Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease
© Skogen et al; licensee BioMed Central Ltd. 2006
Received: 11 April 2006
Accepted: 02 October 2006
Published: 02 October 2006
Huntington's Disease (HD) is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt) is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions.
As such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway. Here, we report that short guanosine monotonic oligonucleotides capable of adopting a G-quartet structure, are effective inhibitors of aggregation. By utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all G-oligonucleotide (HDG) as an active molecule. Subsequent testing in a cell-based assay revealed that HDG was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant Htt fragment and green fluorescent protein (eGFP). Taken together, our results suggest that a monotonic G-oligonucleotide, capable of adopting a G-quartet conformation is an effective inhibitor of aggregation. This oligonucleotide can also enable cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene.
Single-stranded DNA oligonucleotides capable of forming stable G-quartets can inhibit aggregation of the mutant Htt fragment protein. This activity maybe an important part of the pathogenecity of Huntington's Disease. Our results reveal a new class of agents that could be developed as a therapeutic approach for Huntington's Disease.
Huntington's Disease (HD) is an inherited disorder caused by expansions of CAG repeats (polyglutamine- polyQ) at the N-terminus, within exon 1, of the HD protein. The extent of polyglutamine expansion is correlated with the severity of the symptoms and their onset  while the pathology of the disease and neuronal cell death are thought to be associated with protein misfolding and protein aggregation. These aggregates are usually seen in the nucleus but can also be found in the cytoplasm . Protein aggregates develop via a complex biochemical process with intermediates being visible during the process. PolyQ tracts within the pathogenic range induce a protein insolubility whereas Htt with nonpathogenic length maintains a measured degree of solubility [3, 4].
Consistent with the aggregate toxicity hypothesis, inhibition of aggregate formation has been shown to have beneficial effects on the progression of HD in the R6/2 mouse model . The implication of the polyQ aggregates in cytotoxicity validates them as targets for novel therapeutics. Despite the lack of details surrounding the molecular structure of the polyQ aggregates, high throughput screening for compounds that inhibit their formation have produced some promising results. Several compounds, including Congo Red  and Clioquinol , have been reported to inhibit the aggregation process in the R6/2 mouse model but their neurotoxicity tempers enthusiasm. Thus, identifying molecules that show efficacy with minimal toxicity should be an important consideration in the search for HD therapeutics.
Synthetic oligonucleotides (ODNs) provide a model category of reagents that meet some of these requirements. Oligonucleotides are synthetic polymers that are produced in highly purified quantities in a cost-effective way and the technology surrounding ODN synthesis has advanced dramatically in the last 10 years. Recently, Parekh-Olmedo et al. (2004) showed that certain classes of ODNs can inhibit aggregation. One of these groups is the G-rich oligonucleotide (GROs) class which have been used previously as aptamers to block protein function. Specifically, GROs have been shown to bind directly to STAT3 and interact with regions of the protein that enable dimerization  and in another instance, GROs have been shown to block the integration of the HIV into the host chromosome by interacting with the HIV integrase [8, 9]. In both cases, the GRO forms a structure known as a G-quartet which arises from the association of four adjacent G-bases assembled into a cyclic conformation. These structures are stabilized by von Hoogstein hydrogen bonding  and by base stacking interactions. These molecules exhibit a very compact structure which allows them to interact productively with functionally important protein domains.
Much of the focus on developing therapeutics that block aggregate formation comes from a wealth of data associating HD pathogenesis with the presence of cellular inclusion bodies. But, recent evidence from in vitro [11–13] and in vivo [14–16] studies suggest that Htt inclusions may not be toxic to the cell or lead to neuronal degeneration. In fact, Hayden and colleagues have created an exciting mouse model that shows no long term effect of Htt inclusions on behavior or viability . It may be true that inclusion bodies are neuroprotective and eliminating them may actually increase the potential for neurotoxicity.
