ABSTRACT
Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate-ODN conjugates have been reported to improve the cell-specific delivery of ODNs through receptor mediated endocytosis. We tested the anti-HIV activity and biochemical properties of the 5'-end glucose-conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV-1 RNA in cell-based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti-HIV-1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA-RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV-1 dimerisation.
Subject(s)
Adjuvants, Immunologic/chemistry , Anti-HIV Agents/chemistry , Glucose/analogs & derivatives , HIV Infections/drug therapy , HIV-1/drug effects , Oligonucleotides, Antisense/chemistry , Thionucleotides/chemistry , Adjuvants, Immunologic/pharmacology , Anti-HIV Agents/pharmacology , Base Sequence , CpG Islands , Glucose/pharmacology , Humans , Jurkat Cells , Oligonucleotides, Antisense/pharmacology , Thionucleotides/pharmacologyABSTRACT
Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold surfaces. However, the stability of such nucleic acid-gold nanoparticle conjugates in certain conditions may be a limitation due to premature break of the thiol-gold bonds followed by aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides carrying thioctic acid moiety based on the use of several thioctic acid-L-threoninol derivatives containing different spacers, including triglycine, short polyethyleneglycol, or aliphatic spacers. The novel thioctic-oligonucleotides were used for the functionalization of gold nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols than gold nanoparticles prepared with commercially available thiol-oligonucleotides. Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B present in endosomes. In this way this derivative may be interesting for the cellular delivery of therapeutic oligonucleotides as these results provides the basis for a potential endosomal escape.
Subject(s)
Gold/chemistry , Metal Nanoparticles/chemistry , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Thioctic Acid/analogs & derivatives , Thioctic Acid/chemistryABSTRACT
Oligonucleotides carrying thiol groups are useful intermediates for a remarkable number of applications involving nucleic acids. In this study, DNA oligonucleotides carrying tert-butylsulfanyl protected thiol groups have been prepared. A building block derived from threoninol has been developed to introduce a thiol group at any predetemined position of an oligonucleotide. The resulting thiolated oligonucleotides have been used for the preparation of oligonucleotide conjugates and for the functionalization of gold nanoparticles using the reactivity of the thiol groups.
Subject(s)
Amino Alcohols/chemistry , Butylene Glycols/chemistry , Metal Nanoparticles/chemistry , Oligonucleotides/chemistry , Oligonucleotides/chemical synthesis , Sulfhydryl Compounds/chemistry , DNA/chemistry , Gold/chemistry , Nucleic Acid ConformationABSTRACT
Oligonucleotides carrying amino, thiol groups, as well as fluorescein, c-myc peptide sequence and nanogold at internal positions were prepared and used for the assembly of bidimensional DNA arrays.
Subject(s)
DNA/chemistry , DNA/chemical synthesis , Nucleic Acid Conformation , Oligonucleotide Array Sequence Analysis/methods , Amino Acids/chemistry , Base Sequence , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Fluorescein/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Microscopy, Atomic Force , Oligonucleotides/chemistry , Sulfhydryl Compounds/chemistryABSTRACT
Oligonucleotide-peptide conjugates have attracted considerable interest especially for biomedical uses. In the first part of this chapter, we describe protocols for the stepwise synthesis of oligonucleotides carrying peptide sequences at the 3'-end on a single support. The resulting oligonucleotide-peptide conjugates may be used as exogenous effectors for the specific control of gene expression. In the second part of this chapter, detailed postsynthetic conjugation protocols to introduce peptide sequences into oligonucleotide sequences are also presented.
