This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 8971–8973 8971 Tetra-end-linked oligonucleotides forming DNA G-quadruplexes: a new class of aptamers showing anti-HIV activityw Giorgia Oliviero, a Jussara Amato, a Nicola Borbone,* a Stefano D’Errico, a Aldo Galeone,* a Luciano Mayol, a Shozeb Haider, b Olujide Olubiyi, b Bart Hoorelbeke, c Jan Balzarini c and Gennaro Piccialli a Received 28th July 2010, Accepted 5th October 2010 DOI: 10.1039/c0cc02866e The biophysical and biological properties of unprecedented anti-HIV aptamers are presented. The most active aptamer (1L) shows a significant affinity to the HIV protein gp120. Combinatorial chemistry techniques have proven to be particularly valuable in medicinal chemistry because they exploit the molecular diversity to design, prepare, and test new chemical structures in view of the discovery of unprecedented pharmacophores. In this frame, the SELEX (Systematic Evolution of Ligands by EXponential enrichment) techniques, 1 relying on DNA or RNA libraries, come out particularly advantageous since nucleic acid sequences can be automatically synthesized in high yields and in a large variety. Most importantly, the species selected for affinity to the target can be easily amplified and further selected thus obtaining few species (aptamers) endowed with high affinity and selectivity for the target. Aptamers can adopt a number of conformational arrangements. Among these, the four-stranded DNA or RNA structures known as G-quadruplexes have emerged as nucleic acid architectures of remarkable significance 2 due to their dramatic thermal stability. An important class of potentially therapeutic aptamers are those endowed with antiviral activity. They can adopt monomolecular (anti-HIV integrase aptamer, T30695), 3 bimolecular (anti-HIV integrase aptamer, 93del) 4 and tetramolecular parallel (anti-HIV glycoprotein gp120 aptamer, ODN phosphorothioate ISIS5320) 5 G-quadruplex structures. Among these, the tetramolecular parallel ones, thanks to their shorter sequences and simpler strand arrangements, could be potentially more prone to be modified in order to improve their pharmacokinetic and pharmacodynamic properties in view of their use in therapy. In 1994 Hotoda et al. reported the anti-HIV-1 activity of a 6-mer ODN having the sequence TGGGAG and bearing a dimethoxytrityl group linked to its 5 0 hydroxyl function. 6 Subsequently, other modified ODNs were prepared and tested and the 6-mer bearing a 3,4-dibenzyloxybenzyl group at the 5 0 -end and a 2-hydroxy- ethylphosphate at the 3 0 -end (R-95288) showed the most potent activity and the least cytotoxicity. 7,8 CD investigations on R-95288 and similar ODNs suggested that they form parallel tetramolecular quadruplexes. 8 In a further study, the biophysical properties of the most interesting 5 0 -modified ODNs were investigated and it was established that the aromatic groups at the 5 0 -position of TGGGAG dramatically enhance the equilibrium and the rate of formation of the quadruplex complexes. 9,10 Furthermore, the overall stability of the investigated complexes was found to correlate with the reported anti-HIV activity, thus strongly suggesting that the G-quadruplex structures are the species responsible for the biological activity. 11 In 2004, we described the synthesis and characterization of a new quadruplex structure (tetra-end- linked oligonucleotides: TEL-ODNs) formed by a cluster of four d(TG 4 T) hexanucleotides linked together by their 3 0 -ends through a tetra-branched linker. 12 Subsequently, the new structure has been proven to possess a remarkable thermal stability compared to its natural counterpart. 13 In following studies, the effects on structural properties of different orientations of the sequences and linker size have been investigated. 14 In this communication we propose some TEL-ODNs containing the sequence TGGGAG as new modified aptamers and provide data on their properties and evidences for their noteworthy anti-HIV activity. Fig. 1 shows the ODNs involved in this study. TEL-ODNs were prepared according to the approach previously reported. 11 Fig. 1 Schematic structures of the investigated ODNs. a Dipartimento di Chimica delle Sostanze Naturali, Universita ` degli Studi di Napoli Federico II, Via D. Montesano 49, I-80131 Napoli, Italy. E-mail: galeone@unina.it, borbone@unina.it b CRUK Biomolecular Structure Group, The School of Pharmacy, University of London, 29–39 Brunswick Square, London, UK c Rega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium w Electronic supplementary information (ESI) available: Synthesis and characterization of TEL-ODNs, as well as CD, SPR and docking studies and biological evaluation assays. See DOI: 10.1039/c0cc02866e COMMUNICATION www.rsc.org/chemcomm | ChemComm Downloaded by Katholieke Universiteit Leuven on 29 November 2010 Published on 23 November 2010 on http://pubs.rsc.org | doi:10.1039/C0CC02866E View Online