Because of the known biological activity of GROs in interacting with specific protein domains, we tested this type of oligonucleotide in assays that measure the aggregation activity of mutant Htt fragment. We report that G-rich oligonucleotides, which form G-quartets, inhibit aggregation of mutant htt fragment in biochemical and cell-based assays. Overall, our results suggest that G-rich oligonucleotides could be used to examine the relationship between cellular aggregates and toxicity in various model systems.
Biochemical analyses of GROs
Inhibition of aggregate formation in HEK293 cells
Discussions and conclusion
Intracellular aggregates of Htt have long been considered phenotypic evidence of the neurodegenerative disorder Huntington's Disease. It is, however, not clear how the appearance of such inclusion bodies relates to the pathogenesis of the disease. A number of model systems have been designed to screen for therapeutic agents that can inhibit aggregation. Some of these assays measure the inhibition of fusion protein aggregation, proteins containing a fragment of Htt (here, GST-Q58-Htn) and a marker/reporter protein, often eGFP. The Htt component of this fusion protein harbors an expanded polyQ stretch.
We have examined the capacity of G-rich oligonucleotides that can adopt a G-quartet conformation to block aggregation. A well-established biochemical assay was used to examine GROs blockage of aggregation. Molecules T40216 and T30923 that are known to form intermolecular G-quartets were found to be effective inhibitors of aggregation. Both of these GROs, which were used previously to inhibit Stat3, adopt conventional G-quartet structure with the G residues (quartets) in the center and a loop domain at the top and bottom . The GRO HDG, which exhibits the highest level of activity in the aggregation assays, can also adopt a stable G-quartet structure and further studies to elucidate the details of the G-quartet structure adopted by HDG are currently being performed. This molecule also may prevent or delay neurotoxicity in PC12 cells.
The GRO, HDG, is unique among monotonic oligonucleotides containing 20 bases. None of the related 20-mers, HDA, HDC or HDT show reproducible inhibitory activity in the biochemical or cell-based assays. Furthermore, HDG displays a dose response with concentrations as low as 1 μM exhibiting substantial levels of aggregate reduction. It is effective when added at the start of the Q58-Htn aggregation reaction but much less so when added after the process has begun (Skogen et al., in preparation). Thus, it is likely to block the nucleation phase of aggregation rather than the elongation phase, although our experiments were not designed to discriminate between these two phases [26, 27].
HDG is also quite active in blocking aggregation of the Httexon1-eGFP fusion protein aggregation in HEK293 cells. In this system, the fusion protein is produced from an expression plasmid and co-transfection with HDG prevents the appearance of green fluorescent foci in a dose-dependent inhibition. Importantly, the well-known aggregation inhibitor, Congo Red, was used as a positive control and displayed effects similar to HDG. MTT viability assays reveal no cell toxicity or negative effects on cell growth as a function of oligonucleotide addition (data not shown). This result is not surprising since ODNs used as antisense or antigene therapy have been found to be practically inert in human cells with regard to cytotoxicity. A number of clinical trials using ODNs have taken place and while the efficacy of such treatments may be questioned, significant adverse effects on cells or patients were not observed. The lack of serious side effects from oligonucleotides is a virtue in the development of these molecules for use in HD patients. For example, while the effective levels for GRO actvitiy presented in this work are higher than those used for traditional pharmaceuticals, oligonucleotides are particularly well-tolerated in humans. The level herein is not unusual and levels exceeding 50 mg/kg have been found to be both efficacious and nontoxic in various antisense therapies. The higher amounts may be required because delivery to target cells or penetration into the cells may be less efficient than other drug treatments.
The potency of the G-quartet structure of HDG is demonstrated further in the activity of the modified HDGs. Using a type of reverse genetics strategy, we created several "mutant" HDGs; HDG 20/7 wherein each 7th G was replaced with a T, HDG 20/3 wherein each 3rd G was replaced with a T and HDG 20/2 wherein every other G was substituted with a T residue. None of these molecules were found to be effective inhibitors of aggregation. The results presented in Figure 11 most clearly illustrate the importance of the HDG G-quartet structure while support for this notion is also gained when T30923 could not fully substitute for HDG in the HEK293 assay.