Subject(s)
Biomedical Research/methods , Oligonucleotides/chemistry , Oligonucleotides/chemical synthesis , Peptides/chemistry , Biological Transport , DNA/chemical synthesis , DNA/chemistry , DNA/metabolism , Guanidine/chemistry , Oligonucleotides/metabolism , Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/metabolism , RNA/chemical synthesis , RNA/chemistry , RNA/metabolism , Sulfhydryl Compounds/chemistryABSTRACT
An efficient route for the synthesis of the phosphoramidite derivative of 5-methylcytosine bearing a tert-butylsulfanyl group protected thiol is described. This building block is used for the preparation of oligonucleotides carrying a thiol group at the nucleobase at the internal position of a DNA sequence. The resulting thiolated oligonucleotides are useful intermediates to generate oligonucleotide conjugates carrying molecules of interest at internal positions of a DNA sequence.
Subject(s)
5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/chemistry , Chemistry, Organic/methods , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Base Sequence , Chromatography, High Pressure Liquid , Fluorescent Dyes/chemistry , Iodine/chemistry , Molecular Sequence Data , Oligodeoxyribonucleotides/genetics , Solutions , tert-Butylhydroperoxide/chemistryABSTRACT
Oligonucleotides carrying novel fluorescent compounds with a dipolar isoquinoline imidazo[1,2-a]azine core were prepared. Analysis of the melting curves demonstrates that DNA duplexes carrying these fluorescent labels at their ends have a slight increase in DNA duplex stability. The UV absorption and fluorescent properties of the oligonucleotide conjugates were analyzed. The fluorescent label is sensitive to duplex formation, as cooperative melting curves are also observed at 366 nm and fluorescence has a large increase upon denaturation. Cell uptake studies allow observation of these fluorescently labeled oligonucleotides internalized into HeLa cells.
Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , HeLa Cells/pathology , Imidazoles/chemistry , Isoquinolines/chemistry , Oligonucleotides/chemical synthesis , Base Sequence , DNA/metabolism , HeLa Cells/metabolism , Humans , Nucleic Acid Denaturation , Oligonucleotides/chemistry , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Transition TemperatureABSTRACT
Oligonucleotide conjugates carrying carbohydrates at the 5'-end have been prepared. Glucose, fucose, and saccharides containing glucose at the nonreducing end were attached to DNA strands using the classical phosphoramidite chemistry. Two types of spacers and a dendron scaffold helped to obtain a diversity of sugar presentations in the DNA conjugates. Cellular surface adsorption and cellular uptake of carbohydrate oligonucleotide antisense sequences were measured using flow cytometric analysis. Conjugates with the glucose moiety linked through long spacers (15 to 18 atom distances) were internalized better than those with short linkers (4 atom distance) and than DNA control strands without sugar modification. Conjugates with tetravalent presentation of glucose did not improve cell uptake.
Subject(s)
Cell Membrane/metabolism , DNA/metabolism , Fluorescent Dyes/chemistry , Glucose/metabolism , Glycoconjugates/chemical synthesis , Glycoconjugates/metabolism , Adsorption , Binding Sites , Biological Transport , Cells, Cultured , DNA/chemistry , Flow Cytometry , Glucose/chemistry , Glycoconjugates/chemistry , HeLa Cells , Humans , Microscopy, Fluorescence , Molecular Structure , Oligonucleotides/chemistry , Organophosphorus Compounds/chemistry , StereoisomerismABSTRACT
The impact of the presence of nonnatural bases on the properties of oligodeoxynucleotides has been studied. First, oligodeoxynucleotides carrying 2'-deoxyzebularine were prepared, and the stability of duplexes carrying this analogue was determined by DNA melting experiments. Melting temperatures and thermodynamic data indicated the preference of 2'-deoxyzebularine for 2'-deoxyguanosine, which behaves as a 2'-deoxycytidine analogue, forming a less stable base pair due to the absence of the amino group at position 4. Moreover, the duplex-hairpin equilibrium of a self-complementary oligodeoxynucleotide carrying several natural and nonnatural bases including 2'-deoxyzebularine as a central mispair, was studied. Depending on the base present in the middle of the sequence, it is possible to affect the stability of the bimolecular duplex modulating the duplex-hairpin equilibrium. Magnesium ions were shown to stabilize preferentially the bimolecular duplex form. The results indicate the importance of the modifications and the role of cations in shifting the structural equilibrium.
