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Hotoda’s Sequence and HIV prevention

A modified guanine-rich phosphodiester oligonucleotide prevents HIV-1 infection.

The 6mer sequence d(TGGGAG) is commonly called “Hotoda’s sequence”.

In the 1990s Japanese researchers studied oligodeoxynucleotides (ODNs) in which the 5’-end was covalently linked to a 4,4’-dimethoxytriphenylmethyl (DMT) residue for their ability to inhibit HIV-1 infection. The guanine-rich phosphodiester oligonucleotide with a dimethoxytrityl (DmTr) residue on its 5'-terminal, DmTr-TGGGAGGTGGGTCTG (SA-1042), was found as an inhibitor of HIV-1 infection in vitro.

SA-1042 interfered with the attachment of gp120 to the CD4 receptor and prevented the subsequent entry of the virus into cells. Furukawa et al. showed that guanine nucleosides at the 5'-terminal and modification of the 5'-terminal with DmTr are essential for anti-HIV-1 activity. The substitution of the guanine nucleoside close to the 5'-terminal with other nucleotides prevented antiviral activity. At least three consecutive guanine nucleotides adjacent to the 5'-terminal are required for the activity and modification of the 5'-terminal is essential for the activity. As a result of the study the hexanucleotide, DmTr-TGGGAG, was identified as a potent inhibitor of HIV-1 infection. The hexamer is capable of inhibiting the binding of the gp120 protein to its receptor the CD4 molecule. Furthermore, the hexamer inhibits the accessibility of anti-V3 monoclonal antibody to its ligand V3 peptide.

Sequences rich in guanosine nucleotides are known to form nonlinear structures known as guanine quadruplexes (G4s) stable under physiological conditions. A variety of G4 topologies are also known with variations in strand stoichiometry and polarity as well as different lengths of loop structures and locations the guanines within the sequence. Like aptamers, G4 structures are single-stranded DNA oligonucleotides that specifically bind to their target as a result of their unique 3-dimensional structure. Furthermore, G4 sequences appear to have a role in gene regulation, are important for telomerase maintenance and are also found in genomes, including mammalian genomes as well as the human genome. Several G-quadruplex-forming oligonucleotides with in vitro anti-HIV activity have already been discovered while using either the Systematic Evolution of Ligands by Exponential enrichment (SELEX) process or rational design approaches such as by using Synthetic Unrandomization of Randomized Fragments (SURF). Often evolved or natural occurring aptamers can be improved by chemical modification. Improvements to aptamers can be made by conjugation of large hydrophobic groups to terminal ends, the replacement of phosphodiester linkages with phosphorothioate bonds, as well as the addition of modified nucleic acids such as bridged nucleic acids (BNAs) at selected nucleotide position.

Several G-quadruplex forming aptamers are now known as HIV inhibitors. These quadruplexes function by inhibiting (i) virus binding and entry into the target cell, (ii) HIV reverse transcription, or (iii) virus integration, by interaction with HIV proteins such as envelope proteins, reverse transcriptase and integrase.

The Japanese researchers found that both the G4 structure and the cluster of large aromatic groups at the 5′-ends are essential for the anti-HIV activity. Oligonucleotide R-95288 with a 3,4-dibenzyloxybenzyl (DBB) residue at the 5’-end and a 2-hydroxyethylphosphate residue on the 3′-end of the d(5′TGGGAG3′) sequence was the most potent ODN identified.

With the goal to improve the G4 folding kinetics further Oliviero et al. reported the synthesis of stabilized, tetra-end-linked oligonucleotides (TEL-ODN) that form mononuclear G4s based on Hotoda's sequence. Figure 1 shows the molecular model of a TEL-ODN.

Figure 1:G-quadruplex structure of a monomolecular TEL-ODN. The TEL-ODN was modifed at the 5'-ends with a TBDPS group and the 3'-ends were connected using a tetra-end-linker. The adenosines were replaced with cytosines which resulted in a G4 molecule with a potent anti-HIV activity. The observed and reported EC50 was 39 nM. 

The discovery of the high activity of the unmodified Hotoda's sequence revealed a potent G-quadruplex anti-HIV agent. However, the supramolecular structure is complex and more research maybe needed for it to become a sucessful HIV drug.


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