Inhibition of HIV‐1 replication by a two‐strand system (FTFOs) targeted to the polypurine tract

Abstract

Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3P P-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.z 1999 Federation of European Biochemical Societies.

Silencing of HIV by hairpin‐loop‐structured DNA oligonucleotide

“…A triple‐helix has been described previously in vivo for a subregion of the PPT DNA of HIV, however, only after cross‐linking and stabilization of the structure before insertion into the cell [16]. Based on our previous in vivo observations [11,12] and a later report [18], we performed studies with a number of mutants of the ODN and on the time course of the effect. Even in cases where the antisense strand was identical to that of ODN A and fully complementary to the viral RNA no antiviral effect was observed, suggesting some mechanism not only based on a classical antisense effect, since the second arm is also important.…”

Inhibition of human immunodeficiency virus type 1 replication by P‐stereodefined oligo(nucleoside phosphorothioate)s in a long‐term infection model

“…We also chose a random sequence as the control oligonucleotide ([Mix]‐S‐ODN‐28‐ran). The MOLT‐4 clone 8 (3×10 5 ml −1 ) cells were incubated with HIV‐1 IIIB at an MOI of 0.01 for 2 h to allow absorption [33]. The cells were then washed to remove the virus from the medium, and the oligonucleotides (1 μM) were added with fresh medium.…”

“…At the time points indicated, an aliquot of the culture supernatant was removed for p24 antigen analysis and was replaced by fresh medium. Every 4 days, viable cells were counted and passed at 3×10 5 cells per ml [33].…”

Biological investigation using a shear horizontal surface acoustic wave sensor: Small “Click generated” DNA hybridization detection

“…Non-natural oligonucleotides have attracted a great deal of interest among researchers working with antisense and anti-gene technologies (Hiratou et al, 1999;Paillart et al, 2004;Sanghvi and Cook, 1994), triple helix therapies (De Mesmaeker et al, 1995;Uhlmann and Peyman, 1990), and nucleic acid nanotechnology (Gold et al, 1995). Among the various modifications of DNA strands, base or sugar changes (Eppacher et al, 2000;Lee and Kool, 2009;Perez-Rentero et al, 2009;Roig and Asseline, 2009), those of internucleosidic linkers are very promising, since they render the backbone virtually insensitive to acid/alkaline or enzymatic hydrolysis.…”