title: Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide.
abstract: We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.
authors.affiliation: Aronex Pharmaceuticals, Inc., The Woodlands, Texas 77381, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Bishop
authors.forename: J K
authors.initials: JK
authors.lastname: Guy-Caffey
authors.forename: J O
authors.initials: JO
authors.lastname: Ojwang
authors.forename: S R
authors.initials: SR
authors.lastname: Smith
authors.forename: M E
authors.initials: ME
authors.lastname: Hogan
authors.forename: P A
authors.initials: PA
authors.lastname: Cossum
authors.forename: R F
authors.initials: RF
authors.lastname: Rando
authors.forename: N
authors.initials: N
authors.lastname: Chaudhary
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001482:Base Composition; D001483:Base Sequence; D001692:Biological Transport; D003196:Computer Graphics; D004352:Drug Resistance, Microbial; D015726:Giant Cells / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013873:Thionucleotides
chemical_list: D000998:Antiviral Agents; D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides