We have determined the stability of intramolecular DNA quadruplexes in which the four G(3)-tracts are connected by non-nucleosidic linkers containing propanediol, octanediol or hexaethylene glycol, replacing the TTA loops in the human telomeric repeat sequence. We find that these sequences all fold to form intramolecular complexes, which are stabilized by lithium < sodium < potassium. Quadruplex stability increases in the order propanediol < hexaethylene glycol < octanediol. The shallower shape of the melting profile with propanediol linkers and its lower dependency on potassium concentration suggests that this complex contains fewer stacks of G-quartets. The sequence with octanediol linkers displays a biphasic melting profile, suggesting that it can adopt more than one stable structure. All these complexes display melting temperatures above 310 K in the presence of 10 mM lithium, without added potassium, in contrast to the telomeric repeat sequence. These complexes also fold much faster than the telomeric repeat and there is little or no hysteresis between their melting and annealing profiles. In contrast, the human telomeric repeat sequence and a complex containing two hexaethylene glycol groups in each loop, are less stable and fold more slowly. The melting and annealing profiles for the latter sequence show significant differences, even when heated at 0.2 degrees C min(-1). CD spectra for the oligonucleotides containing non-nucleosidic linkers show positive maxima at 264 nm, with negative minima approximately 244 nm, which are characteristic of parallel quadruplex structures. These results show that the structure and stability of intramolecular quadruplexes is profoundly influenced by the length and composition of the loops.

The role of platelets in hemostasis and thrombosis is clearly established; however, the mechanisms by which platelets mediate inflammatory and immune pathways are less well understood. Platelets interact and modulate the function of

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.

Lysyl oxidase (LO), a secreted protein, was recently identified within the nuclei of vascular smooth-muscle cells (SMC) and 3T3 fibroblasts. A possible pathway by which LO can enter cell nuclei was explored in the present study. SMC were incubated with purified 32-kDa bovine aorta LO that had been fluorescently labeled with rhodamine (TRITC-LO). TRITC-LO entered the cytosol and then rapidly concentrated within the nuclei of preconfluent cultures of these cells, whereas carbonic anhydrase, a protein of similar molecular weight and similarly labeled, did not enter the cells under these conditions. LO that had been reductively methylated at lysine residues with [(14)C]HCHO was also taken up into the cytosolic and nuclear compartments. Intracellular uptake and intracellular distribution were not altered by inhibiting LO activity with beta-aminopropionitrile. An excess of native LO but not of carbonic anhydrase competitively inhibited the uptake of the isotopically labeled enzyme. Thus, once secreted and proteolytically processed, mature LO can enter the cells and concentrate within nuclei in a manner that appears to be specific and independent of its catalytic activity.

No abstract