Artificial Sandwich Base for Monitoring Single-Nucleobase Changes and Charge-Transfer Rates in DNA.

Developing a convenient method to discriminate among different types of DNA nucleotides within a target sequence of the human genome is extremely challenging. We herein report an artificial ferrocene-base (Fe-base) that was synthesized and incorporated into different loci of a DNA strand. The Fe-base replacement on a nucleobase can interact with DNA bases and efficiently discriminate among A, T, G, and C DNA bases of the complementary locus on the basis of interacting electrochemical properties. Furthermore, cyclic-voltammetry (CV) studies demonstrated the electrochemical stability of DNA strands incorporated with Fe-bases and the reversibility of the incorporation. Square-wave voltammetry (SWV) was performed to measure current changes between Fe-bases and bases of interest in the DNA duplex. The changes in the charge-transfer rates appeared to be correlated with the position of the Fe-base in the DNA strand, allowing rapid and efficient sensing of single-nucleobase changes in DNA and showing promise for the design of Fe-oligomer chip technology as a tool for DNA sequencing.

[Chemical constituents from flower buds of Lonicera japonica].

Eighteen compounds were isolated by a combination of various chromatographic techniques including column chromatography over macroporous resin, MCI gel, silica gel, and sephadex LH-20 and reversed-phase HPLC. Their structures were elucidated by spectroscopic data analysis as adinoside A (1), stryspinoside (2), benzyl alcohol beta-glucopyranoside (3), benzyl 2-o-beta-D-glucopyranosyl-2,6-dihydroxybenzoate (4) , gentisic acid 2-O-beta-D-glucopyranoside (5), eugenyl beta-D-glucopyranoside (6) , eugenyl-P-xylopyranosyl-(1-->6)-beta-glucopyranoside (7), (-)-lyoniresinol 9-O-fP-D-glucopyranoside (8) , (+)-lyoniresinol 9-O-beta-D-glucopyranoside (9) , apigenin-7-O-L-rhamnopyranoside (10), luteolin-3 '-O-L-rhamnoside (11) , ursolic acid (12) , beta-sitosteryl-3beta-glucopyranoside-6'-O-palmitate (13), abscisic acid (14), guanosine (15), 5-methyluracil (16), trans-cinnamic acid (17), and 4-hydroxybenzaldehyde(18). These compounds were obtained from this plant for the first time.

[Determination of nucleosides in Rhizoma Pinelliae by high performance liquid chromatography].

A high performance liquid chromatographic (HPLC) method was established to determine nucleosides in Rhizoma Pinelliae, which is a dried stem tuber of Pinellia pedatisecta Schott in Pinellia plant belonging to Araceae family and has multiple efficiencies about down-bear counterflow and check vomiting, eliminating dampness and phlegm, etc. The separation of adenine, hypoxanthine, xanthine, uridine, thymine, adenosine and guanosine was achieved on a Lichrospher C18 column (150 mm x 4.6 mm, 5 microm) with the detection at 254 nm and gradient elution by acetonitrile-water containing 0.1% formic acid as the mobile phase. The linear ranges were from 1.6 mg/L to 50 mg/L for adenine, hypoxanthine, xanthine, uridine and guanosine, while from 1.2 mg/L to 40 mg/L for thymine and adenosine with correlation coefficients above 0.999 5. The average recoveries were between 98.9% and 101.2% with the relative standard deviations below 3%. The results of methodological study demonstrated that the method met the requirements of the determination. The nucleosides in Rhizoma Pinelliae from different districts were determined. The method is convenient and accurate with good reproducibility and can be used to evaluate the quality of Rhizoma Pinelliae.

[Determination of thymine, hypoxanthine and uracil contents in medical pipefish by HPLC].

OBJECTIVE: To establish a RP-HPLC method for simultaneous determination of thymine, hypoxanthine and uracil contents in medical pipefish. METHOD: Samples were extracted with distilled water by ultrasonic wave and separated on Waters C18 column eluted with a mobile phase of 0.05 mol x L(-1) KFI2PO4-acetonitrile (97:3). The flow rate was 0.6 mL x min(-1). The determination wavelength was 260 nm and the column temperature was set at 40 degrees C. RESULT: The method had good linearity in the range of 0.033-0.660 (r = 0.9996), 0.620-12.400 (r = 0.9999), 0.048-0.960 microg (r = 0.9995), with average recoveries of 98.67% (RSD 1.6%), 99.03% (RSD 0.74%), 98.65% (RSD 1.8%), for thymine, hypoxanthine and uracil respectively. CONCLUSION: The simultaneous determination method of thymine, hypoxanthine and uracil in medical pipefish is established by RP-HPLC for the first time. The contents of the three constituents in different kinds of medical pipefish are significantly different. The method is simple, rapid and sensitive, and can be used for control the quality of medical pipefish.

Qualitative and quantitative analyses of nucleosides and nucleobases in Ganoderma spp. by HPLC-DAD-MS.