The mechanism by which HDG exhibits its effect in these assays is not clear, but several paths can be suggested. G-quartets created either by natural processes or by exogenous addition, are reactive structures in the cell. They were first recognized in studies aimed at understanding the mechanism of cellular aging  occurring naturally near the ends of chromosomes. These telomeric structures are composed of repetitive blocks of the double-stranded DNA sequence (TTAGGG)n with the guanines forming an overhang at the end of the telomere. In humans, these sequence blocks can become extremely long, increasing the probability that they adopt secondary (G-quartet) structures. Such structures are known to inhibit telomerase activity and may explain why telomerase replication/activity in transformed cells is often absent or reduced [29, 30].
G-quartets formed within GROs have also been shown to inhibit protein dimerization of such molecules as STAT3 . They exert their activity by binding to specific domains within STAT3 with a high degree of precision. Since mutant Htt aggregation relies on a nucleation phase in which the mutant protein begin to assemble, HDG could block the transition between nucleation and elongation as aggregation (dimerization) begins. Alternatively, HDG could block other enzymes involved in the development of the pathogenic phenotype, such as caspases which cleave the native protein perhaps producing a toxic fragment [31, 32]. Bates and colleagues have shown that certain GROs can bind to nucleolin in a variety of cancer cells with a high degree of specificity . In all of these cases, direct interactions with cellular proteins would be required and such a reaction is a well-documented characteristic of GROs. Studies are now underway to determine which proteins are binding to HDG and if there is a difference between the composition of protein-GRO complexes in mutant and wild-type cells.
Biochemical aggregation assay
To analyze the inhibition of aggregation by GROs, a biochemical assay was employed (Figure 1). The fusion protein GST-Q58-Htn  was incubated for 45 minutes at room temperature with thrombin (1 U/1 μg protein) at a concentration of 10 μg/ml in a buffer of 50 mM Tris-HCl, pH 8, 100 mM NaCl, 2.5 mM CaCl2, and 1 mM EDTA, to cleave between the huntingtin and GST. As indicated by Wang et al. , this fragment consists of the amino terminal 171 amino acids with a tract of 58 glutamine residues fused to GST. The protein mix was then centrifuged at 30,000 × g at 4°C for 35 minutes to remove any aggregates that had already formed. The protein was added to wells containing 0.5–60 μM GROs or control ODNs, 10 μM Congo Red, or no treatment in the buffer detailed above with 100 mM KCl replacing NaCl. The 0-hour control was stopped immediately and after 24 hours incubation at room temperature the remaining reactions were stopped by adding 10% SDS/50 mM 2-mercaptoethanol and heating to 99°C for five minutes. The mixture was diluted in 1X PBS and then filtered through a cellulose acetate membrane (Osmonics) using the Easy-Titer ELIFA system (Pierce) followed by a 2% SDS wash. After blocking in 5% milk/1X PBS-0.05% Tween, the membrane was incubated with a specific anti-huntingtin antibody (HP1, 1:1000 dilution), followed by incubation with a peroxidase-conjugated goat anti-rabbit antibody (Sigma, 1:40,000 dilution) and chemiluminescence reagent (ECL-Plus, Amersham). Signals from the aggregates retained on the filter were scanned and quantified using ImageQuant image analysis software (Molecular Dynamics). Aggregates were quantified by optical density and statistical differences were determined by one way ANOVA with Tukey's post hoc analysis using Statistical Package for the Social Sciences (SPSS). Significance was determined by a p < 0.05 as compared to Congo Red (control).
