Nucleolipids of the cancerostatic 5-fluorouridine: synthesis, adherence to oligonucleotides, and incorporation in artificial lipid bilayers.

5-Fluorouridine (1a) was converted to its N(3)-farnesylated nucleoterpene derivative 8 by direct alkylation with farnesyl bromide (4). Reaction of the cancerostatic 1a with either acetone, heptan-4-one, nonadecan-10-one, or hentriacontan-16-one afforded the 2',3'-O-ketals 2a-2d. Compound 2b was then first farnesylated (→5) and subsequently phosphitylated to give the phosphoramidite 6. The ketal 2c was directly 5'-phosphitylated without farnesylation of the base to give the phosphoramidite 7. Moreover, the recently prepared cyclic 2',3'-O-ketal 11 was 5'-phosphitylated to yield the phosphoramidite 12. The 2',3'-O-isopropylidene derivative 2a proved to be too labile to be converted to a phosphoramidite. All novel derivatives of 1a were unequivocally characterized by NMR and UV spectroscopy and ESI mass spectrometry, as well as by elemental analyses. The lipophilicity of the phosphoramidite precursors were characterized by both their retention times in RP-18 HPLC and by calculated log P values. The phosphoramidites 6, 7, and 12 were exemplarily used for the preparation of four terminally lipophilized oligodeoxynucleotides carrying a cyanine-3 or a cyanine-5 residue at the 5'-(n-1) position (i.e., 14-17). Their incorporation in an artificial lipid bilayer was studied by single-molecule fluorescence spectroscopy and fluorescence microscopy.

Nucleoterpenes of thymidine and 2'-deoxyinosine: synthons for a biomimetic lipophilization of oligonucleotides.

2'-Deoxyinosine (1) and thymidine (7) were N-alkylated with geranyl and farnesyl moieties. These hydrophobic derivatives, 3a and 3b, and 9a and 9b, respectively, represent the first synthetic biomimetic nucleoterpenes and were subsequently 5'-protected and converted into the corresponding 3'-O-phosphoramidites, 5a and 5b and 11a and 11b, respectively. The latter were used to prepare a series of lipophilized oligonucleotide dodecamers, a part of which were additionally labelled with indocarbocyanine fluorescent dyes (Cy3 or Cy5), 18-23. The insertion of the lipooligonucleotides into, as well as duplex formation at artificial lipid bilayers was studied by single-molecule fluorescence spectroscopy and fluorescence microscopy.

Specific DNA duplex formation at an artificial lipid bilayer: towards a new DNA biosensor technology.

A novel technique is described which comprises a base-specific DNA duplex formation at a lipid bilayer-H(2) O-phase boundary layer. Two different probes of oligonucleotides both carrying a double-tailed lipid at the 5'-terminus were incorporated into stable artificial lipid bilayers separating two compartments (cis/trans-channel) of an optically transparent microfluidic sample carrier with perfusion capabilities. Both the cis- and trans-channels are filled with saline buffer. Injection of a cyanine-5-labeled target DNA sequence, which is complementary to only one of the oligonucleotide probes, into the cis-channel, followed by a thorough perfusion, leads to an immobilization of the labeled complementary oligonucleotide on the membrane as detected by single-molecule fluorescence spectroscopy and microscopy. In the case of fluorescent but non-complementary DNA sequences, no immobilized fluorescent oligonucleotide duplex could be detected on the membrane. This clearly verifies a specific duplex formation at the membrane interface.

Identification of new channels by systematic analysis of the mitochondrial outer membrane.

The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are ß-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.

Distinct Pores for Peroxisomal Import of PTS1 and PTS2 Proteins.

Two peroxisomal targeting signals, PTS1 and PTS2, recognized by cytosolic receptors Pex5 and cooperating Pex7/Pex18, direct folded proteins to the peroxisomal matrix. A pore consisting of the PTS1 receptor Pex5 and the docking protein Pex14 imports PTS1 proteins. We identified a distinct PTS2-specific pore, which contains the PTS2 co-receptor Pex18 and the Pex14/Pex17-docking complex as major constituents. The estimated maximal pore size of ∼ 4.7 nm is large enough to allow import of folded PTS2 proteins. PTS2 cargo proteins modulate complex gating, open probability, and subconductance states of the pore. While the PTS1 channel is transiently activated by arriving receptor-cargo complexes, the reconstituted PTS2 channel is constitutively present in an open state. However, the cargo-loaded PTS2 channel is largely impermeable to solutes and ions. Our results demonstrate that import of PTS1 and PTS2 proteins does not converge at the peroxisomal membrane as previously anticipated but is performed by distinct pores.