A high-performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS) analytical method was developed for detection of the nucleosides and nucleobases in two species of Lingzhi, the dried sporophore of Ganoderma lucidum and G. sinense. The method, combining advantages of both DAD and MS, was successfully used to qualitatively identify for six nucleosides namely, adenosine, cytidine, guanosine, inosine, thymidine, uridine and five nucleobases namely, adenine, guanine, hypoxanthine, thymine and uracil in Lingzhi samples. Quantitative analyses showed that uridine was the most abundant nucleoside in these Lingzhi samples and the contents of nine target analytes were found to be different in pileus and stipes of the fruiting bodies and among the different species of G. spp. The established method might apply as an alternative approach for the quality assessment of Lingzhi.

The different mobility of complementary strands depends on the proportion AC/GT.

The electrophoretic mobility of DNA fragments on denaturing polyacrylamide gel depends on various factors. One of these is the base composition of a single-stranded DNA (ssDNA). We confirmed that one strand and its complementary strand of polymerase chain reaction (PCR) products migrated with different mobilities in all alleles detected at 12 out of the 13 short tandem repeat (STR) loci studied. The mobility differences between complementary strands (MD) were also observed regardless of end-polishing with Pfu DNA polymerase. MD was therefore not influenced by additional nucleotides to each strand of the PCR products. We then analyzed the relation between MD and the base composition using one representative allele at each of the 13 loci. The results indicated that MD was affected by the adenine plus cytosine (AC) content in the ssDNA and was proportional to the values of the AC content divided by the guanine plus thymine (GT) content in the AC-rich strand (the proportion AC/GT). When the proportion AC/GT was well-balanced, MD decreased. The same tendency was observed even in the end-polished strands. In this study, the electrophoretic mobility of an ssDNA on denaturing polyacrylamide gels was shown to depend on the proportion AC/GT. Unless the same side of the PCR products is labelled in the context of a PCR-based STR typing, distinct alleles may be mistaken for identical ones because of the different mobility of complementary strands. Accordingly, the labelled strand should be described if only one strand of the PCR products is detected. When using an allelic ladder marker as a size standard, the labelled side should be unified between STR alleles and the allelic ladder alleles.

8-Methoxypsoralen photoadduct formation in complementary oligonucleotides containing a cross-linkable site.

The complete profile of 8-methoxypsoralen photoadduct formation in complementary oligonucleotides (5'-GAGTATGAG and 5'-CATAC) has been determined. Equimolar solutions of the oligonucleotides were irradiated at 4 degrees C in order to stabilize the mini-double helix. Photomodified oligonucleotides were separated by reversed phase chromatography on a Vydac C4 column. Photoadduct formation favored the 5'TAT site in the 9mer over the 5'ATA site in the 5mer by a factor of two. Split-dose studies showed that the monoadducts formed on GAGTATGAG were preferentially converted to cross-links by an additional UVA exposure.

Biased distribution of adenine and thymine in gene nucleotide sequences.

We analyzed occurrences of bases in 20,352 introns, exons of 25,574 protein-coding genes, and among the three codon positions in the protein-coding sequences. The nucleotide sequences originated from the whole spectrum of organisms from bacteria to primates. The analysis revealed the following: (1) In most exons, adenine dominates over thymine. In other words, adenine and thymine are distributed in an asymmetric way between the exon and the complementary strand, and the coding sequence is mostly located in the adenine-rich strand. (2) Thymine dominates over adenine not only in the strand complementary to the exon but also in introns. (3) A general bias is further revealed in the distribution of adenine and thymine among the three codon positions in the exons, where adenine dominates over thymine in the second and mainly the first codon position while the reverse holds in the third codon position. The product (A1/T1)x(A2/T2)x(T3/A3) is smaller than one in only a few analyzed genes.

Direct radiation action of heavy ions on DNA as studied by ESR-spectroscopy.

The effects of heavy ions on the mechanisms of free radical formation in DNA-constituents as compared to low-LET irradiation are investigated by means of Electron Spin Resonance (ESR) spectroscopy. Dose-yield curves were measured at low (T < 100 K) and ambient temperatures in order to obtain the G-value, that is number of radicals formed per 100 eV absorbed energy. These G-values show a characteristic LET-dependence and are one to two orders of magnitude lower than for low-LET irradiation. Measurements on 2'Deoxycytidine at 300 K using combined heavy ion and X-ray irradiation methods suggested that this effect can be partially explained by a destruction of radicals during the irradiation.

A conserved and unique (AT)-rich segment in yeast mitochondrial DNA.

The mtDNA of the cytoplasmic petite mutant of yeast RD1A consists mainly of a perfect head-to-tail repetition of a known sequence of 66 consecutive AT and 2 GC base pairs. We have hybridized complementary RNA made on RD1A mtDNA with the mtDNAs of four different wild-type Saccharomyces strains that differ markedly in restriction fragmentation pattern. The tm's of the four heteroduplexes are identical to the tm of the homoduplex of RD1A mtDNA with complementary RNA of one repeat length. With all four wild-type mtDNAs this complementary RNA hybridizes mainly to a single restriction fragment of about 300 base pairs. This shows the conservation and individuality of at least one (AT)-rich segment in yeast mtDNA. The 300 base pair fragment has been mapped in the vicinity of the oxi-2 locus. The possible role of the (AT)-rich segment in the processing of the primary transcript of this region is discussed.

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi.

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.