HEK-293T cell-based aggregation assay
Human embryonic kidney cells, HEK293T, were grown in low glucose DMEM supplemented with 10% FBS. Cells were seeded at 0.5 – 1 × 106 cells/well on 6-well plates. The cells were transfected with 1 μg of the plasmid pcDNA3.1-72Httexon1-eGFP (p72Q) and 150 – 750 nM GRO or control ODN using 2.5 μl Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection cells were viewed to determine the approximate number of green fluorescent foci using an Olympus IX50 microscope.
Circular dichroism spectroscopy
Circular dichroism spectra of 15 μM oligonucleotide samples in 10 mM KCl were recorded on an Aviv model 202 spectrometer. Measurements were performed at 24°C using a 0.1 cm path-length quartz cuvette (Hellma). The CD spectra were obtained by taking the average of two scans made at 1 nm intervals from 200 to 320 nm and subtracting the baseline value corresponding to that of buffer alone. Spectral data are expressed in units of millidegree.
PC12 viability assay
Rat pheochromocytoma cells, PC12, were grown in high glucose DMEM with 10% horse serum and 5% FBS while under selection with G418 (0.05 mg/mL) and Zeocin (0.1 mg/mL) (Invitrogen). This cell line, Htt14A2.6, expresses a truncated form of expanded repeat Htt exon 1 protein containing 1–17 amino acids and 103 polyglutamine tract fused to eGFP. The promoter was induced with muristerone resulting in the expression of the Htt exon 1 with expanded 103 CAG polyglutamine (103Q) region. Cells were seeded at 3 × 104 cells/well on a 24-well plate and transfected with a ratio of 0.8 μg HDG 20 to 2 μL Lipofectamine 2000 (Invitrogen) depending on the desired HDG concentration. After a 4-hour treatment, the transfection media was removed, whole media was added for 1-hour, and then the cells were induced using 5 μM muristerone for 24 hours. The Promega CellTiter-Glo Luminescent cell viability assay was then used. The control cells using only Lipofectamine 2000 were counted and plated at 2 × 104 cells in at least 6 wells of a 96-well plate. The same volume of cells used in this control at 24-hours, was used in the following treatments at that time point and the remaining 48 and 72-hour time points. After the cells were replated, an equal amount of cell viability substrate was added to each well, according to protocol. After the substrate is added, the plate was placed on a rocker for 2 mins then incubated for 10 mins. Finally, the plate was read 3 times per treatment on a Victor3V 1420 Multilabel counter and analyzed using the Wallac 1420 software.
This work was supported by research grant from the Huntington's Disease Society of America (HDSA) and express gratitude to Drs. James Pearson (Duke University) and Jin Wang (Harvard University) for advice on the screening assays.
- Landles C, Bates GP: Huntingtin and the molecular pathogenesis of Huntington's disease. Fourth in molecular medicine review series. EMBO Rep. 2004, 5: 958-963. 10.1038/sj.embor.7400250.PubMed CentralView ArticlePubMedGoogle Scholar
- DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N: Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science. 1997, 277: 1990-1993. 10.1126/science.277.5334.1990.View ArticlePubMedGoogle Scholar
- Scherzinger E, Sittler A, Schweiger K, Heiser V, Lurz R, Hasenbank R, Bates GP, Lehrach H, Wanker EE: Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology. Proc Natl Acad Sci U S A. 1999, 96: 4604-4609. 10.1073/pnas.96.8.4604.PubMed CentralView ArticlePubMedGoogle Scholar
