The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5 and 3 ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2-20°C at 1 M dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC 50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.telomere ͉ DNA structure ͉ G-quartet T elomerase was first identified in ciliates (1). This enzyme is an essential factor in immortalization and tumorigenesis (2-4). Furthermore, telomerase is active in most human tumor cells and inactive in most somatic cells and is therefore an attractive target for the design of anticancer agents. Most human telomeric DNA is double-stranded and contains (TTAGGG͞CCCTAA) n repeats except for the extreme terminal part where the 3Ј region of the G-rich strand is singlestranded (5). For human cells, these 3Ј overhangs are surprisingly long (averaging 130-210 bases in length), exist during most of the cell cycle, and are present on all chromosomal ends. The G-rich single-stranded DNA can adopt an unusual four-stranded DNA structure involving G-quartets (6-8) (see Fig. 1) or it might fold back and displace one strand of a telomeric duplex to form a so-called T-loop (9). Optimal telomerase activity requires the nonfolded single-stranded form of the primer and G-quartet formation has been shown to directly inhibit telomerase elongation in vitro (10). Therefore, a drug that stabilizes quadruplexes could interfere with telomerase and telomere replication (11-13).The peculiar geometry of the quadruplex structure should allow its specific recognition by small synthetic ligands, and previous experiments have shown that this assumption is correct (14). Many of the G4 ligands were shown to have antitelomerase activity in vitro, with IC 50 in the low micromo...

Two synthetically modified nucleoside triphosphate analogues (adenosine modified with an imidazole and uridine modified with a cationic amine) are enzymatically polymerized in tandem along a degenerate DNA library for the combinatorial selection of an RNAse A mimic. The selected activity is consistent with both electrostatic and general acid/base catalysis at physiological pH in the absence of divalent metal cations. The simultaneous use of two modified nucleotides to enrich the catalytic repertoire of DNA-based catalysts has never before been demonstrated and evidence of general acid/base catalysis at pH 7.4 for a DNAzyme has never been previously observed in the absence of a divalent metal cation or added cofactor. This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.

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A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A-T base pairs to form TA-T base triplets and guanines recognize a run of G-C base pairs to form G-G-C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single GC base pair in a run of AT base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.Short oligonucleotides can bind to the major groove of double-stranded DNA at homopurine-homopyrimidine sequences. This was first demonstrated by sequence-specific cleavage with azidoproflavine used to photoinduce crosslinking of the oligonucleotide followed by alkaline cleavage (1) or EDTA-Fe to induce cleavage under reducing conditions (2). Oligonucleotides containing thymine and cytosine bind in a pH-dependent manner with a parallel orientation with respect to the homopurine strand of homopurinehomopyrimidine sequences on double-stranded DNA (3-12). Cytosine methylation was shown to stabilize these triple helices (5, 6) as previously observed on polydeoxynucleotides with alternating sequences (13). Purine-rich oligonucleotides can also bind to homopurine-homopyrimidine sequences (14, 15). The third strand binds in an antiparallel orientation with respect to the homopurine sequence in contrast to homopyrimidine oligonucleotides. This orientation was also found with oligonucleotides containing guanine and thymine or guanine, thymine, and adenine (14). However, energy minimization studies in our laboratory have suggested that third-strand orientation might depend on the number of ApG and GpA steps in the homopurine sequence (16, 17). Here we show that an oligonucleotide containing three bases (thymine, cytosine, and guanine) binds in a parallel orientation with respect to the homopurine sequence of a homopurine-homopyrimidine target of human immunodeficiency virus (HIV) proviral DNA (5'-A4GA4G6A-3' for the purine strand). Binding of this oligonucleotide to its target sequence depends very little on p...

DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them.

Because of their role in the control of the topological state of DNA, topoisomerases are ubiquitous and vital enzymes, which participate in nearly all events related to DNA metabolism including replication and transcription. We show here that human topoisomerase I (Topo I) plays an unexpected role of 'molecular matchmaker' for G-quartet formation. G-quadruplexes are multi-stranded structures held together by square planes of four guanines ('G-quartets') interacting by forming Hoogsteen hydrogen bonds. Topo I is able to promote the formation of four-stranded intermolecular DNA structures when added to single-stranded DNA containing a stretch of at least five guanines. We provide evidence that these complexes are parallel G-quartet structures, mediated by tetrads of hydrogen-bonded guanine. In addition, Topo I binds specifically to pre-formed parallel and anti-parallel G4-DNA.