Production of cis-syn thymine-thymine cyclobutane dimer oligonucleotide in the presence of acetone photosensitizer

cis-syn Cyclobutane pyrimidine dimer (CPD) oligonucleotide was produced by UV irradiation in the presence of acetone photosensitizer. Acetone could enhance the productivity but evidently induced the photocleavage of oligonucleotide under a long time irradiation. A statistical approach of orthogonal design was applied to optimize the preparation condition for the production of the modified oligonucleotide. Optimal conditions for maximal cis-syn CPD oligonucleotide productivity were determined based on three factors: acetone concentration, initial oligonucleotide concentration, and irradiation time at several different levels. The optimal modified oligonucleotide that this optimization could produce was 32.7%. Through analysis of 20% polyacrylamide gel electrophoresis, it was found that modified oligonucleotide migrated slightly more slowly than the parent oligonucleotide. The photoreactivation of cis-syn thymine-thymine dimer oligonucleotide displayed the selectivity of the substrate specificity of DNA photolyase with high-performance liquid chromatography (HPLC) analysis.     

Acid binding and detritylation during oligonucleotide synthesis.

Under the conditions normally used for detritylation in oligonucleotide synthesis, the haloacetic acid binds strongly to the oligonucleotide. Acetonitrile also forms a complex with the deblocking acid, in competition with the oligonucleotide, and drastically slows detritylation. Incomplete removal of acetonitrile during the deblock step may slow the kinetics enough to result in incomplete detritylation of the oligonucleotide. Acid binding to the growing oligonucleotide causes striking chromatographic effects in the presence of high oligonucleotide mass densities. In packed-bed column reactors, at low linear velocities, the acid binding almost completely depletes free acid from the deblocking solution. This results in an advancing zone within which the oligonucleotide is saturated with acid. Detritylation occurs mostly in a narrow band at the front of the advancing saturated zone. Increasing the DCA concentration in order to achieve quick saturation can give faster and more complete detritylation while minimizing the exposure time of the oligonucleotide to acid.     

Synthesis and properties of oligonucleotide derivative with P(V) porphyrin

Oligonucleotide derivatives with a P(V)tetraphenylporphyrin at the internucleotidic phosphodiester linkage were synthesized. Their interactions with the complementary oligonucleotide and pyrene-labeled oligonucleotide were investigated. Fluorescence from the porphyrin moiety was strongly quenched by the addition of the pyrene-labeled oligonucleotide and the template oligonucleotide.     

Specific oligonucleotide invasion into an end of a DNA duplex

A new phenomenon was described: a double-stranded DNA fragment interacted with a single-stranded oligonucleotide complementary to the terminal region of one strand of the duplex to yield a complex with oligonucleotide invasion. Generation of Holliday junctions by homologous linear DNA fragments was less efficient in the presence of single-stranded oligonucleotides complementary to duplex ends. The effect depended on the oligonucleotide concentration, size, and complementarity to a duplex strand. Sequence-specific complexes with single strand invasion were detected in mixtures containing radiolabeled oligonucleotides and duplexes. A single-stranded oligonucleotide invaded a duplex even when its concentration was far lower than the duplex concentration. Complexes with single strand invasion were analyzed by chemical cleavage of noncanonical base pairs. Analysis showed that an oligonucleotide interacts with the complementary region of one strand of the duplex, gradually displacing the other strand. The extent of oligonucleotide invasion into the duplex considerably varied. Oligonucleotide invasion into duplexes became more efficient with increasing oligonucleotide size.     

Generation of amplifiable genome-specific oligonucleotide probes and libraries

Here we describe a process for the generation of oligonucleotide libraries representative of a given nucleic acid. Starting from at random pool of DNA oligonucleotides, the technique selects only those that hybridize to the nucleic acid template. This selection yields a highly specific library that represents an oligonucleotide image of the chosen template. The novel quality of this approach is the generation of amplifiable oligonucleotide probes that are of unique length and are easily subjected to differential selection. Here we apply this technique to produce different genomic oligonucleotide libraries and show that these genomic oligonucleotide libraries do not cross-hybridize. Differential selection of these genomic oligonucleotide libraries produces oligonucleotides that can be used in the identification, characterzation, and isolation of nucleic acids.     

