNA, followed by HPLC prepurification with subsequent GC-MS analysis of the nucleosides. This assay detected an increase in the levels of 2'-deoxyinosine in DNA when commercial salmon testis DNA was treated with nitrous acid. We also used it to measure levels in various rat tissues of both normal and endotoxin-treated rats, but could not find increased 2'-deoxyinosine formation in tissues even though *NO production was substantially increased.     

Fluorescence properties and base pair stability of oligonucleotides containing 8-aza-7-deaza-2'-deoxyisoinosine or 2'-deoxyisoinosine

The fluorescence and the base pairing properties of 8-aza-7-deaza-2'-deoxyisoinosine (1) are described and compared with those of 2'-deoxyisoinosine (2). The corresponding phosphoramidites (11, 12) are synthesized using the diphenylcarbamoyl (DPC) residue for the 2-oxo group protection. The nucleosides 1 and 2 base pair with 2'-deoxy-5-methylisocytidine in DNA duplexes with antiparallel chain orientation and with 2'-deoxycytidine in a parallel DNA. These base pairs are less stable than the canonical dA-dT pair and that of 2'-deoxyinosine (4) with 2'-deoxycytidine. The fluorescence of the nucleosides 1 and 2 is quenched (approximately 95%) in duplex DNA. The residual fluorescence is used to determine the Tm-values, which are found to be the same as determined UV-spectrophotometrically.     

Polynucleotides. XLV Synthesis and properties of poly(2''-azido-2''-deoxyinosinic acid).

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality. In solutions of high Na+ ion concentration, poly (Iz) forms a multi-stranded complex and its Tm at 1.0M Na+ ion concentration was 43 degrees. Upon mixing with poly (C), poly (Iz) forms a 1:1 complex having a Tm lower than that of poly (I)-poly (C) complex in the same conditions. The effect of substitution at the 2'-position of the poly (I) strand was discussed in relation to the interferon-inducing activity.     

Human erythrocytic purine nucleoside phosphorylase: reaction with sugar-modified nucleoside substrates

The kinetic parameters (Km and Vmax) of sugar-modified analogues of inosine and guanosine have been determined with human erythrocytic purine nucleoside phosphorylase (PNP). Steric alterations at the 2' and 3' positions greatly lessened or abolished substrate activity. However, the 5'-deoxy- and 2',5'-dideoxy-beta-D-ribofuranosyl and the alpha-L-lyxosyl analogues were good substrates, indicating that the 5'-hydroxyl and the orientation of the 5'-hydroxy-methyl group are not important for binding. The sugar phosphate analogue, 5-deoxyribose 1-phosphate, was synthesized from 5'-deoxyinosine with immobilized PNP, and its presence was verified by using it in the enzymic synthesis of 5'-deoxyguanosine. The adenosine versions of the 5'-modified analogues were also found to react with adenosine deaminase, albeit at less than 1% of Vmax.     

Anopheles gambiae purine nucleoside phosphorylase: catalysis, structure, and inhibition

The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 10(7), and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 A to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP.DADMe-ImmH.PO4 complex than in HsPNP.DADMe-ImmH.SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.     

Synthesis and properties of oligodeoxynucleotides containing a C-2 polyamine-bearing deoxyguanosine residue as artificial ribonuclease.

Oligodeoxynucleotides containing a 2-fluoro-2'-deoxyinosine residue substituting normal 2'-deoxyguanosine residue were synthesized. Upon treating with ethanol solution of polyamine, the fluorine atom in the oligomers were readily substituted with the polyamine. The thermal stabilities of the duplexes consisted of the polyamine-bearing oligomers and their cDNAs as well as their RNA cleaving activity were investigated.     

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.     

Nucleoside analogue substitutions in the trinucleotide DNA template recognition sequence 3'-(CTG)-5' and their effects on the activity of bacteriophage T7 primase

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.     

Convenient synthesis of oligodeoxynucleotides containing 2'-deoxy-6-thioinosine

A facile synthesis of oligodeoxynucleotides (ODN) containing 2'-deoxy-6-thioinosine (dI6S) based on the convertible nucleoside O6-phenyl-2'-deoxyinosine is presented. After standard solid-phase DNA synthesis and removal of the cyanoethyl protecting groups with DBU treatment with aqueous sodium hydrogen sulfide introduces the sulfur functionality, deprotects the other nucleobases and cleaves the ODN from the solid support in a one-pot reaction. In addition, the extinction coefficient of 2'-deoxy-6-thioinosine is determined by enzymatic fragmentation of the resulting ODN in the presence of adenosine deaminase.     

Factors that contribute to efficient catalytic activity of a small Ca2+-dependent deoxyribozyme in relation to its RNA cleavage function

Recently, we found a small Ca(2+)-dependent deoxyribozyme (unmodified), d(GCCTGGCAG(1)G(2)C(3)T(4)A(5)C(6)A(7)A(8)C(9)G(10)A(11)GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA downward arrow UGCCAGGC) ( downward arrow denotes the RNA cleavage site), in the presence of Ca(2+) and developed a functional SPR sensor chip with this deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769-2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified deoxyribozyme, RNA cleavage reactions were carried out using 24 mutant deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2'-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm(5)C (5-methyl-2'-deoxycytosine), or d(P)C (5-propynyl-2'-deoxycytosine). The K(m) values (Michaelis constants) with the mutants that lacked N7 and O6 of G(1) and O6 of G(2) were 4.5 and 6.6 times that of the unmodified one, respectively. The k(cat) value (cleavage rate constant) with the mutants that lacked O6 of G(10) was 0.025 times that of the unmodified one. The results of UV melting curves, SPR kinetics, and CD spectra supported the quantitative idea that the catalytic activity of the unmodified form was achieved using Ca(2+). On the basis of these results, a preliminary model for two G(1) x A(8) and G(2) x A(7) mismatched base pairs such as G(anti) x A(anti) formed in the catalytic loop is proposed. The factor of 10 increase in the k(cat)/K(m) value of the mutant deoxyribozyme, which has C(9) substituted with d(P)C, suggests that the base stacking interaction between the substituted propynyl group in dC and the nearest-neighbor base grew stronger. Thus, substituting d(P)C for dC in the catalytic loop would be one of the best ways to increase the catalytic activity of the deoxyribozyme.     

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus)

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.     

Synthesis and properties of oligonucleotides containing 2''-deoxynebularine and 2''-deoxyxanthosine.

The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product.     

A study on the inhibition of adenosine deaminase

Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K(I) (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.     

Inosine and N1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNA(Ala) are major epitopes for anti-PL-12 myositis autoantibodies.

Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.     

A convenient synthesis of adenylyl-(2'----5')-adenylyl-(2'----5')-adenosine (2-5A core)

A simple synthesis of adenylyl-(2'----5')-adenylyl (2'----5')-adenosine (2-5A core) has been achieved on the basis of selective 3'-O-silylation of 5'-O-p-monomethoxytrityladenosine and chemo-selective formation of the 2'-5' internucleotide linkage using N-unprotected nucleosides.