DOI: 10.1002/cbic.201200481 Structural Investigations on the Anti-HIV G-Quadruplex- Forming Oligonucleotide TGGGAG and Its Analogues: Evidence for the Presence of an A-Tetrad Antonella Virgilio, Veronica Esposito, Giuseppe Citarella, Luciano Mayol, and Aldo Galeone* [a] Introduction The oligonucleotide-based therapeutic approach has provided unprecedented opportunities for the discovery and develop- ment of new drugs. In fact, therapeutic oligonucleotides [1] as antisense [2, 3] or antigene [3] oligodeoxynucleotides, siRNA, miRNA, ribozymes, and aptamers [4, 5] are the subject of a ple- thora of investigations in several research fields, from chemis- try to molecular biology and pharmacology. Among these, ap- tamers are considered particularly promising because of their dramatic affinity and specificity towards given targets. A re- markable example is Pegaptanib, an anti-VEGF (vascular endo- thelial growth factor) aptamer for ocular vascular disease, recently approved by the FDA. [6] As the structural stability of an aptamer is a key factor (often associated with its biological activity), several aptamers, discovered both fortuitously and by advanced selection techniques, adopt G-quadruplex DNA structures, [7] which have proven to be among the most stable structural motifs that nucleic acid sequences can form. [8] A noteworthy class of therapeutically promising aptamers are those with anti-HIV activity. [9] Aptamers targeting the HIV pro- teins gp120 and integrase have been developed. Although they all form G-quadruplexes, their structures are characterized by different molecularities. For example, the anti-integrase ap- tamers T30695 [10] adopts a monomolecular quadruplex, where- as 93del [11] adopts a bimolecular quadruplex. The bimolecular structure adopted by the aptamer 93del was investigated by NMR in 2005, [12] whereas the quadruplex topology of T30695 was established only recently. [13] In contrast, the sequences of the anti-gp120 aptamers ISIS5320 [14, 15] and “Hotoda’s apta- mer” [16] contain only a G-run, and thus prefer to adopt parallel tetramolecular quadruplex structures. Surprisingly, although Hotoda’s aptamer was one of the first anti-HIV aptamer to be discovered [17] and has recently been the topic of much re- search, [18, 19] no detailed information about its structure is known. It should be emphasized that information concerning the structural details of aptamers is particularly important to gain a better comprehension of the factors involved in the interactions with the targets. This information is particularly useful in designing new derivatives aimed both to improve their biological activity and to increase their resistance in bio- logical environments (for employment as therapeutic agents). In this paper we report our investigations, based on UV, CD, NMR, and electrophoretic techniques on Hotoda’s aptamer and its analogues with related sequences (Table 1). Results and Discussion The original (and shortest) version of Hotoda’s aptamer is a hexamer ODN of sequence TGGGAG that bears a 4,4’-dimeth- oxytrityl group at the 5’-end (LH). [20] Subsequently, other groups have been linked to the 5’-hydroxyl function. [18, 19] Al- though the CD profile of LH (Figure 1) is typical of a tetramo- [a] Dr. A. Virgilio, + Prof. V. Esposito, + Dr. G. Citarella, Prof. L. Mayol, Prof. A. Galeone Dipartimento di Chimica delle Sostanze Naturali Università degli Studi di Napoli Federico II Via D. Montesano 49, I-80131 Napoli (Italy) E-mail : galeone@unina.it [ + ] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201200481. Several anti-HIV aptamers adopt DNA quadruplex structures. Among these, “Hotoda’s aptamer” (base sequence TGGGAG) was one of the first to be discovered. Although it has been the topic of some recent research, no detailed structural investiga- tions have been reported. Here we report structural investiga- tions on this aptamer and analogues with related sequences, by using UV, CD, and NMR spectroscopy as well as electropho- retic techniques. The addition of a 3’-end thymine has allowed us to obtain a single, investigable quadruplex structure. Data clearly point to the presence of an A-tetrad. Furthermore, the effects of the incorporation of an 8-methyl-2’-deoxyguanosine at the 5’-end of the G-run were investigated. Table 1. Oligonucleotides synthesized and their apparent melting tem- peratures. Name Sequence 5’-3’ T 1/2 [8C] Name Sequence 5’-3’ T 1/2 [8C] H TGGGAG 59 LHT L-TGGGAGT [a] 75 HT TGGGAGT 59 HM TMGGAG [b] 64 LH L-TGGGAG [a] 75 HTM TMGGAGT [b] 64 [a] L = DMT group. [b] M = 8-methyl-2’-deoxyguanosine. ChemBioChem 2012, 13, 2219 – 2224  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2219