Amide Protection in Oligodeoxynucleotide Synthesis

Abstract

Several O⁶ -protected deoxyguanosine- as well as O⁴ -protected thymidine-phosphoramidites were prepared according to the Mitsunobu reaction and Michael addition and were tested in a solid-phase automated DNA synthesizer.     

Synthesis of Circular Oligodeoxynucleotide Conjugates VIA Transient Abasic Sites

Abstract

New chemical ligation or cyclisation reactions, using high reactivity of abasic sites with amines, are reported for the synthesis of oligonucleotide clamps and singlestranded circular oligonucleotides. Thermal denaturation experiments show that these molecules display very high binding affinities for complementary DNA oligomer by forming triple-helical complexes.     

Synthesis and Cellular Uptake of Fluorescently Labeled Oligodeoxynucleotide Prodrugs

Abstract

In this paper we described the synthesis on solid support of 5′-fluorescein labeled dodecathymidylates having interucleoside phosphodiester or phosphorothioate linkages temporary masked with enzymolabile S-pivaloyl-2-thioethyl (tBuSATE) groups.     

Depurination of benzo[a]pyrene-diolepoxide treated DNA

Rat liver DNA was treated in vitro with benzo[a]pyrene-diolepoxide (BPDE), the ultimate carcinogenic metabolite derived from the polycyclic hydrocarbon benzo[a]pyrene. On incubation of the reacted DNA, apurinic sites developed which gave rise to strand breakage in alkaline solution. The reduction in molecular weight produced by these breaks was measured by analytical ultracentrifugation. In the case of anti-BPDE this depurination was shown to occur in two stages. The first was mainly due to attack at the 7-position of guanine, to yield an adduct which was lost from the DNA within a few hours. The second stage was due to much slower loss of the major N2-guanine adduct. The separated enantiomers, (+)- and (-)-anti-BPDE, and syn-BPDE all caused depurination to various extents. It is argued that although these processes are important in a study of the action of BPDE on DNA in vitro, their contribution to the biological activity of BPDE is probably negligible.     

Pharmacologic Disposition of a Phosphorothioate Oligodeoxynucleotide in Mice

Abstract

G3139, an 18mer phosphorothioate (S-labeled), was administered to mice by single i.v. bolus or continuous S.C. infusion. The latter schedule resulted in reduced liver metabolism and urinary excretion together with greater tissue accumulation, in particular to the liver, kidney and bone marrow, probably reflecting the dose received.     

Diamino-Antraquinone: A New Intercalating Agent. Synthesis and Linking to Oligodeoxynucleotide

Abstract

Phosphoramidite derivative of 1,4-diamino antraquinone was synthesized, characterized, and incorporated into oligonucleotides. Intercalative interaction between the dye and the nucleic acid was confirmed by CD spectroscopy.     

体外反搏降低心脏后负荷的机理及治疗过程的安全防护

介绍体外反搏降低心脏后负荷的机理,分析影响治疗安全的原因,提出反搏治疗的操作要点。     

Effect of Decreasing Growth Temperature on Cell Yield of Escherichia coli

Studies of the relationship between yield coefficient and growth rate, as affected by temperature of growth, in Escherichia coli have shown that, over a wide range of temperature, yield is relatively constant until the specific growth rate falls below about 0.2 hr(-1), at which point the yield begins to fall off precipitously. No intermediates of glucose metabolism in a form utilizable at higher temperatures could be found in the medium, and no toxic product was produced which limited growth. At 10 C, 37% of the carbon from glucose-UL-(14)C was assimilated into cellular material, whereas, at 30 C, 53% was assimilated. Cells grown at 10 C contained more carbohydrate than did cells grown at 37 C, and the glycogen-to-protein ratio of cells grown at 10 C was approximately three times higher than that of cells grown at 37 C. Adenosine triphosphatase activities of cells grown at 10 and 35 C were similar. Growth rates on glucose, glycerol, and succinate were quite similar at 10 C, but at 35 C growth was most rapid on glucose and slowest on succinate. The data suggest that the decrease in yield with decrease in temperature is a result of uncoupling of energy production from energy utilization.     

