We have investigated the interaction of ohgonucleotides 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 (<1 jAM), 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.Antisense oligodeoxynucleotides and their derivatives have been shown to be specific inhibitors ofgene expression. They have been considered as a potential new generation of drugs, capable perhaps of inhibiting various pathogens and of regulating specific gene expression by inhibiting the translation of mRNA molecules in a highly specific manner (1-4). However, a prevailing view is that cells are not very permeable to oligonucleotides. Considerable efforts have been made to design nonionic membrane-permeable analogs (5) and to develop special delivery techniques (6, 7). Nevertheless, it has also been shown that normal unsubstituted oligonucleotides can cause hybridization arrest of specific mRNAs and viruses in cell culture (8-11). These observations provide evidence that oligodeoxynuicleotides do indeed enter cells. Recently, the ability of oligonucleotides to enter mammalian cells has been proved experimentally (9,(11)(12)(13)(14).The present investigation studied the interaction of oligodeoxynucleotide derivatives with mammalian cells. We examined the efficiency of oligomer binding and the time course of oligomer internalization under various conditions. We also investigated the stability of the internalized oligomers. Experiments involving the reactive 4-[N-(2-chloroethyl)-N-methyl]aminobenzyl phosphamide derivative of oligodeoxynucleotides [general formula ClRCH2NHpTTFr..., where R = -CH2CH2N(CH3)C6Hr---(1)]t were also undertaken: reagents of this type have recently been used by us as inhibitors of the influenza and tick-borne encephalitis viruses (4,15). In the present study, these reagents were used to chemically modify the putative cellular receptors binding oligonucleotides. MATERIALS AND METHODSOligodeoxynucleotides and Their Derivatives. The oligodeoxyribonucleotides pT, (n = 8, 9, 10, 16)

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

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.

The use of synthetic oligonucleotides as possible drugs for human therapy is usually hampered by their low in vivo stability and capacity to achieve high concentrations at their cellular targets. To overcome this, it has been suggested that they be modified chemically. Among the various options, conjugation with short- and long-chain polyethylene glycols (PEGs) has several advantages: PEG is nontoxic and very soluble, reduces immunogenic reactions, and increases the stability of the conjugated molecules. PEG is generally joined to oligonucleotides while bound to the insoluble solid-phase supports, after their synthesis, which does not allow for their being easy scaled up. The use of the liquid-phase approach as an alternative synthetic process, utilizing the PEG polymer both as a soluble, inert synthetic support and a stabilizing agent of the oligonucleotide, is proposed. A new protocol has been set up, characterized by a stable phosphate bond between the support and the oligonucleotide. This method has been tested on a 12mer previously investigated as an antisense agent against HIV. The PEG-conjugated 12mer was efficiently synthesized and purified. It was found that the high-molecular mass PEG chain results in enzymatic stability and does not interfere with the formation of the duplex with its nucleic acid target.

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Partitioning mediators into water-immiscible ionic liquids protects laccase from inactivation.