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 M, 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 t 1 ⁄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 (t 1 ⁄2 from ϳ3 to 7 min), low cellular uptake, and no sequencespecific 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.Guanine-rich nucleic acid strands, under physiological salt and pH conditions, can adopt a higher order, thermodynamically stable conformation containing square-planar arrangement of four guanines that are hydrogen-bonded in the Hoogsteen manner and stabilized by a monovalent cation (1-4). Depending upon the base composition, sequence, and concentration of the nucleic acids, guanine quartet-containing structures (or G-quartets) 1 can be generated from DNA or RNA, either by the intramolecular folding of a single G-rich strand, or by the association of multiple strands (1-7). Believed to be ubiquitous in nature, G-quartets are proposed to participate in diverse biological processes including the modulation of telomere activity, dimerization of HIV RNA, and site-specific genetic recombination in immunoglobulin switch regions (5-9). In addition, using a combination of rational drug design and combinatorial screening methods, several biologically active oligonucleotides have been described, each of unique specificity and the potential to form G-quartet motifs (10 -15). In particular, we have identified a family of deoxyguanosine-and thymidine-rich (deoxyribo)oligonucleotides that are potent inhibitors of HIV-1 expression in standardized cell culture-based assays (16,17). One such inhibitor is T30175, a 17-mer oligonucleotide synthesized with a natural phosphodiester backbone (Table I). A more potent version, T30177, has the same sequence, but contains a single phosphorothioate internucleosid...

The effect of CpG-oligodeoxynucleotides with different backbone structures and 3' hexameric deoxyriboguanosine run conjugation on the treatment of asthma in mice*1

“…PE*-CpG-dG6 inhibited airway hyperresponsiveness like PS-CpG, decreased BAL fluid eosinophilia, increased serum allergen specific IgG2a and Th1 cytokine production by splenocytes. Two PS bonds located at the 5′ terminus can contribute to resistance against exonuclease and do not affect the G-quartet structure (12). PE*-CpG-dG6 was also more effective at preventing murine allergic asthma than PE-CpG-dG6 in previous report (4).…”

“…We have shown that the failure of PE-CpG to inhibit asthmatic responses could be overcome by 3′ dG6-run conjugation when PE-CpG-dG6 is injected at the time of sensitization (4). Runs of dG residues can form a four stranded conformation, which is called the G-quartet, which confer nuclease resistance and facilitate binding to the scavenger receptors of macrophages (11,12). Accordingly, 3′ dG6- run may extend the half-life of PE-CpG and allow the efficient targeting of PE-CpG by APCs, presumably via a scavenger receptor (4).…”

Nucleic acid sensing by T cells initiates Th2 cell differentiation

“…It has been shown that the poly(dG) motif not only protects against DNase degradation 16 but also enhances the cellular uptake of the ODN 17 . Indeed, the uptake of GpC-poly(dC) by T cells was lower than GpC, indicating that GpC-poly(dC) could not induce costimulation due to its poor uptake (Fig.…”

Biodistribution of antisense nanoparticles in mammary carcinoma rat model

“…1343-1362: 5′-CTAACTTAAAAAACAAAAGA -3′) was internally labeled with 33 P ATP (Bishop et al, 1996). Briefly, for OPN AS radio-labeling two short (10 bp long each) oligonucleotides corresponding to 5′-half (sequence A) and 3′-half (sequence B) of the final product, and third oligonucleotide complementary to oligos A and B (sequence T, 20 bp long) were synthesized (Alpha DNA, Montreal, Quebec, Canada).…”