Oligodeoxynucleotide stability in subcellular extracts and culture media

Oligodeoxynucleotide degradation was studied in four systems in order to assess the importance of degradation in hybridization arrest experiments dependent on oligodeoxynucleotides complementary to mRNA sequences. Oligodeoxynucleotides were not detectably degraded over 2 h at 37 degrees C in rabbit reticulocyte lysate or Dulbecco's modified essential medium with 5% fetal calf serum, but were degraded over 2 h in HeLa cell postmitochondrial cytoplasmic extract, and were degraded within 15 min in bovine calf serum.     

Depurination and imidazole ring-opening in nucleosides and DNA alkylated by styrene oxide

Styrene-7,8-oxide was reacted with guanosine and deoxyguanosine and four isomeric 7-alkylguanosines were isolated, two of each being substitutions through the alpha and beta carbon of styrene oxide. The diastereomeric adducts imidazole ring-opened at an identical rate but the alpha- and beta-adducts differed (half-lives 90 and 56 min, respectively, pH 10, 24 degrees C). The 7-beta alkyl-deoxyguanosine derivatives ring-opened at a six times slower rate, which was similar to 7-methyldeoxyguanosine. The diastereomeric guanosine products also depurinated at the same rate but the beta-derivatives depurinated faster than the alpha-derivatives (t1/2 35 vs. 79 min, respectively, pH 1, 70 degrees C). The differences in the ring-opening and depurination of the alpha- and beta-isomers corresponded to their respective pK alpha values (7.31-7.32 vs. 7.16-7.19). The 7-alkyldeoxyguanosine derivatives of styrene oxide depurinated equally fast as 7-methyldeoxyguanosine. By contrast, the depurination of 7-alkylguanine was 15 times slower in the single-stranded DNA and 55 times slower in the double-stranded DNA.     

Denaturing Reversed-Phase HPLC Using a Mobile Phase Containing Urea for Oligodeoxynucleotide Analysis

Denaturing reversed-phase (RP) high performance liquid chromatography (HPLC) is usually achieved by elevating column temperature. In this article, an alternative method involving using a mobile phase that contains urea and performing HPLC at room temperature is described. The efficacy of the new method was demonstrated by analyzing a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs. The multiple peaks of the 61-mer ODN under normal conditions merged into one under the denaturing conditions. The broad single peaks of dsODNs under normal conditions were split into two sharp peaks.     

Thermodynamic and Kinetic Characterization of Antisense Oligodeoxynucleotide Binding to a Structured mRNA

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit β-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.     

Cholecystokinin Stimulates Dopamine Synthesis in Synaptosomes by a Cyclic AMP-Dependent Mechanism

The effects of different fragments of cholecystokinin (CCK) on dopamine synthesis were studied in synaptosomal preparations from the striatum, substantia nigra, and frontal cortex. In striatal synaptosomes, dopamine synthesis rate measured by dopamine accumulation was 12.5% lower than that measured by 3,4-dihydroxyphenylalanine (DOPA) accumulation; however, K+-accelerated synthesis was the same for both methods. Synthesis rate was independent of exogenous tyrosine levels. In the three regions studied, the combined stimulatory effects of 8-Br-cyclic AMP and high K+ were additive. CCK-5, CCK-3, CCK-27-33, and CCK-8 (sulphated) enhanced synthesis, CCK-5 being the most potent fragment. The nonsulphated octapeptide had no effect. In all three regions, CCK-5 and high K+ had an additive effect on dopamine synthesis; CCK-5 and 8-Br-cyclic AMP together produced the same enhancement of synthesis as CCK-5 alone. CCK-5 produced similar dose-dependent increases in dopamine synthesis and cyclic AMP accumulation in striatal synaptosomes, and both effects were blocked by the CCK antagonist proglumide.     

