Driving in vitro selection of anti-HIV-1 TAR aptamers by magnesium concentration and temperature.

In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.

Alternative approach to the numerical synthesis of the dense-ion-beam focusing systems.

An alternative approach has been developed to the numerical synthesis of ion-beam focusing systems that prepare the dense laminar ion beams with the required profile of the ion trajectories. Conventionally an ill-posed synthesis problem arises in such cases, and only low-density beams with no magnetic field are treated. Instead, we compute both the electric and magnetic fields by considering two well-posed problems, the first for the equivalent potential Q and the second for the electric potential U. An analytical solution for Q for the specific case of an axial system has been found and a self-consistent method of satisfying the axial boundary conditions for Poisson's equation in U is described. It is shown that the beam can be focused while preserving its laminar structure and the required profile when applying various superpositions of both the electric and magnetic fields.

Endogenous oligonucleotides of human milk and their possible biological function.

Oligonucleotides (ON) 4 to 60 nucleotides in length were isolated by ion-exchange chromatography on a column with Fractogel TSK DEAE-650 (M) from human milk which was hydrolyzed with proteinase K. ON from 60 to 16 nucleotides were degraded by RNase A but were resistant to DNase I, and, thus, they were ribooligonucleotides. In the presence of [gamma-32P]ATP, ON and heparin inhibited the phosphorylation of 38- and 20-kD milk proteins and failed to affect the phosphorylation of a 76-kD protein. Human milk is believed to contain polyanion-dependent and polyanion-independent protein kinases. The influence of the ON on the activity of the cytotoxic fraction of human milk alpha-lactalbumin towards human mammary gland carcinoma MCF-7 cells was studied. The ON inhibited the cytostatic and cytotoxic effects of alpha-lactalbumin. Synthetic oligonucleotides and heparin had similar effects. The endogenous ON are suggested to be involved in the regulation of cytotoxic activity of human milk.

DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes.

In vitro selection was performed in a DNA library, made of oligonucleotides with a 30-nucleotide random sequence, to identify ligands of the human immunodeficiency virus type-1 trans-activation-responsive (TAR) RNA element. Aptamers, extracted after 15 rounds of selection-amplification, either from a classical library of sequences or from virtual combinatorial libraries, displayed an imperfect stem-loop structure and presented a consensus motif 5'ACTCCCAT in the apical loop. The six central bases of the consensus were complementary to the TAR apical region, giving rise to the formation of RNA-DNA kissing complexes, without disrupting the secondary structure of TAR. The RNA-DNA kissing complex was a poor substrate for Escherichia coli RNase H, likely due to steric and conformational constraints of the DNA/RNA heteroduplex. 2'-O-Methyl derivatives of a selected aptamer were binders of lower efficiency than the parent aptamer in contrast to regular sense/antisense hybrids, indicating that the RNA/DNA loop-loop region adopted a non-canonical heteroduplex structure. These results, which allowed the identification of a new type of complex, DNA-RNA kissing complex, demonstrate the interest of in vitro selection for identifying non-antisense oligonucleotide ligands of RNA structures that are of potential value for artificially modulating gene expression.

Antisense oligonucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin.

Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170-11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs.

Control of gene expression in viruses and protozoan parasites by antisense oligonucleotides.

Chemically-modified oligonucleotides are now routinely used to prevent gene expression in cell-free media and in cultured cells. The binding of an antisense sequence to a complementary RNA target may lead to the selective inhibition of the encoded information. This may occur at different levels: splicing; transport of the mature RNA from the nucleus to the cytoplasm; translation. Antisense oligonucleotides constitute an interesting tool to shed some light on gene function. They are also potential new therapeutic agents against pathogenic organisms. This review discusses the rules that guide the design of an antisense oligomer and the choice of a target sequence. Examples of the potential use of antisense oligonucleotides in the fields of virology and parasitology, in particular in relation to trypanosomatids, are described.

Mechanism of oligonucleotide uptake by cells: involvement of specific receptors?

We have investigated the interaction of oligonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (less than 1 microM), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single- and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.

Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3'-terminus and their interaction with DNA within mammalian cells.

5'-[32P]-labelled alkylating decathymidylate [4-(N-2-chloroethyl)N-methylaminobenzyl]-5'-phosphamide derivatives containing cholesterol or phenazinium residues at their 3'-termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3'-termini. The presence of the phenazinium residue at the 3'-terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.

Nucleotide and oligonucleotide derivatives as enzyme and nucleic acid targeted irreversible inhibitors. Biochemical aspects.

Sequence specific modification of nucleic acids with reactive oligonucleotide derivatives, complementary addressed modification, can provide an efficient approach for specific inactivation of certain cellular nucleic acids. In experiments with ascites tumor Krebs II cells and alkylating oligothymidylate derivatives it was found that alkylating oligonucleotide derivatives enter the living cell and modify complementary sequences in cellular nucleic acids with high efficiency. Complementary addressed modification of poly(A) sequences in cellular RNA with oligothymidylate derivatives was investigated in detail. The results of experiments on alkylation of cellular nucleic acids are consistent with complementary addressed modification of poly(A) sequences in cellular DNA. These results are supported by experiments on modification of chromatin DNA in which it was found that chromatin DNA interacts with oliogothymidylate derivatives more readily than the isolated double stranded DNA. It was found that alkylating oligonucleotide derivatives complementary to a sequence in immunoglobulin mRNA of MOPC 21 cells arrest the cellular immunoglobulin synthesis. Alkylating oligonucleotide derivatives complementary to RNAs of fowl plague virus inhibit virus multiplication in cell culture.