Enhanced Cellular Uptake of G-Rich Oligonucleotides

Abstract

Sequence-dependency of cellular uptake of oligonucleotides into Vero cells has been studied. Cellular uptake of 5′-[³⁵S]-labelled homopolymers decreased in the order (dG)₁₆ >> (dT)₁₆> (dA)₁₆ > (dC)₁₆. The change of two base-pairs (dG → dA) in a dG-rich antisense oligonucleotide with good antiviral activity dramatically decreased cellular uptake and abolished antiviral activity.     

Uptake and Intracellular Distribution of Oligonucleotides Vectorized by a PAMAM Dendrimer

Abstract

We studied the uptake and intracellular distribution of an FITC labelled phosphodiester oligodeoxynucleotide (ODN) vectorized by a dendrimeric structure in cell culture.     

DNA Recognition by Non-Natural Oligonucleotides

Abstract

Non-natural oligonucleotides were synthesized by modified phosphoramidite procedure and tested in triple helix-mediated DNA recognition.     

Mini-antisense Oligonucleotides

Abstract

A new strategy of selective DNA target modification was proposed. The using of reactive derivatives of short oligonucleotides in the presence of flanking effector pair allows one to modify DNA target only when the perfect complementary complex of DNA target and oligonucleotide tandem is formed.     

Strong Binding of Single-stranded DNA by Stem-loop Oligonucleotides

Abstract

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA_gwere studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr · pur · pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 °C higher than Watson- Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.     

Translation Arrest by RNase h Incompetent Antisense Oligonucleotides

Abstract

Most second-generation ON antisense analogs do not activate RNase H. Alternative strategies to arrest translation in a reticulocyte cell-free assay programmed by VSV mRNAs have been explored.     

Gene Correction by RNA‐DNA Oligonucleotides

An oligonucleotide composed of a contiguous stretch of RNA and DNA residues has been developed to facilitate the correction of single‐base mutations of episomal and chromosomal targets in mammalian cells. The design of the oligonucleotide exploited the highly recombinogenic RNA‐DNA hybrids and featured hairpin capped ends avoiding destruction by cellular helicases or exonucleases. The RNA‐DNA oligonucleotide (RDO) was designed to correct a point mutation in the tyrosinase gene and caused a permanent gene correction in mouse albino melanocytes, determined by clonal analysis at the level of genomic sequence, protein and phenotypic change. Recently, we demonstrated correction of the tyrosinase gene using the same RDO in vivo, as detected by dark pigmentation of several hairs and DOPA staining of hair follicles in the treated skin of albino mice. Such RDOs might hold a promise as a therapeutic method for the treatment of skin diseases. However, the frequency of gene correction varies among different cells, indicating that cellular activities, such as recombination and repair, may be important for gene conversion by RDOs. As this technology becomes more widely utilized in the scientific community, it will be important to understand the mechanism and to optimize the design of RDOs to improve their efficiency and general applicability.     

Spectroscopic Comparison of Different DNA Structures Formed by Oligonucleotides

Abstract

Six different nucleic acid structures including duplex, triplex and quadruplex are formed by oligonucleotides. Their structural properties are studied in detail by four spectroscopic techniques, i.e. CD, UV, NMR and fluorescence. Results are: CD Spectra: The common characteristics is a negative band at 240 nm, and the spectra are different from each other in the range 260–300 nm. Many factors such as chain direction, sugar puckering, orientation of the glycosyl bond, base stacking and sequence can effect their conformation and then show diversity and complexity in the spectra. UV Spectra: The UV spectra of all forms are quite similar, all of them exhibit a sharp positive peak around 210 nm and a broad positive band in the region of 240–280 nm. Although the bands are different in absorbance, the spectra are not characteristic enough to distinguish these forms. In addition, their thermal denaturation is also observed by UV spectrum, different melting curves and points are shown and some thermodynamic information is provided. NMR Spectra: Since the G residues in the six samples all participate in hydrogen bond, the imino proton can not exchange with the solvent freely so as to allow an observable resonance to arise. The resonance number and chemical shift will vary with the change in base-pairing number and mode as well as the whole geometry of its molecule. Fluorescence Spectra: The interaction mechanisms between EB and these structures are different. B type duplex and triplex adopt an intercalative mode in which the efficiency of energy transfer is relatively high and the fluorescence of EB can not be quenched easily. While for the parallel duplex, outside binding is predominant in which energy transfer can hardly happen and most of its fluorescence can be quenched. As for the quadruplex, groove binding is possible, so the efficiency of energy transfer is higher than that in outside binding, but lower than that in intercalative binding, and fluorescence is quenched partly.     

Generation of Oligonucleotides Under Hydrothermal Conditions by Non-enzymatic Polymerization

We previously reported that 5′-mononucleotides organized within a multilamellar lipid matrix can produce oligomers in the anhydrous phase of hydration–dehydration (HD) cycles. However, hydrolysis of oligomers can occur during hydration, and it is important to better understand the steady state in which ester bond synthesis is balanced by hydrolysis. In order to study condensation products of mononucleotides and hydrolysis of their polymers, we established a simulation of HD cycles that would occur on the early Earth when volcanic land masses emerged from the ocean over 4 billion years ago. At this stage on early Earth, precipitation produced hydrothermal fields characterized by small aqueous pools undergoing evaporation and refilling at elevated temperatures. Here, we confirm that under these conditions, the chemical potential made available by cycles of hydration and dehydration is sufficient to drive synthesis of ester bonds. If 5′-mononucleotides are in solution at millimolar concentrations, then oligomers resembling RNA are synthesized and exist in a steady state with their monomers. Furthermore, if the mononucleotides can form complementary base pairs, then some of the products have properties suggesting that secondary structures are present, including duplex species stabilized by hydrogen bonds.     