Improved hybridization assays employing tailed oligonucleotide probes: a direct comparison with 5'-end-labeled oligonucleotide probes and nick-translated plasmid probes

Terminal deoxynucleotidyl transferase was used to add labeled dAMP residues to the 3' end of oligonucleotide probes that hybridize to the 5' end of the neomycin phosphotransferase II gene. Southern hybridization conditions were described in which the sensitivity per unit of exposure time was about 30-fold greater for the tailed probe as compared to the 5'-end-labeled probe. The tailed oligonucleotide probe had the sensitivity per unit of exposure time comparable to that of a nick-translated probe of high specific activity: in 3 h of autoradiographic exposure both easily detected an amount of target equivalent to a single-copy gene in 10 micrograms of human DNA. The thermal dissociation profiles of 5'-end-labeled and tailed oligonucleotide probes were virtually identical and the tailed oligonucleotide probe was as allele specific as the 5'-end-labeled oligonucleotide probe. The useful lifetime of a 32P-tailed probe was about 1-2 weeks. Finally, by adding 50 35S-labeled nucleotides to the 3' end, we prepared a stable oligonucleotide probe with a sensitivity per unit of exposure time comparable to that of the unstable 5'-32P-labeled oligonucleotide probe.     

Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates.

Oligodeoxynucleotide-containing phosphorothioate backbones have been used to regulate viral as well as cellular gene expression. The studies carried out in tissue culture have shown promising results on the use of oligonucleotide phosphorothioates as antiviral agents and, at present, study is underway to develop these oligonucleotide analogues as chemotherapeutic agents. To analyze and purify oligonucleotide analogues, high-performance liquid chromatography using weak anion exchange column has been described. The separation of oligonucleotide phosphorothioate is found to be length dependent.     

Detection of dairy Leuconostoc strains using the polymerase chain reaction

This paper reports the design of a Leuconostoc -specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc -specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc -specific oligonucleotide was used also as a specific hybridization probe.     

Evaluation of some properties of a phosphorodithioate oligodeoxyribonucleotide for antisense application.

An all phosphorodithioate oligodeoxyribonucleotide (PS2; 17-mer) complementary to the coding region of the rabbit beta-globin mRNA was compared with the normal (PO2) and phosphorothioate (POS) oligonucleotide of the same size and sequence with respect to physicochemical properties and antisense activity in cell-free systems. The melting temperature (Tm) of the PS2-cDNA duplex was reduced by 17 degrees C relative to the PO2-cDNA duplex, compared to 11 degrees C for the POS-cDNA duplex, suggesting a decreased stability of the duplex with an increasing sulfur substitution. Like the POS-derivative, the PS2 oligonucleotide is quite stable against exonucleases, but these modified oligonucleotides showed different stability towards endonucleases and also towards different sub-cellular fractions of MCF-7 cells. During in vitro protein binding studies, the PS2 oligonucleotide showed similar binding (10-20%) to that of the PO2 oligonucleotide, while the POS oligonucleotide bound 60%. In cell-free translation, the PS2 oligonucleotide produced slightly higher specific translation inhibition of rabbit beta-globin mRNA compared to that of the PO2 oligonucleotide, and this was true only at concentration below 2 mM. The POS-derivative, except at 10 mM concentration, always showed higher translation arrest of the rabbit beta-globin mRNA compared to that of the other two oligonucleotides. The present study suggests that the PS2 oligonucleotide offers very little advantage over the POS oligonucleotide for use as an antisense analog.     

寡核苷酸芯片技术用于检测过氧化物酶体活化物激活受体基因多态性

 通过寡核苷酸芯片技术检测PPARα基因Leu162Val、Val227Ala多态性和PPARγ Pro12Ala的基因多态性,建立一种快速、简便、准确的方法,为研究非酒精性脂肪性肝病的发病机制、临床诊断和治疗提供依据.收集人体外周血标本,提取DNA进行PCR扩增,设计相应的探针和引物,制备检测芯片,PCR产物与芯片杂交后,扫描芯片并分析结果.PCR产物进行测序验证.寡核苷酸芯片技术检测PPARα基因Leu162Val、Val227Ala多态性和PPARγ Pro12Ala基因多态性结果与测序结果一致.寡核苷酸芯片技术检测非酒精性脂肪性肝病(NAFLD)密切相关的PPAR基因多态性快速、准确,值得临床推广和应用.     