Oligodeoxynucleotide folding in solution: loop size and stability of B-hairpins

The secondary structures of the synthetic DNA fragments d(CGCGCGTTTTTCGCGCG) (T5), d(CGCGCGAAAAACGCGCG) (A5), d(CGCGCGTACGCGCG) (TA), and d(CGCGCGATCGCGCG) (AT) were investigated in a combined electrophoretic and spectroscopic study. All the oligomers exist, at low temperature and over a wide range of ionic strength (0.5-100 mM salt) and of nucleotide concentration [0.1-2.0 mM (phosphate)], as a mixture of two slowly interconverting species, identified as the dimeric duplex and the monomeric hairpin structure. The thermodynamic parameters for hairpin denaturation of T5, A5, TA, and AT and for duplex denaturation of d(CGCGCG) show that (a) the hairpins are more stable than the reference hexamer duplex at all accessible nucleotide concentrations; (b) the loop contributes favorably to the enthalpy change of hairpin denaturation in the four DNA fragments; (c) the base composition of the loop (A vs T) and the size of the loop (A5/T5 vs TA/AT) do not appreciably influence the enthalpic contents of the hairpins; (d) hairpins TA and AT, with two AT bases intervening in the CG self-complementary part of the molecule, exhibit a markedly higher thermal stability than hairpins T5 and A5, which is entropic in origin. These findings are consistent with the presence of two-residue loops in the tetradecamers TA and AT.     

Oligodeoxynucleotide 5mers containing a 5'-CpG induce apoptosis through a mitochondrial mechanism in T lymphocytic leukaemia cells

A chimeric methylphosphonodiester/phosphodiester 15mer oligodeoxynucleotide of randomly selected sequence was observed to rapidly induce apoptosis in MOLT-4 and Jurkat E6 T lymphocytic leukaemia cells following intracytoplasmic delivery. A series of further methylphosphonate substitutions and mutations and truncations of the oligodeoxynucleotide served to establish that the phosphodiester-linked sequence CGGTA present in the 15mer was responsible for this biological activity. End-protected CpG oligodeoxynucleotide 5mers of sequence type CGNNN exhibited a range of apoptosis-inducing potencies, with CGTTA being the most active. The latter was shown to significantly reduce the rate of RNA synthesis in MOLT-4 cells within 1 h; DNA laddering and redistribution of phosphatidylserine to the outer surface of the plasma membrane were marked by 160 min and mitochondrial transmembrane potential collapsed over roughly the same time scale. Pro-caspase 8 was reduced within 130 min and the proteolytically activated caspase 8 substrate Bid was also down by this time, implicating release of cytochrome c from mitochondria by the active 15 kDa fragment of Bid. Substantial proteolytic activation of pro-caspase 3 was relatively delayed. These findings support a mitochondrial amplification mechanism for apoptosis triggered by CpG 5mers.     

Cytidine and Uridine Increase Striatal CDP-Choline Levels Without Decreasing Acetylcholine Synthesis or Release

Summary Aims: Treatments that increase acetylcholine release from brain slices decrease the synthesis of phosphatidylcholine by, and its levels in, the slices. We examined whether adding cytidine or uridine to the slice medium, which increases the utilization of choline to form phospholipids, also decreases acetylcholine levels and release. Methods: We incubated rat brain slices with or without cytidine or uridine (both 25–400 μM), and with or without choline (20–40 μM), and measured the spontaneous and potassium-evoked release of acetylcholine. Results: Striatal slices stimulated for 2 h released 2650±365 pmol of acetylcholine per mg protein when incubated without choline, or 4600±450 pmol/mg protein acetylcholine when incubated with choline (20 μM). Adding cytidine or uridine (both 25–400 μM) to the media failed to affect acetylcholine release whether or not choline was also added, even though the pyrimidines (400 μM) did enhance choline`s utilization to form CDP-choline by 89 or 61%, respectively. The pyrimidines also had no effect on acetylcholine release from hippocampal and cortical slices. Cytidine or uridine also failed to affect acetylcholine levels in striatal slices, nor choline transport into striatal synaptosomes. Conclusion: These data show that cytidine and uridine can stimulate brain phosphatide synthesis without diminishing acetylcholine synthesis or release.