DNA Synthesizer Research Articles

Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.

Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.

The Synthesis of N4‐(6‐Aminopyridin‐2‐yl)‐2′‐deoxycytidine for Recognizing the CG Base Pair at Neutral pH by Oligodeoxyribonucleotide‐Directed Triple Helix Formation

AbstractThe sequence‐specific recognition of double‐helical DNA by oligodeoxyribonucleotide‐directed triple helix (triplex) formation is limited mostly to purine tracts. To interrupt the purine tract in a target sequence, a non‐natural deoxyribonucleoside N4‐(6‐aminopyridin‐2‐yl)‐2′‐deoxycytidine (pC) was designed to interact with the C base in the CG base pair. The protected phosphoramidite synthon of pC was synthesized in seven steps and then was incorporated into an oligodeoxyribonucleotide by an automatic DNA synthesizer. Two 22‐mers, designated as C2 and P, with a common sequence of 5′‐d‐TmCTXTmCTTCTGTCTCCAGACAG were synthesized in this study. mC is 5‐methyl‐2′‐deoxycytidine and X is either 2′‐deoxycytidine (C) or pC for C2 and P, respectively. C2 is able to form a paper clip type triplex with one C · CG mismatched base triad in slightly acidic conditions but not at the neutral pH. On the other hand, P forms a stable triplex under both acidic and neutral conditions. This indicates that pC is able to form a pC · CG base triad in the triplex. Their physical properties were studied by UV thermal melting experiments and circular dichorism spectroscopy (CD). The thermal melting results imply that the pC · CG base triad is as stable as the C+ · GC triad at pH 6.0, and pC helps the triplex formation preferably at neutral to acidic pH. In addition to the hydrogen bonding interaction with the CG base pair, the hydrophobic interaction of pC may also play an important role in stabilizing the triplex formation of oligodeoxyribonucleotides. In the presence of spermine at either pH 5.0 or pH 6.0, the melting temperature of the third strand of P was elevated about 30 and 21 °C, respectively. Thus, spermine can enhance the stability of the triple‐helical structure.

Rapid esterification of nucleosides to solid-phase supports for oligonucleotide synthesis using uronium and phosphonium coupling reagents.

Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 micromol/g) and rapid coupling reactions (< or = 10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3'-succinyl or 3'-hydroquinone-O, O'-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3'-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.

Synthesis and enzymatic stability of oligonucleotides consisting of isonucleosides

Abstract

Reusable Solid-Phase Supports for Oligonucleotide Synthesis Using Hydroquinone-O,O'-diacetic Acid (“Q-Linker”)

Reusable solid-phase supports for large scale oligonucleotide synthesis have been prepared by converting amino derivatized supports into hydroxyl supports. Rapid nucleo side attachment, via a Q-linker arm, was automatically performed on the DNA synthesizer using HBTU and DMAP as the coupling reagents. All steps were suitable for automation and ∼ 1.5 h was required to prepare the supports for reuse. Up to twelve consecutive syntheses of a 20-mer phosphorothioate were performed on a synthesis column.

Genetic mapping of the thymoma susceptible locus, Tsr1, in BUF/Mna rats.

Genetic mapping of the thymoma susceptible locus, Tsr1, in BUF/Mna rats.

Syntheses and Properties of Novel Conformationally Restrained Nucleoside Analogues.

This account describes our syntheses and conformational analyses of the bicyclic nucleoside analogues, 3'-O, 4'-C-methyleneribonucleosides (4-BC), 3'-amino-3'- deoxy-3'-O, 4'-C-methyleneribonucleosides (aza 4-BC), 2'-O, 4'-C-methyleneribonucleosides (5-BC), and 3'-amino- and 3'-azido-3'deoxy-2'-O, 4'-C-methyleneribonucleosides (aza 5-BC). The nucleoside analogues (4-BC and 5-BC) were effectively introduced into oligonucleotides by using a DNA synthesizer. Furthermore, unprecedented hybridizing ability towards complementary RNA and DNA, potent triplex-forming ability, and sufficient enzymatic stability of these modified oligonucleotides were also confirmed. From these studies, we believe that these conformationally restrained nucleoside analogues are good candidates for practical antisense/antigene molecules.