Introduction of statistical mutations into strictly defined areas of the genome using a modified oligonucleotide]

A method for inducing mutations in the short region of a gene is suggested. The method involves oligonucleotide modification by hydroxylamine derivative, in vitro enzymatic synthesis of double-stranded DNA using modified oligonucleotide as a primer and selection of mutant colonies using the starting unmodified oligonucleotide as a probe.     

A portable generic DNA bioassay system based on in situ oligonucleotide synthesis and hybridization detection

In this study, we present a portable and generic DNA bioassay system based on in situ oligonucleotide synthesis followed by hybridization based detection. The system include two main parts, an oligonucleotide synthesizer and a fluorescence detection system. The oligonucleotide synthesizer is based on microfluidic technology and capable of synthesizing any desired oligonucleotide which can be either used as a primer for PCR based detection (external) or a probe for hybridization based detection (integrated) of a target DNA analyte. The oligonucleotide sequence can be remotely sent to the system. The integrated fluorescence detection system is based on a photodiode to detect Texas Red fluorophore as low as 0.5 fmol. The complete system, integrating the oligonucleotide synthesizer and fluorescence detection system, was successfully used to distinguish DNA from two different bacteria strains. The presented generic portable instrument has the potential to detect any desired DNA target sequence in the field. Potential applications are for homeland security and fast responses to emerging bio-threats.     

R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide

F factor TraI is a helicase and a single-stranded DNA nuclease ("relaxase") essential for conjugative DNA transfer. A TraI domain containing relaxase activity, TraI36, was generated previously. Substituting Ala for Arg150 (R150A) of TraI36 reduces in vitro relaxase activity. The mutant has reduced affinity, relative to wild type, for a 3'-TAMRA-labeled 22-base single-stranded oligonucleotide. While both R150A and wild-type TraI36 bind oligonucleotide, only wild type increases steady-state fluorescence anisotropy of the labeled 22-base oligonucleotide upon binding. In contrast, binding by either protein increases steady-state anisotropy of a 3'-TAMRA-labeled 17-base oligonucleotide. Time-resolved intensity data for both oligonucleotides, bound and unbound, require three lifetimes for adequate fits, at least one more than the fluorophore alone. The preexponential amplitude for the longest lifetime increases upon binding. Time-resolved anisotropy data for both oligonucleotides, bound and unbound, require two rotational correlation times for adequate fits. The longer correlation time increases upon protein binding. Correlation times for the protein-bound 17-base oligonucleotide are similar for both proteins, with the longer correlation time in the range of molecular tumbling of the protein-DNA complex. In contrast, protein binding causes less dramatic increases in correlation times for the 22-base oligonucleotide relative to the 17-base oligonucleotide. Binding studies indicate that R150 contributes to recognition of bases immediately 3' to the DNA cleavage site, consistent with the apparent proximity of R150 and the 3' oligonucleotide end. Models in which the R150A substitution alters single-stranded DNA flexibility at the oligonucleotide 3' end or affects fluorophore-DNA or fluorophore-protein interactions are discussed.     

In vitro selection of hairpin ribozymes activated with short oligonucleotides

We have carried out an in vitro selection to obtain an allosteric hairpin ribozyme, which has cleavage activity in the presence of an exogenous short oligonucleotide as a regulator. Random sequences were inserted in a region corresponding to the hairpin loop of the ribozyme. After 12 rounds of selection, DNA templates were cloned. Of a total of 34 clones, 18 contained the same sequence, and the obtained hairpin ribozymes showed the cleavage activity specifically in the presence of the regulator oligonucleotide. All of the clones contained sequences complementary to the regulator oligonucleotide. The ribozymes with high cleavage activities gained characteristic hairpin loops at the random domain, which were similar to each other. In the absence of the oligonucleotide, the loop domain within the allosteric ribozyme probably forms a slipped hairpin loop, and the complementary sequence, with the regulator oligonucleotide located at the single stranded loop, would allow easy access of the oligonucleotide. The binding of the regulator oligonucleotide triggers a structural change of the hairpin loop to form an active conformation. Furthermore, we constructed an allosteric hammerhead ribozyme by introducing the characteristic hairpin loop. The modified hammerhead ribozyme was also changed to an allosteric ribozyme, which was activated by the addition of the regulator oligonucleotide. The characteristic hairpin loop, which was proved to be regulated by an exogenous oligonucleotide in this report, may be used to control RNA functions in various fields.     