- Scherzinger E, Lurz R, Turmaine M, Mangiarini L, Hollenbach B, Hasenbank R, Bates GP, Davies SW, Lehrach H, Wanker EE: Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell. 1997, 90: 549-558. 10.1016/S0092-8674(00)80514-0.View ArticlePubMedGoogle Scholar
- Sanchez I, Mahlke C, Yuan J: Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature. 2003, 421: 373-379. 10.1038/nature01301.View ArticlePubMedGoogle Scholar
- Slow EJ, Graham RK, Osmand AP, Devon RS, Lu G, Deng Y, Pearson J, Vaid K, Bissada N, Wetzel R, Leavitt BR, Hayden MR: Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions. Proc Natl Acad Sci U S A. 2005, 102: 11402-11407. 10.1073/pnas.0503634102.PubMed CentralView ArticlePubMedGoogle Scholar
- Jing N, Li Y, Xu X, Sha W, Li P, Feng L, Tweardy DJ: Targeting Stat3 with G-quartet oligodeoxynucleotides in human cancer cells. DNA Cell Biol. 2003, 22: 685-696. 10.1089/104454903770946665.View ArticlePubMedGoogle Scholar
- Jing N, De Clercq E, Rando RF, Pallansch L, Lackman-Smith C, Lee S, Hogan ME: Stability-activity relationships of a family of G-tetrad forming oligonucleotides as potent HIV inhibitors. A basis for anti-HIV drug design. J Biol Chem. 2000, 275: 3421-3430. 10.1074/jbc.275.5.3421.View ArticlePubMedGoogle Scholar
- Mazumder A, Neamati N, Ojwang JO, Sunder S, Rando RF, Pommier Y: Inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures. Biochemistry. 1996, 35: 13762-13771. 10.1021/bi960541u.View ArticlePubMedGoogle Scholar
- Sen D, Gilbert W: A sodium-potassium switch in the formation of four-stranded G4-DNA. Nature. 1990, 344: 410-414. 10.1038/344410a0.View ArticlePubMedGoogle Scholar
- Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S: Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature. 2004, 431: 805-810. 10.1038/nature02998.View ArticlePubMedGoogle Scholar
- Kim M, Lee HS, Laforet G, McIntyre C, Martin EJ, Chang P, Kim TW, Williams M, Reddy PH, Tagle D, Boyce FM, Won L, Heller A, Aronin N, DiFiglia M: Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition. J Neurosci. 1999, 19: 964-973.PubMedGoogle Scholar
- Saudou F, Finkbeiner S, Devys D, Greenberg ME: Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell. 1998, 95: 55-66. 10.1016/S0092-8674(00)81782-1.View ArticlePubMedGoogle Scholar
- Klement IA, Skinner PJ, Kaytor MD, Yi H, Hersch SM, Clark HB, Zoghbi HY, Orr HT: Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell. 1998, 95: 41-53. 10.1016/S0092-8674(00)81781-X.View ArticlePubMedGoogle Scholar
- Stenoien DL, Cummings CJ, Adams HP, Mancini MG, Patel K, DeMartino GN, Marcelli M, Weigel NL, Mancini MA: Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone. Hum Mol Genet. 1999, 8: 731-741. 10.1093/hmg/8.5.731.View ArticlePubMedGoogle Scholar
- Bowman AB, Yoo SY, Dantuma NP, Zoghbi HY: Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation. Hum Mol Genet. 2005, 14: 679-691. 10.1093/hmg/ddi064.View ArticlePubMedGoogle Scholar
- Parekh-Olmedo H, Wang J, Gusella JF, Kmiec EB: Modified single-stranded oligonucleotides inhibit aggregate formation and toxicity induced by expanded polyglutamine. J Mol Neurosci. 2004, 24: 257-267. 10.1385/JMN:24:2:257.View ArticlePubMedGoogle Scholar
- Huang CC, Faber PW, Persichetti F, Mittal V, Vonsattel JP, MacDonald ME, Gusella JF: Amyloid formation by mutant huntingtin: threshold, progressivity and recruitment of normal polyglutamine proteins. Somat Cell Mol Genet. 1998, 24: 217-233. 10.1023/B:SCAM.0000007124.19463.e5.View ArticlePubMedGoogle Scholar