A Negative Vitamin D Response DNA Element in the Human Parathyroid Hormone-related Peptide Gene Binds to Vitamin D Receptor Along with Ku Antigen to Mediate Negative Gene Regulation by Vitamin D

We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDREhPTHrP) homologous to a negative vitamin D response element in the human parathyroid hormone gene. It bound to vitamin D receptor (VDR) but not retinoic acid Xalpha receptor (RXRalpha) in the human T cell line MT2 cells. VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP. In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex. The nVDREhPTHrP-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation. On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D. These results indicate that nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.

Solid-phase synthesis of peptidomimetic oligomers with a phosphodiester backbone.

An unnatural biopolymer is described in which amino acid side-chains are presented along a negatively charged phosphodiester backbone. For this purpose, a series of phosphoramidite monomers was prepared from chiral 1,2-diols. These were efficiently converted into oligomers using standard coupling conditions on an automated DNA synthesizer.

Cloning and Expression of a Prokaryotic Enzyme, Arginine Deiminase, from a Primitive Eukaryote Giardia intestinalis

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Molecular Cloning and Functional Expression of Mannitol-1-phosphatase from the Apicomplexan Parasite Eimeria tenella

A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown to be essential for their catalytic activity. Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E. tenella. Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.

A fiber optic biosensor for fluorimetric detection of triple-helical DNA.

A fiber optic biosensor was used for the fluorimetric detection of T/AT triple-helical DNA formation. The surfaces of two sets of fused silica optical fibers were functionalized with hexaethylene oxide linkers from which decaadenylic acid oligonucleotides were grown in the 3'to 5'and 5'to 3'direction, respectively, using a DNA synthesizer. Fluorescence studies of hybridization showed unequivocal hybridization between oligomers immobilized on the fibers and complementary oligonucleotides from the solution phase, as detected by fluorescence from intercalated ethidium bromide. The complementary oligonucleotide, dT10, which was expected to Watson-Crick hybridize upon cooling the system below the duplex melting temperature ( T m), provided a fluorescence intensity with a negative temperature coefficient. Upon further cooling, to the point where the pyrimidine motif T*AT triple-helix formation occurred, a fluorescence intensity change with a positive temperature coefficient was observed. The reverse-Hoogsteen T.AT triplex, which is known to form with branched nucleic acids, provided a corresponding decrease in fluorescence intensity with decreasing temperature. Full analytical signal evolution was attainable in minutes.

FAP52, a Novel, SH3 Domain-containing Focal Adhesion Protein

Src-homology 3 (SH3) domain is a 60-70-amino acid motif present in a large variety of signal transduction and cytoskeletal proteins. We used reverse transcriptase-polymerase chain reaction with degenerate and specific primers and chicken brain mRNA to clone a cDNA that codes for a novel SH3 domain-containing protein. The sequence predicts a 448-amino acid polypeptide with a molecular mass of 51, 971 daltons. In the amino terminus, it shows a very high propensity for alpha-helicity, suggesting coiled-coil and possibly a higher order oligomeric arrangement. In the carboxyl terminus, there is a unique SH3 sequence. In Northern blotting, a major 3.7-kilobase and a minor 7.2-kilobase transcript was detected in most chicken tissues. In immunofluorescence microscopy and immunoelectron microscopy on cultured chicken fibroblasts, the protein was localized to focal adhesions in which it showed a distinct codistribution with the focal adhesion proteins vinculin, talin, and paxillin. Phosphoamino acid analysis showed that in cultured chicken heart fibroblasts, the protein contains phosphoserine, but no phosphothreonine or phosphotyrosine, and that the phosphorylation is not dependent on fibronectin. We propose this protein the name FAP52, for Focal Adhesion Protein of 52 kDa, and suggest that it forms part of the multimolecular complex constituting focal adhesion sites.

Gas-Phase DNA: Oligothymidine Ion Conformers

Gas-Phase DNA: Oligothymidine Ion Conformers

Hydroquinone-O,O'-diacetic acid ('Q-linker') as a replacement for succinyl and oxalyl linker arms in solid phase oligonucleotide synthesis.