Pyranosyl - Oligonucleotides

Abstract

Pentopyranosyl-oligonucleotides, constitutional isomers of RNA containing the aldopentose units in the pyranose (instead of furanose) form and the phosphodiester bridges between the positions 4′ and 2′ (instead of 5′ and 3′) are being studied in our laboratories in the context of a systematic investigation directed towards a chemical etiology of nucleic acid structure. The primary aim of these studies is to collect factual information that may bear on the problem of why Nature, in evolving a genetic system, had chosen RNA and not some alternative system, such as one from RNA′s close structural neighborhood. In addition, investigations of this type are expected to extend insights into the relationships between structure and base pairing properties of the natural nucleic acids.     

Conjugated Antisense Oligonucleotides

Abstract

We have employed chemical modification strategies to improve cellular ahsorption of oligonucleotides. These include the conjugation of various pendant moieties to the oligonucleotide to affect its overall physical properties such as hydrophobicity, charge, and amphipathicity as well as pendants that may mediate absorption by binding to certain cellular receptors which internalize specific ligands. Our laboratory has prepared polyamines, polyethylene glycols and lipidic constituents conjugated to oligonucleotidcs in order to study their effects in enhancing absorption of antisense agents. These conjugates were targeted against human or murine Intercellular Adhesion Molecule- 1 (ICAM-1) mRNA.     

Synthesis of Thymine-Modified Oligonucleotides

Abstract

Oligonucleotide-resins containing N-nitrothymidine residues yield N3-thymine modified oligonucleotides by reaction with a variety of amines followed by the standard ammonia treatment.     

Properties of Triple Helices Formed by Oligonucleotides Containing 8-Aminopurines

Abstract

The synthesis of parallel hairpins carrying 8-aminopurines is described. These hairpins have a high affinity for specific polypyrimidine sequences resulting in the formation of very stable triplexes.     

Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies

The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease.     

Fluorescent Oligonucleotides Can Serve As Suitable Alternatives to Radiolabeled Oligonucleotides

Prolonged exposure to radiation from radionuclei used in medical research can cause DNA damage and mutation, which lead to several diseases including cancer. Radioactivity-based experiments are expensive and associated with specialized training, dedication of instruments, approvals, and cleanup with potential hazardous waste. The objective of this study was to find an alternative to the use of radioactivity in medical research using nucleic acid chemistry. FITC-labeled oligonucleotides that contain wild-type (wt) and modified base (8-oxo-G) at the same position and their complementary unlabeled strand were synthesized. Purified DNA repair enzyme, OGG1, and nuclear lysates from MCF-7 breast cancer cells were incubated with double-stranded FITC-labeled wt and 8-oxo-G oligonucleotide to demonstrate the OGG1 incision assay. We found that FITC-coupled oligonucleotides do not impose a steric hindrance during duplex formation, and the fluorescence intensity of the oligonucleotide is comparable with the intensity of the radioactive oligonucleotide. Moreover, we have seen that the OGG1 incision assay can be performed using these fluorescence oligonucleotides, replacing conventional use of radiolabeled oligonucleotides in the assay. Although the use of fluorescent-labeled oligonucleotides was described in detail for incision assays, the technique can be applied to replace a broad range of experiments, where radioactive oligonucleotides are used, eliminating the hazardous consequences of radiation.     

Uptake of putrescine by Tetrahymena

Summary The uptake of the diamine ³H-putrescine by Tetrahymena pyriformis GL was studied in cultures which were synchronized by heat shocks. An inverse correlation was found between the uptake of putrescine and the acid stability of DNA, but there was also a parallelism between putrescine uptake and the intracellular amount of putrescine. There was no evidence for a transformation of the labeled putrescine to other amino compounds within the cells. Electronmicroscopical autoradiography showed a structure-bound radioactivity localized to nuclear and mitochondrial structures. In the nucleus, both the chromatin and the nucleoli showed labeling.The authors are indebted to fil. kand. Per Arlock, who participated in some preliminary experiments, and to Mrs Siv Nilsson and Mrs Annagreta Petersen for skilful technical assistance. The investigation was financially supported by the Swedish Natural Science Research Council, the Swedish Cancer Society and the C.-B. Nathhorst Scientific Foundation.     

反义寡核苷酸的化学修饰

反义寡核苷酸的功能在很大程度上取决于其稳定性、生物利用度及与靶基因结合或反应的特性．通过特定的化学修饰可以改变这些物理、化学特性,从而增加其抗病毒、抗肿瘤及对其他特定基因的表达抑制活性．     

The Derivatives of Phosphorothioate Oligonucleotides

Abstract

Derivatives of phosphorothioate oligonucleotide analogues bearing alkylating N-methyl-4-(N-2-chloroethyl)-N-methylamino)benzylamine or stabilizing complementary complexes N-(2-hydroxyethyl)phenazinium residues at the 3′-terminal phosphate group were synthesized and investigated.