Deoxyinosine Residues Research Articles

In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V.

Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5' end. Treatment of the PCR products with endonuclease V generates 3' overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.

Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.

A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes.

BackgroundDetection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous.MethodsAssays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput.Results169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays.ConclusionsThis is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.

Electrochemical genotyping and detection of single-nucleotide polymorphisms based on junction-probe containing 2′-deoxyinosine

A novel electrochemical biosensor for the genotyping/detection of single-nucleotide polymorphisms (SNPs) of trace oral-cancer salivary DNA based on junction probes containing 2'-deoxyinosine (dI) residues is described.

Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene.

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.

Application of probes having 2′-deoxyinosine for typing of single nucleotide polymorphisms (SNPs) using DNA microarray

The principle of hybridizing oligonucleotides is employed with single nucleotide polymorphisms (SNPs) typing, but that typing frequently failed. We investigated the SNPs typing of multi-alleles on a chip. Two bases adjacent to the SNPs on the immobilized oligonucleotide probes were substituted for the 2′-deoxyinosine (dI) residues. The biotinylated probes containing the partial sequence of CYP2C9, CYP2C19, GLC1A and ApoE alleles were applied to the streptavidin-embedded plasma-polymerized (PP) DNA microarrays. For the probes containing dI, up to a 2–5-fold difference in the match to a mismatch oligonucleotide fluorescence intensity was found as the probes without dI were 1.5-fold or less. According to the substitution for dI, the hybridization accuracy was enhanced, and then several single-base mismatches were detected on a chip.

Non-canonical hydrogen bonds in genetic information flow

Non-Watson-Crick-type hydrogen bonds involving minor nucleosides such as inosine in anticodon loops of transfer RNA are found in nucleic acids. Inosine contains hypoxanthine as a nucleobase and can form base pairs with various bases in the genetic decoding process. This property was applied to cloning complementary DNA by using oligodeoxyribonucleotide primers which contain deoxyinosine residues. Hypoxanthine is generated by deamination of adenine, one of the major nucleobases in DNA and RNA. Formation of unusual hydrogen bonds derived from damaged nucleobases in nucleic acids is thought to be main cause of disorders in genetic information flow. Mutagenesis and carcinogenesis of damaged bases, including hypoxanthine, 7, 8-dihydro-8-oxoguanine (8-oxoguanine) and pyrimidine photo-dimers in c-Ha-ras genes, were investigated by using synthetic genes containing modified nucleotides. The mode of recognition of unusual base pairing between a thymine photo-dimer and adenine by a repair enzyme was studied by X-ray analysis. (Communicated by Takashi SUGIMURA, M.J.A.)

Duplex Stabilizing Effect and Nuclease Resistant Property of Novel Oligonucleotides Containing C-2 Branched Polyamine-Bearing Deoxyinosine Derivative

Abstract Novel oligodeoxyribonucleotides containing a C-2 branched polyamine-bearing deoxyinosine moiety at either inside-region or the 5′-terminus of the sequence exhibited enhanced nuclease resistant property. Among them, only the oligomers having the modified deoxyinosine residue at inside-region brought about duplex stabilizing effect upon mixing with the complements.

Molecular characterization of human enteroviruses in clinical samples: Comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products

Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.

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.

Serotype-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy.

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

Interaction of Deoxyinosine 3′-Endonuclease from Escherichia coli with DNA Containing Deoxyinosine

By using a band mobility shift assay, deoxyinosine 3'-endonuclease, an Escherichia coli enzyme which recognizes deoxyinosine, AP site, urea residue, and base mismatches in DNA, was shown to bind tightly to deoxyinosine-containing oligonucleotide duplexes. Two distinct protein-DNA complexes were observed, the faster migrating complex (complex I, Kd = 4 x 10(-9) M) contained one molecule of deoxyinosine 3'-endonuclease, while the slower migrating complex (complex II, Kd = 4 x 10(-7) M) contained two molecules of the protein bound to every molecule of duplex DNA. The endonucleolytic activity of deoxyinosine 3'-endonuclease paralleled the formation of the complex I. Interestingly, deoxyinosine 3'-endonuclease exhibited similar affinities for both the substrate and the nicked duplex product and thus remained bound to the DNA after the cleavage reaction. The formation of a stable complex required the presence of a duplex structure 5' to the deoxyinosine residue. DNase I footprinting revealed that deoxyinosine 3'-endonuclease protected 4-5 nucleotides 5' to the deoxyinosine, and when complex II was formed, at least 13 nucleotides 3' to deoxyinosine were protected. Based on these results, a model is proposed for the interaction of deoxyinosine 3'-endonuclease with DNA containing deoxyinosine.