Transfection protocol for antisense oligonucleotides affects uniformity of transfection in cell culture and efficiency of mRNA target reduction

In an effort to optimize the transfection of cell lines with antisense oligonucleotides, we examined cellular accumulation of a labeled oligonucleotide by flow cytometry. We were surprised to observe that a routinely used transfection protocol, a fixed lipid/oligonucleotide ratio, resulted in variable transfection efficiency depending on the concentration of oligonucleotide used. A significant population of cells, especially at lower doses of oligonucleotide and cationic lipid, were untransfected. We investigated lipid/oligonucleotide ratios, different lipid preparations, and different cell types and found that these variables did not alter the percentage of cells transfected at these lower doses of oligonucleotide. However, when lipid-oligonucleotide complexes were formed at the high dose and then diluted into a solution of lipid or a complex of lipid and unlabeled, negative control oligonucleotide, a constant percentage of cells was transfected. Under these conditions, mRNA target reduction dose-response curves were also shifted to lower doses. We hypothesize that poor transfection observed at a low concentration of lipid-oligonucleotide complex when diluted in medium is due to loss of active complexes, either by adsorption to the substrate or by changes in physical characteristics of complexes. By maintaining a constant lipid concentration, more consistent transfection was achieved.     

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.     

Intravenous administration of stabilized antisense lipid particles (SALP) leads to activation and expansion of liver natural killer cells

Stabilized antisense lipid particles (SALP) have been developed for the systemic delivery of oligonucleotides. The impact of intravenous SALP administration was measured with respect to activation of natural killer (NK) and NK1.1+ T (NKT) cells in the livers of immunocompetent mice. Treatment with a SALP containing a highly mitogenic oligonucleotide (INX-6295) generated an increase in NK cytolytic activity and cell number within the liver but did not appear to affect the number of hepatic NKT cells or their cytolytic activity. The same results were observed after intravenous administration of the mitogenic oligonucleotide alone. Interestingly, treatment with a SALP containing a weakly mitogenic oligonucleotide (INX-6300) also activated the liver NK cells, whereas the oligonucleotide alone was unable to elicit these effects. The NK stimulatory activity of a SALP containing INX-6300 required both lipid and oligonucleotide components. These results demonstrate that in addition to modifying the pharmacokinetics and biodistribution of intravenously administered oligonucleotides, SALP possess immunostimulatory activity independent of oligonucleotide mitogenicity, which can serve as an adjuvant to antisense therapies for cancer.     

T1 oligonucleotides that segregate with tropism and with properties of gp70 in recombinants between N- and B-tropic murine leukemia viruses.

We have analyzed large RNase T1-resistant oligonucleotides derived from the genomes of 16 recombinants between N- and B-tropic murine leukemia viruses of BALB/c. The parental viruses, designated SP-N and LP-B, differ in several phenotypic or biochemically defined properties: N- or B-tropism; XC plaque morphology, electrophoretic mobility of three virion proteins (p15, p30, and gp70); ability to induce GIX antigen on infected cells; presence of 6 to 8 (out of 36 to 38 analyzable) large T1 oligonucleotides. One SP-N-specific T1 oligonucleotide was inherited by all 16 N-tropic recombinants and, thus, appears to be linked to N-tropism. This oligonucleotide lies in the 5' third of the oligonucleotide map of SP-N. One LP-B-specific T1 oligonucleotide was inherited by all 11 recombinants whose gp70 has an electrophoretic mobility like that of LP-B gp70 and that, like LP-B, fail to induce GIX antigen. This oligonucleotide lies in the 3' third of the oligonucleotide map of LP-B.