The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.The Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, is associated with the development of different epithelial malignancies (28), including nasopharyngeal carcinoma (NPC). It has also been linked with subsets of other types of carcinomas, including gastric carcinoma and lymphoepithelioma-like carcinoma in salivary glands and thymus (32,44). Several laboratories have reported detection of EBV in a subset of breast tumors (3,10,19,23). However, negative results have also been reported (6,9,14,27). Nevertheless, in most of the studies, a low viral load was detected in breast cancer biopsy specimens but the infected cells were not clearly identified. In the study presented here, we used real-time PCR (Q-PCR) to quantify the copy numbers of the EBV genome in biopsy specimens as well as in microdissected tumor cells. The results show that breast cancer cells harbor the viral genome. However, through microdissection and isolation of pure tumor cells, we now find that even in EBV-positive tumor samples, many tumor cells do not contain EBV genomes and that the breast carcinomas are highly heterogeneous in terms of genome content and distribution. Moreover, using reverse transcription-PCR (RT-PCR), we detected EBNA-1 and BARF-1 transcripts in almost all of the EBV-positive tumors and LMP-1 RNA in 3 of the 15 cases studied. The findings raise the possibility that although EBV is unlikely to have an etiologic role in the genesis of breast cancer, the virus might contribute to tumor progression. Finally, the potential impact of EBV in breast cancer progression was evaluated by estimation of resistance to chemotherapeutic agents on in vitro-infected MDA-MB-231 cells. The resul...

The 3' strand of telomeres is composed of tandem repeats of short G-rich sequences which protrude as single-stranded DNA overhangs. These repeats are G(3)T(2)A in humans and G(4)T(2) and G(4)T(4) in the ciliates Tetrahymena and Oxytricha, respectively. We analyzed different quadruplex-forming sequences derived from telomeric sequences, G(3+k)(T(n+k)G(3+k))(3) and G(3+k)(T(2)AG(3+k))(3), in the presence of Li(+), Na(+), and K(+) through the use of circular dichroism, UV spectroscopy, and electrophoresis, where k = 0 or 1 and n = 1-3. Results obtained under the given conditions can provide more detailed information about the quadruplex structure. The major findings are as follows. (i) G-Repeats in solution form a mix of topologically different structures; only G(3)(T(2)G(3))(3) and G(3)(TG(3))(3) repeats preferentially form the parallel interstrand structure. (ii) The Tetrahymena repeat can form at least two intramolecular conformers with different strand orientations and levels of stability. (iii) G-Quadruplex conformation and molecularity strongly depend on the type and concentration of ions used in the solution. The formation of intramolecular quadruplexes is governed by the length of the loops connecting G-runs. Intermolecular G-quadruplex forms are more likely to form in a higher concentration of ions for sequences where G-runs are separated by only one or two nucleotides.

Infection with human papillomaviruses (HPVs) is one of the most common sexually transmitted infections and can lead to development of head and neck, skin, and anogenital cancer, including cervical cancer, which represents one of the world's most significant health problems. In this study, we analyze G-rich regions in all known HPV genomes in order to evaluate their potential to fold into G-quadruplex structure. Interestingly, G-rich loci fulfilling the criteria for G-quadruplex formation were found in only 8 types of HPV. Nevertheless, viral G-quadruplexes in 7 sequences derived directly from HPVs are confirmed here for the first time. G-rich regions with the capacity to form G-quadruplexes are located in the LCR, L2, E1, and E4 regions of the HPV genome; therefore we assume that regulation processes in viruses could be affected by G-quadruplex formation. Our results represent a starting point for the design of specific ligands with viral G-quadruplex motifs and suggest novel methods for the control of viral replication and transcription.

We herein report on the formation and high-resolution NMR solution-state structure determination of a G-quadruplex adopted by d[G3ATG3ACACAG4ACG3] comprised of four G-tracts with the third one consisting of four guanines that are intervened with non-G streches of different lengths. A single intramolecular antiparallel (3+1) G-quadruplex exhibits three stacked G-quartets connected with propeller, diagonal and edgewise loops of different lengths. The propeller and edgewise loops are well structured, whereas the longer diagonal loop is more flexible. To the best of our knowledge, this is the first high-resolution G-quadruplex structure where all of the three main loop types are present.

G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of G-runs alone is not sufficient to prove that such sequences can form stable G-quadruplexes. While G-rich sequences are essential for G-quadruplex formation, not all G-rich sequences have the propensity to form G-quadruplex structures. In addition, monovalent metal ions, dehydrating agents, and loop sequences connecting the G-runs also play important roles in the topology of G-quadruplex folding. To date, no quantitative analysis of the CD spectra of G-quadruplexes in confrontation with the electrophoretic results has been performed. Therefore, in this study, we use information gained through the analysis of a series of well-known G-quadruplex-forming sequences to evaluate other less-studied sets of aptameric sequences. A simple and cost-effective methodology that can verify the formation of G-quadruplex motifs from oligomeric DNA sequences and a technique to determine the molecularity of these structures are also described. This methodology could be of great use in the prediction of G-quadruplex assembly, and the basic principles of our techniques can be extrapolated for any G-rich DNA sequences. This study also presents a model that can predict the multimerization of G-quadruplexes; the predictions offered by this model are shown to match the results obtained using circular dichroism.

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function.

Abstract. Guanine quadruplex (G-quadruplex) structures are one of a number of structures which are capable of adopting aptamers. G-rich DNA or RNA has an increased propensity to form quadruplex structures which have unusual biophysical and biological properties. G-rich aptamers which form G-quadruplexes have several advantages over unstructured sequences: G-quadruplexes are non-immunogenic, thermodynamically and chemically stable and they have both higher resistance to various serum nucleases and an enhanced cellular uptake. These advantages have led to a number of synthetic oligonucleotides being studied for their potential use as therapeutic agents for cancer therapy and in the treatment of various other diseases. In addition to their suitability in the fields of medicine and biotechnology, these, highly specified, aptameric G-quadruplexes also have great potential in the further development of nano-devices; e.g. basic components in microarrays, microfluidics, sandwich assays and electrochemical biosensors. This review summarizes the biophysical properties of G-quadruplexes and highlights the importance of the stability and recognition properties of aptamers. Examples of the application of aptamers in medical therapy and in biosensors are also discussed.

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