Multinuclear nuclear magnetic resonance studies of Na cation-stabilized complex formed by d(G-G-T-T-T-T-C-G-G) in solution: Implications for G-tetrad structures

There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment and report on multinuclear 1H, 31P and 13C nuclear magnetic resonance studies on the d(G 2T 4CG 2) deoxynonanucleotide with Na cation as counterion in aqueous solution at low temperature. This sequence forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation (see the paper on d(G 2T 5G 2) describing optical and calorimetric measurements by Jin, R., Breslauer, K. J., Jones, R. A. & Gaffney, B. L. (1990), Science, 250, 543–546). The four narrow exchangeable protons detected between 11.5 and 12.0 parts per million (p.p.m.), which are common to the d(G 2T 4CG 2) deoxynonanucleotide and the d(G 2TCG 2) deoxyhexanucleotide sequences, are assigned to deoxyguanosine imino protons hydrogen-bonded to carbonyl acceptor groups. These narrow imino protons are not detected for d(IGN 5IG) and d(I 2N 5G 2), where two deoxyguanosine residues are replaced by two deoxyinosine residues in the deoxynonanucleotide sequences. This implies that the 2-amino protons of deoxyguanosine must also participate in hydrogen bond formation and stabilize the structured conformation of d(G 2T 4CG 2) in Na cation-containing solution. We have completely assigned the base and sugar H1′, H2′,2 , ̋ H3′, and H4′ protons of the d(G 2T 4CG 2) oligomer following analysis of two-dimensional nuclear Overhauser enhancement spectroscopy and two-dimensional correlated spectroscopy data sets in 0.1 m-NaCl, 10 m m-sodium phosphate, 2H 2O solution at 0 °C. The relative magnitude of the nuclear Overhauser enhancements (NOEs) between the base H8 and its own sugar H1′ protons of individual deoxyguanosine residues establishes that G1 and G8 adopt syn orientations while G2 and G9 adopt anti orientations about the glycosidic bond in the d(G1-G2-T3-T4-T5-T6-C7-G8-G9) sequence in both Na and K cation-containing aqueous solution. Consequently, any structure proposed for the tetramolecular complex of d(G 2T 4CG 2) must exhibit alternating G( syn) and G( anti) glycosidic torsion angles within each strand. The directionality and magnitude of the observed NOEs are consistent with the G( syn)-G( anti) steps adopting right-handed helical conformations in solution. We also note that the H8 protons of G1 and G8 (7.35 to 7·45 p.p.m.) in a syn alignment are shifted significantly upfield from the H8 protons of G2 and G9 (8·0 to 8·3 p.p.m.) in an anti alignment. Two-dimensional proton-detected heteronuclear ( 1H 31P and 1H 13C) spectra have been recorded at natural abundance on the d(G 2T 4CG 2) sequence in aqueous solution. The phosphorus resonances are dispersed over 1·5 p.p.m. with the G2-T3 and T6-C7 phosphates shifted to low field and the T4-T5 phosphate shifted to high field of the unperturbed spectral region. These results suggest that the pyrimidine-rich T 4C segment in d(G 2T 4CG 2) is involved either in hairpin loop or in hairpin bulge formation. The base and sugar carbon resonances have been assigned and we note that the base, C-1′, C-3′ and C-4′ carbons of G1 and G8 in a syn alignment resonate to low field of the corresponding carbon atoms of G2 and G9, which adopt an anti alignment. The nuclear magnetic resonance results are discussed in relation to proposed models for G-tetrad alignments in G-quadruplex structures.