- Wang J, Gines S, MacDonald ME, Gusella JF: Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation. BMC Neurosci. 2005, 6: 1-10.1186/1471-2202-6-1.PubMed CentralView ArticlePubMedGoogle Scholar
- Macaya RF, Schultze P, Smith FW, Roe JA, Feigon J: Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. Proc Natl Acad Sci U S A. 1993, 90: 3745-3749. 10.1073/pnas.90.8.3745.PubMed CentralView ArticlePubMedGoogle Scholar
- Heiser V, Scherzinger E, Boeddrich A, Nordhoff E, Lurz R, Schugardt N, Lehrach H, Wanker EE: Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy. Proc Natl Acad Sci U S A. 2000, 97: 6739-6744. 10.1073/pnas.110138997.PubMed CentralView ArticlePubMedGoogle Scholar
- Hardin CC, Henderson E, Watson T, Prosser JK: Monovalent cation induced structural transitions in telomeric DNAs: G-DNA folding intermediates. Biochemistry. 1991, 30: 4460-4472. 10.1021/bi00232a013.View ArticlePubMedGoogle Scholar
- Balagurumoorthy P, Brahmachari SK: Structure and stability of human telomeric sequence. J Biol Chem. 1994, 269: 21858-21869.PubMedGoogle Scholar
- Balagurumoorthy P, Brahmachari SK, Mohanty D, Bansal M, Sasisekharan V: Hairpin and parallel quartet structures for telomeric sequences. Nucleic Acids Res. 1992, 20: 4061-4067.PubMed CentralView ArticlePubMedGoogle Scholar
- Apostol BL, Kazantsev A, Raffioni S, Illes K, Pallos J, Bodai L, Slepko N, Bear JE, Gertler FB, Hersch S, Housman DE, Marsh JL, Thompson LM: A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila. Proc Natl Acad Sci U S A. 2003, 100: 5950-5955. 10.1073/pnas.2628045100.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang W, Dunlap JR, Andrews RB, Wetzel R: Aggregated polyglutamine peptides delivered to nuclei are toxic to mammalian cells. Hum Mol Genet. 2002, 11: 2905-2917. 10.1093/hmg/11.23.2905.View ArticlePubMedGoogle Scholar
- Chen S, Berthelier V, Hamilton JB, O'Nuallain B, Wetzel R: Amyloid-like features of polyglutamine aggregates and their assembly kinetics. Biochemistry. 2002, 41: 7391-7399. 10.1021/bi011772q.View ArticlePubMedGoogle Scholar
- Zakian VA: Telomeres: beginning to understand the end. Science. 1995, 270: 1601-1607.View ArticlePubMedGoogle Scholar
- Sun D, Thompson B, Cathers BE, Salazar M, Kerwin SM, Trent JO, Jenkins TC, Neidle S, Hurley LH: Inhibition of human telomerase by a G-quadruplex-interactive compound. J Med Chem. 1997, 40: 2113-2116. 10.1021/jm970199z.View ArticlePubMedGoogle Scholar
- Fedoroff OY, Salazar M, Han H, Chemeris VV, Kerwin SM, Hurley LH: NMR-Based model of a telomerase-inhibiting compound bound to G-quadruplex DNA. Biochemistry. 1998, 37: 12367-12374. 10.1021/bi981330n.View ArticlePubMedGoogle Scholar
- Bates G: Huntingtin aggregation and toxicity in Huntington's disease. Lancet. 2003, 361: 1642-1644. 10.1016/S0140-6736(03)13304-1.View ArticlePubMedGoogle Scholar
- Ross CA, Poirier MA, Wanker EE, Amzel M: Polyglutamine fibrillogenesis: the pathway unfolds. Proc Natl Acad Sci U S A. 2003, 100: 1-3. 10.1073/pnas.0237018100.PubMed CentralView ArticlePubMedGoogle Scholar
- Dapic V, Abdomerovic V, Marrington R, Peberdy J, Rodger A, Trent JO, Bates PJ: Biophysical and biological properties of quadruplex oligodeoxyribonucleotides. Nucleic Acids Res. 2003, 31: 2097-2107. 10.1093/nar/gkg316.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.