When hydroquinone-O,Ooffiacetic acid is used as a linker arm in solid phase oligonucleotide synthesis, the time for NH4OH cleavage of oligodeoxy- or oligoribonucleotides is reduced to only 2 min. This allows increased productivity on automated DNA synthesizers without requiring any other modifications to existing reagents or synthesis and deprotection methods. The Q-linker may also be rapidly cleaved by milder reagents such as 5% NH4OH, potassium carbonate, anhydrous ammonia, t-butylamine or fluoride ion. However, the Q-linker is sufficiently stable for long-term storage at room temperature without degradation and no loss of material occurs during synthesis. The linker is also reasonably resistant to 20% piperidine/DMF, 0.5 M DBU/pyridine and 1:1 triethylamine/ethanol. The Q-linker can therefore serve as a general replacement for both succinyl and oxalyl linker arms.

Automated Chemical Synthesis of PNA and PNA-DNA Chimera on a Nucleic Acid Synthesizer

Automated chemical synthesis of PNA and PNA-DNA chimera on a DNA synthesizer using the monomethoxytrityl/acyl protecting group strategy is described.

Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. Implications for DNA synthesis and dimerization.

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the beta12-beta13 hairpin (residues 227-235). It has been proposed to play a role in aligning the 3'-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66(W229A)/51 and 23-fold for mutant p66(G231A)/51, and the maximal pre-steady-state rate of nucleotide incorporation, kpol, was reduced 27-fold for p66(W229A)/51 and 70-fold for p66(G231A)/51, respectively. Mutant p66(M230A)/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase.

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.

Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.

Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha2beta2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered alpha(II) subunit from human sources. The mRNA for the alpha(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (alpha(II))2beta2 tetramer was demonstrated in cultured human WI-38 and HT-1080 cells. The type II tetramer was found to represent about 30% of the total prolyl 4-hydroxylase in these cells and about 5-15% in various chick embryo tissues. The results of coexpression in insect cells argued strongly against the formation of a mixed alpha(I)alpha(II)beta2 tetramer. PDI/beta polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/beta polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source for a simple large scale purification of the recombinant enzyme. The properties of the purified human type II enzyme were very similar to those of the type I enzyme, but the Ki of the former for poly(L-proline) was about 200-1000 times that of the latter. In agreement with this, a minor difference, about 3-6-fold, was found between the two enzymes in the Km values for three peptide substrates. The existence of two forms of prolyl 4-hydroxylase in human cells raises the possibility that mutations in one enzyme form may not be lethal despite the central role of this enzyme in the synthesis of all collagens.

Nucleosides and nucleotides. 160. Synthesis of oligodeoxyribonucleotides containing 5-(N-aminoalkyl)carbamoyl-2'-deoxyuridines by a new postsynthetic modification method and their thermal stability and nuclease-resistance properties.

Heptadecadeoxynucleotides containing 5-(N-aminoethyl- or N-aminohexyl)carbamoyl-2'-deoxyuridines (E or H) were synthesized using a newly developed postsynthetic modification method. As a convertible nucleoside unit, 5-methoxycarbonyl-2'-deoxyuridine (1) was initially incorporated into oligodeoxynucleotides (ODNs) according to the phosphoramidite method at various positions using a DNA synthesizer. Fully protected ODNs attached to a solid support were treated with alkyldiamines such as ethylenediamine and 1,6-hexanediamine to give the above modified ODNs. The thermal stability, resistance toward nuclease digestion, and stability in fetal calf serum of the modified ODNs were studied. An increase in the number of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (H) in the ODNs was found to effectively stabilize duplex formation with both the corresponding complementary DNA and RNA and protect against nucleolytic hydrolysis by snake venom phosphodiesterase. In particular, the half-life of ODN 19, which contained four H residues, was about 162 h in the presence of the nuclease. Furthermore, 19 was also stable in medium containing 10% fetal calf serum with a t1/2 of about 48 h, while t1/2 for the corresponding unmodified ODN was 13 min.