Preferential recognition of I·T base-pairs in the initiation of excision-repair by hypoxanthine-DNA glycosylase

Double-stranded synthetic oligonucleotides with a centrally located dIMP residue in a 5'-32P-labeled strand were employed as substrates for hypoxanthine-DNA glycosylase. The enzyme activity was monitored by the generation of a piperidine-sensitive site in the labeled oligonucleotide. The enzyme was purified approximately 5000-fold from calf thymus. The purified enzyme removed efficiently a hypoxanthine base residue from an I.T base pair, but 15-20 times more slowly from an I.C base pair. Similar results were obtained with oligonucleotides in which the deoxyinosine residue was placed in different surrounding nucleotide sequences. The enzyme had no detectable activity on mismatched G.T, A.G or A.C base pairs. The data indicate that hypoxanthine-DNA glycosylase participates in the repair of deaminated adenine residues in DNA.

Identification of I:A mismatch base-pairing structure in DNA.

Deoxyoligonucleotides containing deoxyinosine residues at positions corresponding to ambiguous nucleotides derived from an amino acid sequence have been successfully used as hybridization probes. It is assumed that the hypoxanthine residue can make base pairs with multiple bases. In order to obtain direct evidence for I:A base-pairing, a self-complementary deoxyoligonucleotide, d(G-G-I-A-C-C), was synthesized and its properties were examined by NMR spectroscopy. Three hydrogen-bonded imino proton resonances are observed at low temperatures in H2O suggesting the formation of a self-duplex with complete base pairing. Nuclear Overhauser effect (NOE) experiments showed that a signal at 15.1 ppm originated from the imino proton (H1) of the dI residue (I3) which is hydrogen-bonded to the dA residue (A4). Both the I3 and A4 residues were assumed to have taken an anti glycosidic conformation since irradiating the H1 of I3 gave NOEs both to its own H2 and to that of A4, an NOE also being observed between the H2 protons of I3 and A4. Comparison of the 31P NMR spectra of d(G-G-I-A-C-C) and d(G-G-I-C-C-C) showed the backbone structure of d(G-G-I-A-C-C) to have been disturbed by the presence of purine:purine base pairs in the middle of the hexamer duplex.

An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions.

Two deoxyoligonucleotide probes (23-mer and 26-mer) carrying deoxyinosine residues (I) at positions corresponding to ambiguous nucleotides derived from amino acid sequence have been synthesized by the phosphotriester method using a polymer support. The 23-mer and 26-mer corresponded to the mRNA for 8 amino acids from gastrin and 9 amino acids from cholecystokinin, respectively. The dIs have been used where the base in the third position of the amino acid codon is ambiguous. These deoxyoligonucleotides were used as probes for hybridization with colonies containing the corresponding cDNAs or genes. The hybrid formed between a gastrin clone and the 23-mer that harbors 5 dIs was dissociated at 50-55 degrees C, suggesting that deoxyinosine did not significantly effect the stabilization or destabilization of the DNA duplex. A similar result was obtained using the 26-mer that contains 5 dIs and a phage clone DNA of the cholecystokinin gene. Thus oligonucleotide probes with deoxyinosine residues at ambiguous points seem to be useful as hybridization probes for cloning genes for proteins containing amino acids with degenerate codons.

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.

3′-Deoxyinosine as an anti-leishmanial agent: The metabolism and cytotoxic effects of 3′-deoxyinosine in [formula omitted] promastigotes

3′-Deoxyinosine is a potent inhibitor for growth of the promastigote form of Leishmania tropica . In culture, EC 50 value is 4.43 × 10 −7 M for the promastigote. On the other hand, it is less toxic towards mouse mammary tumor FM3A cells: EC 50 value is 1.25 × 10 −4 M . 3′-Deoxyinosine is metabolized by Leishmania promastigote to give 3′-deoxyinosine-5′-monophosphate and 3′-deoxyadenosine(cordycepin)-5′-mono-, di-, and triphosphates. This metabolic conversion provides a mechanism for the parasite-selective toxicity of 3′-deoxyinosine: Leishmania can aminate the 6-position of 3′-deoxyinosine residue, thereby converting a less toxic nucleoside into the cordycepin nucleotides that are known to be highly toxic to cells.